Download Identification of a novel Endoplasmic Reticulum Stress Response

Identification of a novel Endoplasmic Reticulum Stress Response Element
regulated by XBP1
Michael Misiewicz
260182952
Department of Anatomy and Cell Biology
McGill University, Montreal
August 2012
A thesis submitted to the Graduate Studies and Research of McGill University in partial fulfillment of
the requirements of the degree of Master of Science.
© Michael Misiewicz 2012
Abstract
The Prion protein (PrP), which is the causative agent of scrapie diseases, has a still
unclear physiological function, despite 30 years of research on its nature. However, a
preponderance of evidence is beginning to support the idea that cellular prion protein
(PrPC) has a pro-survival function. Here, we study the regulation of the prion protein
gene (PRNP) in this context, as the regulation of the PRNP gene is not well understood.
By homology, we identified in the PRNP a novel promoter element which bears
similarity to the Endoplasmic Reticulum Stress Response Element (ERSE). This novel
ERSE (“ERSE-26”) is able to regulate PRNP endogenously in response to endoplasmic
reticulum (ER) stress. In order to determine whether or not the ERSE-26 exists elsewhere
in the genome and what is co-regulated with PRNP, we conducted a bioinformatic
search and identified 38 other genes with an ERSE-26. Their expression was confirmed
by treating cultured primary human neurons and MCF-7 cells with the ER stressors
Brefeldin A, Tunicamycin and Thapsigargin and conducting Reverse Transcriptase PCR
or Quantitative PCR. We found that the genes SESN2, GADD45B and PRNP were
significantly upregulated, and others showed an upward trend. Finally, a luciferase
reporter construct containing the ERSE-26 only was used to identify that the ER stress
transcription factor XBP1 is a transcription factor that induces ERSE-26 activity. Finally,
we conducted a literature search to determine what functions of the cell are co-regulated
with PRNP with the ERSE-26. Oxidative stress response and pro-survival genes were
found in the ERSE-26 genes and found to be the most upregulated by the ERSE-26,
strengthening the case for PrPC as a pro-survival gene.
Misiewicz 2
Résumé
La protéine Prion (PrP), qui est l'agent infectieux causant les encéphalopathies
transmissibles, n'a pas toujours un rôle bien identifié dans la cellule, malgré 30 ans de
recherche sur sa fonction physiologique. Cependant, de plus en plus de preuves
commencent à impliquer PrP dans des fonctions de protection dans la cellule. Dans cette
étude, nous avons étudié la régulation peu connue du promoteur du gène qui encode
PrP (PRNP). Par homologie de séquence, nous avons identifié un nouvel élément dans le
promoteur de PRNP qui ressemble à l'Endoplasmic Reticulum Stress Response Element
(ERSE). Ce nouvel ERSE (appelé ERSE-26) est capable de réguler l'expression du PRNP
de manière endogène en réponse au stress dans le réticulum endoplasmique (RE). Pour
savoir si l'ERSE-26 existe ailleurs dans le génome et afin de trouver d'autres gènes régulé
avec PRNP, nous avons fait une recherche bioinformatique dans le génome entier. Nous
avons identifié 38 gènes contenant aussi un ERSE-26 dans leur promoteur. Afin de
confirmer l'expression de ces gènes en réponse au stress ER, nous avons traité des
cultures de neurones primaires humains et des cellules MCF-7 avec les activateurs du
stress RE Brefeldin A, Tunicamycin et Thapsigargin, puis vérifié l'expression par
Transcriptase Inverse PCR (RT-PCR) ou RT-PCR quantitative. Nous avons montré
l'induction des gènes GADD45B, SESN2 et PRNP, et d'autres ont montré une tendance
positive. Ensuite, un plasmide rapporteur luciferase contenant l'ERSE-26 seulement a été
utilisé pour montrer que le facteur de transcription du stress ER XBP1 est un facteur de
transcription responsable pour l'activité de l'ERSE-26. Finalement, nous avons fait une
recherche dans la littérature afin de déterminer la fonction des gènes contenant ERSE-26.
Les gènes répondant au stress oxydant et les gènes pro-survie étaient parmi les gènes
ERSE-26, et aussi ont été le plus induits, soutenant le rôle protecteur du PrP dans la
cellule.
Misiewicz 3
Preface
This thesis focuses on the regulation of the prion protein encoding gene and the
discovery and characterization of a novel DNA motif that regulates prion and other gene
expression in response to endoplasmic reticulum stress. It contains three chapters: a
literature review, the novel research data in a manuscript submitted for publication and
a conclusion.
Manuscript contained in thesis:
Identification of a novel Endoplasmic Reticulum Stress Response Element regulated by
XBP1.
Misiewicz 4
Acknowledgments
I'd like to thank my supervisors, Dr. LeBlanc and Dr. Ruths for their great assistance.
Under their scholarship I have learned a great deal during my Master's, and I am very
grateful for their time and financial support. I'd also like to thank my lab members for
their input, assistance and opinions. In particular, I'd like to thank Dr. Julie Jodoin for
her help during my undergraduate degree and the beginning of my Master’s. I'd also
like to thank Dr. Vikas Kushal for his great assistance in culturing primary human fetal
neurons obtained from the University of Washington Tissue Bank.
Misiewicz 5
Abbreviations
AD
Alzheimer Disease
ADP
Adenosine diphosphate
AMV-RT
Avian myeloblastosis virus reverse transcriptase
ATF4
Activating Transcription Factor 4
ATF6
Activating Transcription Factor 6
ATP
Adenosine triphosphate
Bax
BCL-2 Associated protein X
BCL2
B-Cell Lymphoma 2
BFA
Brefeldin A
BH2
Bcl Homology Domain 2
BiP
Binding Immunoglobin Protein
BLAST
Basic Linear Alignment and Search Tool
bp
Basepairs
BSE
Bovine spongiform encephalopathy
cDNA
Complimentary DNA
ChIP
Chromatin Immunoprecipitation
CHOP
C/EBP homologous protein
CJD
Creutzfeldt–Jakob disease
CNS
Central Nervous System
CyPrP
Cytosolic PrP
DMSO
Dimethyl sulfoxide
eIF2α
Eukaryotic initiation factor 2 alpha
ER
Endoplasmic Reticulum
ERAD
ER associated decay
ERSE
Endoplasmic Reticulum Stress
FFI
Fatal Familial Insomnia
FSA
Finite state automaton/Finite state automata
GABA
γ-Aminobutyric acid
GADD45B
Growth Arrest and DNA Damage Inducible 45 kDa Beta
GPI
Glycophosphatidylinositol
Misiewicz 6
GRC
Genome Reference Commission
GSS
Gerstmann–Sträussler–Scheinker syndrome
HERP
Homocysteine-inducible, endoplasmic reticulum stress-inducible
HMM
Hidden Markov Model
IRE1α
Inositol requiring enzyme 1 alpha
mRNA
Messenger RNA
NF-Y
Nuclear Transcription Factor Y
ORF
Open reading frame
PCR
Polymerase chain reaction
PDI
Protein Disulfide Isomerase
PEI
Polyethylenimine
PERK
PKR-like endoplasmic reticulum kinase
PRNP
Human Prion Protein Gene
Prnp
Mouse Prion Protein Gene
PrP
Prion Protein
PrPC
Prion protein, normal cellular form
PrPSc
Prion protein, scrapie form
qPCR
Quantitative Polymerase Chain Reaction
ROS
Reactive Oxygen Species
RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
S1P
Site 1 Protease
S2P
Site 2 Protease
SDS-PAGE Sodium dodecyl sulfate polyacrylimide gel electrophoresis
sXBP1
Spliced XBP1
TAE
Tris, Acetic Acid, EDTA
Thps
Thapsigargin
TM
Tunicamycin
TNFα
Tumor Necrosis Factor
UPRE
Unfolded Protein Response element
XBP1
X-box binding protein 1
Misiewicz 7
List of figures
Figure 1 – Known regulatory elements in the PRNP promoter
Pg. 17
Figure 2 – Topology and transmembrane forms of PrP.
Pg. 24
Figure 3 – ERSE-26 activity is XBP1 dependent.
Pg. 64.
Figure 4 – Induction of ER stress increases ERSE-26 gene expression Pg. 71.
in cultured primary human neurons.
Figure 5 – MCF-7 cells show induced ERSE-26 gene expression
Pg 74.
following ER stress treatment or sXBP1 transfection
Table 1 – Genes containing an ERSE-26 in their promoter.
Pg. 67.
Supplemental Figure 1 – Activity of secreted luciferase in N2a cells Pg. 65.
transfected with pML2-EL26 and sXBP1.
Supplemental Table I – Primers used in RT-PCR
Pg. 57.
Supplemental Table II – Primers used in qRT-PCR.
Pg. 58.
Supplemental Table III – Genes containing and ERSE in their Pg. 68.
promoter
Misiewicz 8
Table of Contents
Abstract ...................................................................................................................................... 2
Résumé ....................................................................................................................................... 3
Preface ........................................................................................................................................ 4
Acknowledgments ................................................................................................................... 5
Abbreviations ............................................................................................................................ 6
List of figures ............................................................................................................................. 8
Table of Contents ....................................................................................................................... 9
Chapter 1 – Literature Review ............................................................................................... 12
Introduction ............................................................................................................................. 12
1 Introduction to prion disease and the prion gene ........................................................ 13
1.1 The human PRNP gene and Prn locus .................................................................. 15
1.1.1 Structure of the PRNP gene .......................................................................................... 15
1.1.1.1 PRNP promoter structure ....................................................................... 16
1.1.1.2 Intron and exons in PRNP and Prn locus members .............................18
1.1.2 Shadoo and Doppel .......................................................................................................18
1.1.3 Patterns of constitutive PNRP expression .................................................................... 19
1.1.3.1 Signal dependent PRNP expression ...................................................... 20
1.1.3.2 Expression of PRNP in cancer and disease ........................................... 20
1.2 Functions and topology of prion protein .............................................................. 21
1.2.1 Topology of PrP ............................................................................................................... 21
1.2.2 Processing and localization ............................................................................................ 22
1.2.2.1 Signal peptides ......................................................................................... 22
1.2.2.2 Secretion .................................................................................................... 23
1.2.2.3 Post-translational modifications .............................................................23
1.2.3 Protective functions ........................................................................................................ 25
1.2.3.1 Anti-Bax function ..................................................................................... 25
1.2.3.2 Copper metabolism and redox activity ................................................. 26
2 The endoplasmic reticulum: functions and properties ................................................27
2.1 Protein folding .......................................................................................................... 28
Misiewicz 9
2.1.1 Mechanisms of enzymatic activity for protein folding in the ER .............................. 28
2.1.2 Post-translational modifications and sorting ............................................................... 29
2.2 ER Stress .................................................................................................................... 30
2.2.1 Discovery and definition ................................................................................................ 30
2.2.2 ER Stress signal transduction .........................................................................................31
2.2.2.1 IRE1α and XBP1 ....................................................................................... 31
2.2.2.2 Site 1 and Site 2 proteases and ATF6 .....................................................32
2.2.2.3 PKR-like endoplasmic reticulum kinase and ATF4 .............................33
2.2.3 Description of the ER stress response program ........................................................... 33
2.2.3.1 Translational ............................................................................................. 34
2.2.3.2 Transcriptional ..........................................................................................35
2.2.4 Regulation of ER stress mediated transcription ......................................................... 35
2.2.4.1 ERSE .......................................................................................................... 36
2.2.4.2 ERSE II ....................................................................................................... 36
2.2.4.3 ERSE-like ................................................................................................... 37
2.2.4.4 UPRE ......................................................................................................... 37
2.2.5 Exit from ER stress .......................................................................................................... 37
2.2.5.1 Restoration of ER homeostasis ............................................................... 38
2.2.5.2 Activation of apoptosis ............................................................................38
2.2.6 ER Stress and Disease ..................................................................................................... 39
3 Study of the Human Genome ......................................................................................... 40
3.1 Sequencing ................................................................................................................ 40
3.1.1 The current state of the art of genome sequencing ..................................................... 41
3.1.2 The Human Reference Genome .....................................................................................41
3.2 Annotations and the definition of loci and their promoters ...............................42
4 Computational pattern matching algorithms in biological sequences ......................43
4.1 Regular expressions ................................................................................................. 44
5 Methods of detecting gene expression levels ............................................................... 45
5.1 Quantitative Real Time PCR ................................................................................... 45
6 Working hypothesis and research objectives ................................................................ 46
Chapter 2 – Manuscript .......................................................................................................... 49
Preface ...................................................................................................................................... 49
Misiewicz 10
Author Contributions ............................................................................................................. 49
Identification of a novel Endoplasmic Reticulum Stress Response Element regulated by
XBP1 ............................................................................................................................................50
Chapter 3 – General Discussion ............................................................................................ 81
Conclusion ............................................................................................................................... 84
References ................................................................................................................................ 85
Ethics approval ........................................................................................................................ 99
Misiewicz 11
Chapter 1 – Literature Review
Introduction
The prion protein is the causative agent of several fatal and incurable neurodegenerative
diseases. It was originally discovered to be the first transmissible disease that does not
contain nucleic acid, a controversial finding. Although it may also be the cause of
disease, the prion protein also has a normal, non-disease state. Given its conservation
across the animal kingdom, prion protein must have an important role in the cellular
context.
This role has been difficult to precisely define, despite 30 years of research on
physiological as well as disease prion. Nevertheless, a plurality of evidence now depicts
prion as a protective protein in its normal state. Prion protein inhibits activators of
apoptosis, and mice lacking prion show subtle but noticeable defects over time. Prion
has been identified as a binder of copper ions in the cell, and exhibits anti-superoxide
activity. Finally, prion has been identified as a gene upregulated in response to oxidative
stress.
However,
the regulation of prion expression is not well understood. The
promoter of the gene has been poorly studied, and a better understanding of the
promoter could provide insight into the activation and function of prion protein. In
order to address this question, our lab has investigated the prion protein promoter. We
identified a novel Endoplasmic Reticulum Stress Response element which is capable of
transactivating prion expression in response to Endoplasmic Reticulum Stress. In this
thesis, I will describe the search for this element in the entire human genome as well as a
characterization of the transcription factors that activate it. Finally, by examining what
genes contain this novel element, insight into the function of prion is gained.
Misiewicz 12
1
Introduction to prion disease and the prion gene
Prion diseases are progressive fatal neurodegenerative diseases with no known cure.
Prion diseases can be inherited, arise sporadically, and even be transmitted between
hosts due to ingestion of contaminated tissues or iatrogenically through tissue transfers.
Many different prion diseases have been described, each with distinct symptoms and
presentations (Norrby, 2011).
Prion diseases have been well known in livestock for some time, since the mid
18th century (Parry, 1962). Scrapie in sheep and Bovine Spongiform Encephalopathy
(BSE) have been widely reported in the media and are a concern for many public health
officials due the apparent transmissible nature of these diseases. In humans, the most
common is Creutzfeldt–Jakob disease (CJD), which can be contracted genetically,
sporadically, or through ingestion of contaminated food. However other known scrapies
in humans include Fatal Familial Insomnia (FFI), Kuru, and Gertsman Strankler
Strausser syndrome (GSS). In cervids, a current area of concern is Chronic Wasting
Disease, which affects deer and moose in the western part of North America. While each
prion disease presents different symptoms, all are neurodegenerative diseases which
result in death (Norrby, 2011).
One of the most surprising aspects of scrapies is the apparent lack of traditional
infectious agents. It was not until 1982 that the causes of scrapies were demonstrated to
be an infectious protein, which when mis-folded became protease resistant and able to
catalyze the conversion of normal cellular prion protein (PrP C) into infectious scrapie
prion (PrPSc) (Prusiner, 1982). This proteinaceous infectious particle (hence “prion”) was
proven to be devoid of any nucleic acids or any other hallmarks of bacteria or viruses.
Because it seemed to contradict the germ theory of transmissible disease, this discovery
was quite controversial initially but it has been subsequently accepted and Dr. Stanley
Prusiner has won a Nobel prize for his work.
Unusually, many of these diseases can be transmitted by eating or otherwise
ingesting contaminated tissue. The most well known example of this phenomenon is the
Misiewicz 13
consumption of scrapie meat, which causes a variant of CJD to occur. Additionally,
iatrogenic transmission of prions is possible through hormone transfusions and
improper medial instrument sterilization, a mode of transmission that was more
common before there was a broad understanding of the causes of scrapies (Norrby,
2011). In laboratories, experimentally, prion diseases can be transmitted by injecting
animals with brain homogenates from afficted animals. The catalytic activity of scrapie
can even be reproduced in vitro through an assay called protein misfolding cyclic
amplification. Although the effects and modes of transmission are well identified, the
exact mechanisms by which PrPSc enters the body, crosses the blood brain barrier and
induces misfolding of normal prion remains unclear.
In addition to its role as the infection particle in scrapies, prion has a normal,
physiological function. Prion's function has remained difficult to define despite 30 years
of research. However, it has come to be implicated in the protection of cells from
apoptosis induced by BCL-2 associated protein X (Bax). It has been associated with the
response to oxidative stress, protection against serum deprivation, anti-apoptotic
responses and a responder to endoplasmic reticulum stress. Prion's role in cancer due to
its pro-survival properties is currently an area of active research (Westergard et al., 2007;
Linden et al., 2008). PrP has been identified to be highly expressed in central nervous
system (CNS) tissues, where it has been additionally implicated in the sleep-wakefulness
cycle, synapse function, and memory formation. Finally, PrP is thought to be important
in copper ion metabolism, and there is a line of evidence to suggest that PrP evolved
from a family of metal ion transporters.
Although PrP has been associated with many different cellular functions, one
area of prion research that remains poorly defined is its regulation and response to
various cellular states. There has been some research in this area, but it remains largely
unknown how and why physiological prion expression is controlled in the cell.
Understanding the processes that regulate the prion gene (PRNP) expression could lead
to treatments for cancers and scrapie diseases (the severity of these two disease appears
to be exacerbated by high PrP levels) and provide a better understanding of the ways in
which cells cope with various stresses throughout their lifetimes. In this thesis, I aim to
lay out the known mechanisms of PrP regulation, general mechanisms of pro-survival
Misiewicz 14
responses in the cell, and how PrP is regulated by these responses. Our results show that
the PRNP promoter contains a novel Endoplasmic Stress Response Element (ERSE)
which enables it and other genes in the human genome to respond to perturbations in
the homeostasis of the endoplasmic reticulum. This new element is a new mechanism
for the regulation of PRNP expression, helps better explain the functionality of the PRNP
promoter, and provides a better understanding of the broad cellular response to stress.
1.1
The human PRNP gene and Prn locus
Human PrP is encoded by the PRNP gene. Originally mapped in 1986, it is located on
chromosome 20, at 20p13 (Basler et al., 1986). In addition to PRNP, two other prion-like
genes have been discovered, Doppel (PRND) and Shadoo (SPRN). PRNP and PRND are
adjacent, and comprise what is sometimes referred to as the prion locus. PRNP is well
conserved in vertebrates, showing conservation in mammals and existence in fish and
birds (Rivera-Milla et al., 2006). The strong conservation and deeply branching nature of
PRNP suggests that it has a very important role in animals.
PRNP encodes a 2.7 kb mRNA (Sayers et al., 2012), with a 761 bp open reading
frame (ORF). There are several variants of the PRNP mRNA (5 have been confirmed),
but they all encode the same protein. The protein contains two signal peptides, and is
almost entirely processed in the ER (Caughey et al., 1989; Yedidia et al., 2001).
1.1.1
Structure of the PRNP gene
Human PRNP contains two exons and a 13kb intron. The open reading frame is located
entirely in exon two. The large intron is unique to humans. Although the structure is
well conserved in primates, other mammals have different numbers of introns and
Misiewicz 15
exons. For example, the mouse Prnp gene contains three exons and two introns, although
like PRNP the coding sequence is contained entirely in the last exon.
1.1.1.1
PRNP promoter structure
The PRNP promoter contains several confirmed transcription factor binding sites,
although generally not much is known about the function of the PRNP promoter (Figure
1). PRNP is expressed as a housekeeping gene: its promoter contains a SP1 site, no TATA
box, high G/C content and a CCAAT box leading to constitutive PRNP expression and
its classification as a housekeeping gene (Basler et al., 1986). In addition to its
constitutive expression, the PRNP promoter also contains conditionally activated
elements. Putative binding sites for heat shock elements, p53 binding sites, AP-2, Nkx2-5
and Myo-D, IL-6, NF-AT, Ets-1, metal responsive element binding sites, and cell
membrane dependent AP-1 have been identified (Bellingham et al., 2009; Funke-Kaiser
et al., 2001; Mahal et al., 2001). Finally, several elements of unknown function are well
conserved in mammalian PRNP promoters (Mahal et al., 2001). However, of these
putative sites, only Sp1, p53, metal transcription factor 1 and heat shock elements have
been confirmed to regulate PRNP expression (Qin et al., 2009; Liang et al., 2007;
Bellingham et al., 2009; Vincent et al., 2009; Shyu et al., 2002). There is evidence that
PRNP might be regulated by p53, but also that PRNP might itself regulate p53; the
relationship is unclear (Vincent et al., 2009; Paitel et al., 2003) The elements are located
in two clusters around the transcription start site (TSS) of PRNP: from up to -800 bp to
0bp and within exon I (Mahal et al., 2001).
Although many putative elements have been identified, a full understanding of
the function of PrP will require a complete understanding of the PRNP promoter, and
much remains to be discovered.
Misiewicz 16
TSS
-1000
p53
HSE
-755 -737 -700 -680
EL26
-231 -196
MRE
-90 -84
SP1
-62
-57
MRE
+200
SP1
-39 -33 0bp
6
Figure 1 – Known regulatory elements in the PRNP promoter. Binding sites for p53,
Heat Shock Element (HSE), the ERSE-26 (EL26), Metal Responsive Element (MRE) and
SP1 binding site (SP1) have been shown to be functional in the PRNP promoter.
Misiewicz 17
1.1.1.2
Intron and exons in PRNP and Prn locus members
PRNP contains two exons and one intron of 13 kb. Exon I does not encode any part of
the open reading frame, the protein is entirely encoded by exon II. PRND, which is
located downstream of PRNP, contains a similar structure: two exons, separated by a
large intron. The entire mRNA is 4 kbp, and the ORF is contained entirely in the first
part of the second exon, like in PRNP. Next in the Prn locus following PRND is PRNT, a
testis-specific version of PRNP. It does not appear to be protein coding however,
although it also retains a similar structure to PRNP and PRNP. On chromosome 10,
outside of the Prn locus is SPRN. It encodes a 3.2 kb intron, which like PRNP, PRND,
and PRNT is comprised of two exons separated by a large intron. The open reading
frame is located in the beginning part of the first exon.
1.1.2
Shadoo and Doppel
SPRN and PRND are two genes that are homologous to PRNP and form the prion family.
Doppel was originally discovered accidentally in Prnp knockout mice, when ataxia and
Pukinje cell death was observed in aged mice (Sakaguchi et al., 1996). These results were
surprising, because Prnp knockout mice normally do not show symptoms, however
upon sequencing and further investigation, it was determined that the deletion used to
produce the PRNP knockout mouse actually put the PRND gene under the PRNP
promoter (Moore et al., 1999). In mice, Doppel is normally expressed in the testis only
after birth, and the Prnp promoter led to aberrant expression in the CNS. Doppel was
subsequently found to be very similar to the C-terminal end of PRNP, and its expression
has been confirmed in many organisms and in various tissue types (Westaway et al.,
2011). In addition, Doppel has an important role in spermatogenesis, being primarily
Misiewicz 18
expressed in the male reproductive tract (Westaway et al., 2011). But its exact function
remains mysterious.
Shadoo was discovered by searching nucleotide databases for sequences with
homology to PRNP. It is a short protein at 98 residues, and contains a great deal of
homology to the N-terminal half of PrP, and the gene is known to be protein-coding in
the central nervous system. Shadoo's function in the CNS is unclear, however (Westaway
et al., 2011). Although the exact function of these prion-like genes is not precisely known,
they might provide new insight into prion function and indicate the presence of a prionlike family of proteins, a relatively new and exciting development.
1.1.3
Patterns of constitutive PNRP expression
Human PRNP is expressed in most tissues of the body. Its highest levels of expression
are in the CNS, where in mice, it can be detected at embryonic day 8.5, when the
transition to oxidative respiration occurs (Miele et al., 2003).
In addition to its expression in CNS tissue, PrP has been found in most tissues of
the human body (Westergard et al., 2007; Linden et al., 2008). PrP expression appears to
be higher in immune system tissues, particularly lymphocytes, which typically have
higher expression of binding of immunoglobulin protein (BiP) and other chaperones due
to their large secretory load (Haas and Wabl, 1983; Li et al., 2001). Additionally, there is
some evidence to suggest that PRNP may play a role in immune system signaling, and is
implicated in lymphocyte development (Li et al., 2001).
PRNP knockout mice do not show major phenotypes, however. There are defects
in cell survival (Prnp -/- neurons die faster when deprived of serum in culture), memory
formation, and vulnerability to chemically induced seizure. PRNP knockout mice are
immune to prion infection (Linden et al., 2008).
Misiewicz 19
1.1.3.1
Signal dependent PRNP expression
Despite the putative and confirmed regulatory elements of PRNP, the regulation of
PRNP remains unclear. There is evidence that PrP expression is increased in breast
cancer carcinoma cells subjected to the ER stressors BFA and Bisphenol A in MCF-7 and
TTE3 cells respectively (Tabuchi et al., 2006; Jodoin et al., 2007). Furthermore, during
development of the hamster, injection of neuron growth factors induces expression
PRNP mRNA expression (Mobley et al., 1988). PRNP expression has been observed in
ischemia and in response to oxidative stress. Hydrogen peroxide and hyperbaric oxygen
both upregulate PRNP expression. Prnp knockout mice show increased markers for
oxidative stress, suggesting that PRNP has an important role in oxidative stress (Shyu et
al., 2004; Liang et al., 2007).
However, the functionality of the PRNP promoter and its properties remains an
area of active research.
1.1.3.2
Expression of PRNP in cancer and disease
Another disease role for PrP is in cancer. In the breast cancer carcinoma line MCF-7,
cells which gain resistance to cell death by Tumor Necrosis Factor alpha (TNFα) also
show large increases in both PRNP mRNA and PrP levels. Furthermore, overexpression
of PrP in TNFα-sensitive MCF-7 cells results in a reduction in their sensitivity to TNFα
treatment. Additionally, silencing PRNP in MCF-7 cells had the effect of re-sensitizing
them to TNF-related apoptosis-inducing ligand mediated apoptosis (Diarra-Mehrpour et
al., 2004).
Given that cancer is fundamentally a disease of inappropriate cell survival and
growth, there is currently great interest in better understanding PrP's pro-survival role
in these contexts as it may lead to more effective treatments for cancers.
Misiewicz 20
1.2
Functions and topology of prion protein
While the exact details are still up for debate, a growing ensemble of evidence collected
so far tends to indicate that PrP plays a protective role in the cell (Diarra-Mehrpour et al.,
2004; Bounhar et al., 2001; Roucou et al., 2005; Jodoin et al., 2007). Due to its localization
at the cell surface and synapse, PrP is thought to play a role in synaptic function as well.
PrP knockout mice do not show very noticeable symptoms, however they are prone to
seizures and have a shortened lifespan (Walz et al., 1999). Furthermore, PrP knockout
mice are immune to infection with PrPSc, consistent with PrP's infectious protein
property (Büeler et al., 1993). In addition, PrP has been associated with memory
formation, the sleep-wakefulness cycle, synaptic function, immune system function and
differentiation, and as a possible receptor or scaffold for intercellular signaling
(Westergard et al., 2007; Linden et al., 2008).
1.2.1
Topology of PrP
PrP is a 253 aa, 26 kDa protein that is primarily expressed as a glycosyl
phosphatidylinositol (GPI)-anchored glycoprotein at the cell surface. It contains a
disulfide bridge and two glycosylation sites (Figure 2A). There is a highly conserved
octapeptide repeat region, a signal peptide and a GPI anchor sequence. Additionally,
there is a transmembrane domain, which has lead to the discovery of two different forms
of transmembrane PrP. One isoform, termed PrP Ctm, is found with the C-terminus in the
ER lumen, and the N-terminus in the cytosol. PrP Ntm has also been identified, which is
the inverse: the N-terminus is found in the lumen, and the C-terminus is in the cytosol
(Figure 2B) (Hegde et al., 1998). In the C-terminal globular region of the protein, there
are three alpha helices and two beta sheets. Although it was originally thought that PrP's
Misiewicz 21
demonstrated ability to inhibit Bax activity (see below) would be conferred by the
conserved octapeptide repeat region, it was determined that helix three is necessary and
sufficient for this purpose (Laroche-Pierre et al., 2009).
1.2.2
Processing and localization
The majority of PrP is present at the cell surface in lipid rafts, although there are several
other isoforms that exists. A cytosolic, retrotranslocated form the protein, without any
signal peptides exists(“CyPrP”, Figure 2) (Roucou et al., 2003). Since signal peptide
cleavage is required for entry into the ER, the lack of signal peptide in CyPrP indicates
that retrotranslocation must have occurred. Additionally, full-length PrP with a signal
peptide can be found in the cytosol, indicating that the peptide failed to enter the ER.
Retrotranslocation of PrP has been shown to be important for protection of cells against
apoptotic insults (Lin et al., 2008), and disease causing mutations in PrP affect the ability
of PrP to retrotranslocate and exert its protective effect (Jodoin et al., 2007, 2009). PrP has
also been detected in nuclei, but the reasons and purpose of PrP in the nucleus remain
unclear (Hosokawa et al., 2008).
1.2.2.1
Signal peptides
PrP contains two signal peptides. The first N-terminal signal peptide is an ER signal,
which recruits PrP to the translocon for translocation into the secretory pathway. This
peptide is not considered 'strong', explaining why sometimes PrP fails to enter the
secretory pathway (Rane et al., 2004). The C-terminal signal peptide is a GPI anchor
signal, targeting PrP to GPI rafts for secretion (Caughey et al., 1989). These signal
peptides are cleaved during maturation of the prion protein (Roucou et al., 2003).
Misiewicz 22
1.2.2.2
Secretion
PrP's C-terminal GPI signal sequence is cleaved by the enzyme GPI protein
transamidase, and a glycolipid complex is added to the new C-terminus of the protein.
This glycolipid anchor remains embedded in the phospholipid bilayer, and when
attached to a protein may indicate protein maturity and readiness for ER exit. The GPIanchored protein is trafficked to the Golgi in a mechanism which is independent of
COP-I and COP-II vesicles normally used for transmembrane protein exocytosis. The
exact details remain unclear, but once PrP reaches the Golgi, it is secreted like other GPIanchored proteins (Harris, 2003). Once PrP reaches the cell surface, it tends to
accumulate in lipid rafts with other GPI proteins (Caughey et al., 1989; Campana et al.,
2005).
1.2.2.3
Post-translational modifications
Human PrP exists in several post-translationally modified states. A disulfide bridge is
present between residues 179 and 214. In addition, PrP is detectable in various stages of
the glycoprotein maturation process (i.e. immature glycosylated, and mature)
(Haraguchi et al., 1989). These modifications can occur at residues 181 and 197. It
appears that glycosylation may affect PrP's ability to form aggregates, although this
remains unclear (Lawson et al., 2005).
Misiewicz 23
A
PrP, full length
Disulfide bridge
TMD
Signal peptide
GPI anchor
179
N
22
112
181 197 214
136
C
231
253
Polysaccharide
Side chains
CyPrP
TMD
179
N
181 197 214
C
23
112
136
231
23
B
PrP
Lumen
PrPNtm
PrPCtm
231
GPI
231
23
GPI
23
Cytosol
231
N
CyPrP
C
Figure 2 – Topology and transmembrane forms of PrP. A. Schematic diagram of PrP
isoforms, both cytosolic and secreted forms. B. Localization of PrPCtm, PrPNtm, normal PrP
and CyPrP.
Misiewicz 24
1.2.3
Protective functions
Prion protein appears to be important in protecting cells against apoptotic insults
(Brown et al., 1999; Kuwahara et al., 1999; Bounhar et al., 2001; Roucou et al., 2003;
Diarra-Mehrpour et al., 2004). There appear to be two ways in which this occurs. The
first is by reduction of reactive oxygen species (ROS). PrP has been observed to possess
superoxide dismutase activity, which may be important in neutralizing reactive oxygen
species in cells (Brown et al., 1999). PrP also protects against oxidative stress. When PrP
deficient cells are subjected to various oxidative stresses, their survivability is reduced
(Shyu et al., 2004; Brown et al., 1997b; Wong et al., 2001). PrP's other protective effect is
to exert a negative effect on the pro-apoptotic protein Bax. This activity is impaired
when PrP contains disease causing familial prion disease mutations, possibly explaining
the mechanism for neurodegeneration in familial scrapie diseases (Bounhar et al., 2001;
Jodoin et al., 2007, 2009). Finally, PrP has been shown to protect cells against TNFα
mediated apoptosis, which possibly implicates PrP in cancer progression (DiarraMehrpour et al., 2004).
1.2.3.1
Anti-Bax function
Bax is a 218 aa protein that oligomerizes at the surface of mitochondria and ultimately
allows membrane permeabilization leading to caspase dependent cytochrome-c
dependent apoptosis (Oltvai et al., 1993). In order to cause cytochrome c release, Bax
must oligomerize at the mitochondria surface to solubilize the membrane (Antonsson et
al., 2000). Control of Bax is therefore very tight, normally regulated by ubiquitination
and proteolysis. PrP's role in Bax activation first came to light when it was noted that the
octapeptide repeat region of PrP contains a Bcl2 homology (BH2) domain (Yin et al.,
Misiewicz 25
1994; Wang and Snyder, 1998). Additionally, in S. cerevisiae, which has no endogenous
PRNP or Bax equivalent, PrP rescued against Bax-induced cell cycle arrest and toxicity
(Bounhar et al., 2006). Through an unknown partner, PrP appears to indirectly regulate
Bax activity. Only mature CyPrP has this effect (Lin et al., 2008). In the presence of CyPrP
only (i.e. not other forms), Bax shows reduced oligomerization activity (Roucou et al.,
2005). Importantly, PrP that contains GSS, FFI or CJD mutations shows reduced or
eliminated Bax inhibition, suggesting one possible way in which these mutations can
lead to disease (Jodoin et al., 2007, 2009). The unknown factor that binds with CyPrP to
inhibit Bax oligomerization remains unknown. However, by using expoxymicin to
inhibit protein retrotranslocation or by introducing a known familial disease-causing
mutation to PrP, anti-Bax activity can be eliminated, implicating CyPrP specifically in the
protective process (Jodoin et al., 2007).
1.2.3.2
Copper metabolism and redox activity
ROS are thought to be one of the major causes of aging and damage in cells. ROS have
been implicated in oxidization of organic molecules, DNA damage, and cellular
dysfunction. Therefore it is important that cells have mechanisms in place to reduce or
control ROS, particularly due to their production during aerobic respiration (Lenaz,
2012). Many metal ions produce ROS if not properly chaperoned. Cu2+ is one such ion,
and having a system to chaperone and sequester these ions is important (Lenaz, 2012). In
vitro and in vivo, PrP has been shown to be able to reduce Cu2+ ions. The octapeptide
repeat region of the PrP has been shown to bind copper, and this region of the protein is
one of the best conserved regions of the protein (Brown et al., 1997a; Stöckel et al., 1998;
Brown et al., 1999). Based on homology and alignment studies, PrP and its related family
members Shadoo and Doppel are thought to have descended from ion chaperones
(Schmitt-Ulms et al., 2009). Due to its reductase activity and homology, it is thought this
is one of PrP's major functions.
Misiewicz 26
2
The endoplasmic reticulum: functions and properties
The ER is the organelle of the cell where secreted proteins are folded and processed into
their final functional confirmations. In addition to its function in protein folding, the ER
also has the following critical functions: providing appropriate enzymes for protein
folding, being a sensitive sensor for errors in this process and mediating disposal of
proteins which have failed to properly assemble.
Three major classes of enzymes enable protein folding: chaperones, foldases and
lectins (Schröder and Kaufman, 2005). The most well known protein folding enzymes
are chaperones, which provide a hydrophobic folding environment for client proteins.
They allow the protein to more rapidly cycle through all possible configurations in
space. Foldases are ATP-dependent enzymes that accelerate protein folding by
accelerating covalent modifications that must take place during protein maturation
(such as disulfide bridge formation). Lectins “read” glycoproteins' carbohydrate side
chains and direct them to the Golgi or retain them in the ER if they need more time to
fold. If a protein remains attached to quality control enzymes for too long, this will
signal the activation of the protein disposal mechanisms. A pathway called Endoplasmic
Reticulum Associated Decay (ERAD) is able to recognize proteins that have remained
associated with folding enzymes for too long, and removes them to the cytosol where
they are subject to proteasomal degradation.
In addition to folding enzymes, the ER contains transferases which induce the
binding and processing of complex carbohydrates as proteins mature, allowing secreted
glycoproteins to attain their correct forms. Glycoproteins receive their immature
carbohydrate side chains in the ER, and are then released to the Golgi for cleavage and
maturation. Failure to achieve proper glycosylation results in retention in the ER and
ultimately activation of ERAD.
Finally, the solute content of the ER is carefully controlled by the organelle, in
order to assure proper ion concentrations and the efficient production of secreted
proteins. In addition to its role in protein synthesis and ion concentration, the ER is the
site of sterol and lipid synthesis, about which very little is known, particular in regards
Misiewicz 27
to ER stress. Finally, the ER is an intracellular store of calcium ions, which are needed in
many cellular functions in or out of the ER.
2.1
Protein folding
Protein function is determined by its shape, thus in order to assure function, structure
must be assured. In order to reach these structures, cells employ enzymes which provide
the proper conditions to catalyze the needed bonds. There are three properties of a
protein that give rise to its final conformation. First, is its primary structure, which is
determined by its amino acid sequence. Second, a protein's secondary structure consists
of beta sheets, alpha helices and coiled coils, structures which are formed due to
hydrogen bonding between the various amino acids. Third, the tertiary structure which
is more complex and represents how these structures all interact together to form a
larger globular protein.
2.1.1
Mechanisms of enzymatic activity for protein folding in the ER
The three major classes of protein folding enzymes in the ER. Each kind of enzyme has a
different mode of action to assist client proteins assume their correct confirmations. The
best known class of protein folding enzymes are the chaperones, and best known of
these is BiP (GRP78), although many have since been discovered. Next there are the
foldases, and the best known foldase in the ER is Protein Disulfide Isomerase (PDI)
which helps establish disulfide bridges between cysteine amino acids. Finally, there are
the lectins, and the best known lectins are Calnexin and Calreticulin.
The chaperone BiP is an ATP dependent enzyme. ADP-BiP has a high affinity for
unfolded client proteins and binds them. In this confirmation, the ADP is exchanged for
Misiewicz 28
an ATP, and BiP becomes tightly attached to the client protein which is now able to
undergo a folding step. Finally, the ATP is hydrolyzed to ADP, and BiP returns to a lower
affinity state for the client protein. This cycle causes protein folding to become an ATP
dependent process, and as the protein folds, ATP is consumed. Depleting ATP can
inhibit protein folding. The purpose of BiP activity is to provide for greatly accelerated
protein folding, because protein folding steps proceed faster when BiP is present (Flynn
et al., 1989).
Lectins are responsible for ensuring the quality of glycoproteins. The best known
lectin proteins are Calnexin and Calreticulin, which bind to newly-glycosylated proteins.
While the carbohydrate remains immature (not cleaved to a smaller, more mature form),
it is deglycosylated and re-glycosylated and retained by calnexin and calreticulin.
Several cycles of this occur, which allows for sufficient folding to take place before
cleavage to the mature form occurs. Furthermore, calnexin and calreticulin have the
important function of keeping unfolded proteins out of the Golgi, since immature
glycoproteins remained retained until proper cleavage occurs (Schröder and Kaufman,
2005; Wang et al., 2012a).
Finally, there are foldases. The best known foldase is protein disulfide isomerase
(PDI), which catalyzes the formation of a disulfide bridge between two cysteine
residues. This requires the formation of a covalent bond between the two cysteines to
form cystine. Bonds must be formed between the correct cysteines, and reduction of -SH
groups on the cysteine amino acid must take places. PDI ensures that all of these
activities are carried out correctly and efficiently. Since the discovery of PDI, other
additional foldases have been discovered with various functions (Wilkinson and Gilbert,
2004).
2.1.2
Post-translational modifications and sorting
The ER and Golgi are the sites of post-translational protein modification and sorting to
their destinations. GPI anchor attachment occurs in the ER, which leads to anchored
Misiewicz 29
proteins being targeted to the cell surface. Immature glycosylation occurs in the ER as
well, where nitrogen (typically on an asparagine residue) and oxygen (typically on
serine or threonine residues) are covalently linked to large carbohydrates. In the Golgi
apparatus, these glycans are trimmed, indicating protein maturity. Other modifications
such as protein sialylation can occur in the Golgi as well. Following the appropriate
glycosylation, proteins exit the secretory pathway for their final destinations (Alberts et
al., 2008).
2.2
ER Stress
Fundamentally, stress is the response to a perturbation in the environment. The response
to stress occurs as the cell responds to that pertuburation to restore optimal
functionality: either by adjusting the environment or altering its own functional
properties to function most efficiently. Restoration of optimal functionality constitutes
the exit from stress. In the context of the ER, stress impairs the ability of the organelle to
complete its function. In order to respond to this change, a complex program has
evolved in mammalian cells that enables the restoration of homeostasis.
2.2.1
Discovery and definition
ER stress is vaguely defined. It was originally noticed that cells upregulated certain
proteins when deprived of oxygen or glucose, or in normal tissue that secreted large
amounts of protein. This led to the discovery of chaperones, and the idea that high
expression of chaperones constitutes a stress (Haas and Wabl, 1983; Munro and Pelham,
1986; Hetz, 2012). Generally, ER stress is defined as an excess of unfolded proteins in the
ER, which results in higher than normal chaperone activity.
Misiewicz 30
2.2.2
ER Stress signal transduction
The cellular response to ER stress entails increasing folding enzyme (e.g. chaperone)
activity and altering protein expression levels, both transcriptionally and translationally
(Hetz, 2012). However, the status of the contents of the ER cannot be easily signaled to
the rest of the cell, since the ER is a membrane bound, closed structure. As a result,
unique mechanisms are needed to inform the rest of the cell of the current status of the
ER. Three pathways have evolved to transmit the ER stress signal across the ER
membrane. All of them are dependent on the level of BiP associated to transmembrane
sensors, which is lower when BiP is bound to an excess of unfolded client proteins.
Therefore, BiP acts as both a measurement of the amount of unfolded proteins, as well as
the tool to reduce the pool of unfolded clients.
2.2.2.1
IRE1α and XBP1
The first transducer of ER stress signals discovered was Inositol Requiring Enzyme 1
(IRE1α) and the transcription factor X Box Binding Protein 1 (XBP1). XBP1 was initially
discovered in yeast as Hac1, where it is the only transducer of ER stress mediated gene
transactivation; the mammalian version was discovered by homology (Mai and Breeden,
1997). When high levels of unfolded client proteins are present in the ER, BiP dissociates
from a transmembrane splicing enzyme Inositol Requiring Enzyme 1 alpha (IRE1α).
When
IRE1α
is
no
longer
associated
with
BiP,
it
homodimerizes
and
autophosphorylates, which results in the activation of endonuclease activity (Li et al.,
2010). IRE1α excises a 26bp intron from the XBP1 mRNA, which removes a stop codon.
The shortened message encodes the highly active transcription factor sXBP1, which
translocates to the nucleus where it acts as a transcription factor for ER stress mediated
transactivation (Yoshida et al., 2001). In addition to its endonuclease activity, IRE1α also
has the ability to activate JNK signaling through the adapter protein TRAF2. Through
this mechanism, IRE1α appears to be able to regulate signaling for apoptosis and
Misiewicz 31
autophagy (Hetz and Glimcher, 2009).
2.2.2.2
Site 1 and Site 2 proteases and ATF6
ATF6 was the second ER stress transcription factor identified after yeast Hac1p. It exists
in two different isoforms, a longer 90 kDa isoform that is retained in the ER, and a
shorter 55 kDa isoform which can be found in the nucleus as a highly active
transcription factor. In conjunction with the general transcription factor NF-Y, it binds to
DNA and is able to recruit the needed transcription machinery, resulting in high levels of
transactivation during ER stress (Haze et al., 1999).
In order to be converted from the longer ER resident form to the soluble 55 kDa
form, ATF6 is cleaved by two transmembrane Golgi-resident proteases Site 1 Protease
and Site 2 Protease (Ye et al., 2000). Normally, ATF6 is retained in the ER, bound to BiP.
However, as with XBP1, when BiP dissociates to fold client proteins, ATF6 is able to
advance in the secretory pathway to the Golgi. Upon reaching the Golgi, ATF6 is cleaved
by Site 1 Protease (S1P) and Site 2 Protease (S2P), resulting in a 55 kDa fragment and a 36
kDa fragment which are able to activate transcription in the nucleus (Yoshida et al.,
2000).
In non stress conditions, ATF6 is normally fully glycosylated, due to three wellconserved glycosylation sites in its lumenal half. However, during disruptions to ER
homeostasis, ATF6 becomes under glycosylated. The less glycosylated form of ATF6
loses affinity for calreticulin, which normally retains ATF6 in the ER during non-stress
conditions. No longer retained by calnexin/calreticulin, ATF6 is transported more
quickly to the Golgi, where it can be cleaved by S1P and S2P. Thus, ATF6 is also able to
act as a sensor for protein glycosylation: improper or impaired protein glycosylation in
the ER increases the transcriptional activity of ATF6 by making more of it available for
cleavage activation (Hong et al., 2004).
Misiewicz 32
2.2.2.3
PKR-like endoplasmic reticulum kinase and ATF4
The final ER stress mediated transcription factor is ATF4. Similar to XBP1, ATF4 is
regulated on a translational level. Its mRNA is not transcribed under normal conditions
(Vattem and Wek, 2004). Upon activation of the ER stress response however, ATF4
translation is initiated and the active transcription factor is able to transactivate gene
expression in the nucleus.
During ER stress, cells attempt to reduce the ER load by attenuating the rate of
new protein translation, which allows the chaperones, foldases and lectins to clear the
unfolded proteins from the ER. In order to achieve this, a transmembrane serine
threonine
kinase
known
as
PKR-like
endoplasmic
reticulum
kinase
(PERK)
phosphorylates the translation initiation factor eIF2α, which arrests nearly all translation
in the cell (Harding et al., 2000). Similar to the other ER stress sensors, PERK is normally
bound to BiP when the ER does not contain an excess of unfolded proteins. During ER
stress, BiP dissociates from PERK in favor of client proteins. PERK dimerizes and
autophosphorlates, which causes PERK to gain kinase activity (Bertolotti et al., 2000).
When eIF2α is phosphorylated, the majority of protein translation is inhibited due to a
failure of the ribosome to find the initiation site. However, messages containing a second
open reading frame after the first are efficiently translated when eIF2α is
phosphorylated. ATF4 mRNA contains one of these sites, resulting in high translation of
ATF4 mRNA, the active transcription factor, during ER stress (Vattem and Wek, 2004).
2.2.3
Description of the ER stress response program
The cell's response to ER stress is both transcriptional and translation. Fundamentally,
Misiewicz 33
the goal of the cell's response to ER stress is to reduce the load of unfolded proteins by
attenuating translation, and by properly processing client proteins in the ER. Thus, the
genes that are regulated transcriptionally and translationally in the ER stress response
help achieve these functions. If the response to ER stress is not sufficient to clear the
blockage, cells activate an apoptotic program to initiate caspase-dependent cell death
(Hetz, 2012). In addition to its role in protein folding, ER stress can also affect the output
and processing of other ER resident molecules, such as lipids and ions, which are
produced or processed in the ER.
During ER stress, translation of most new proteins is inhibited by the cell due to
phosphorylation of eIF2α. However, there are several mRNAs which avoid this
translational arrest by containing an additional special ORF. The best example of this is
the ATF4 mRNA, which contains two ORFs. The first contains a sequence which
prevents appropriate attachment of translation initiation factors when eIF2α is not
phosphorylated. However, when eIF2α is phosphorylated, the first inhibitory ORF is not
recognized during ribosomal scanning of the mRNA, and instead a second, downstream
ORF is recognized and translated. This mechanism allows the cell to impair translation
of most new client proteins, while increasing the level of chaperones and transcription
factors needed to restore ER homeostasis (Vattem and Wek, 2004).
2.2.3.1
Translational
Protein chaperones are amongst the molecules that are most induced by ER stress.
Originally, protein chaperones were identified as proteins which appeared on gels when
cells were either deprived of glucose or subjected to heat shock (Munro and Pelham,
1986). It was also discovered that certain toxic chemicals (such as a tunicamycin) would
also cause Grp78 induction, leading to the idea that this protein did more than just
respond to heat and glucose deprivation. Independently, BiP was identified as a protein
in the ER of B-cells, and it was identified as a binding partner of secreted proteins (Haas
and Wabl, 1983). Eventually, cloning and mapping of BiP and Grp78 lead researchers to
Misiewicz 34
the realization that they were actually the same protein (Munro and Pelham, 1986).
In addition to BiP, other proteins increased during ER stress have been identified.
These include ER enzymes such as HERP, PDI6, Calnexin/Calreticululin, and Grp94.
Additionally, cells upregulate the expression of nutrient transporters, such as amino acid
transporters and sugar transporters in order to help synthesize proteins and the
appropriate glycans for their correct modification (Oyadomari and Mori, 2004; Hetz,
2012).
2.2.3.2
Transcriptional
By way of the above mentioned transcription factors, cells are able to activate a diverse
transcriptional program. The mRNA levels of chaperones are strongly increased during
ER stress due to an increase in chaperone gene transcription levels (Munro and Pelham,
1986). Following excessive ER stress signaling, and especially if IRE1α signaling
becomes diminished and PERK signaling increases, pro-apoptotic genes start to be
transcribed, activating a cell death program (Oyadomari and Mori, 2004; Hetz, 2012).
2.2.4
Regulation of ER stress mediated transcription
ER stress-mediated transcription in higher eukaryotes is regulated by a series of DNA
promoter motifs that transactivate gene expression. Originally identified by examination
of the promoters of ER stress responsive genes, these ER Stress Response Elements
(ERSE) have been identified in many locations throughout the genome and have several
different mechanisms of activation.
Misiewicz 35
2.2.4.1
ERSE
The first ERSE was identified in 1998 by examining the promoters of several ER stress
regulated genes (HSPA5, HERP and PDI6). It contains the sequence CCAAT-N9CCACG, where the middle nucleotides are non-specific (Yoshida et al., 1998). It can
function in either a forward or a reverse orientation. The element was identified in a one
hybrid screen, which identified the transcription factors ATF6 and XBP1 as being the
transcription factors able to activate this element (the ATF6-NF-Y complex recruits the
general transcription factors needed to initiate transcription). The spacing between
CCAAT and CCACG appears to be important, mutation experiments showed that
changing the variable region to either 8 or 10 nucleotides resulted in attenuation of
transcriptional activity (Yoshida et al., 1998). The general transcription factor NF-Y binds
the CCAAT part of the ERSE, while the transcription factor ATF6 binds the CCACG part.
The cleavage of ATF6 and its translocation to the nucleus is dependent on ER stress,
meaning that the CCACG section provides transcriptional specificity for ER stress
(Yoshida et al., 2000).
XBP1 is able to bind to the ERSE, and was identified in the original yeast one
hybrid screen, however in vivo it does not appear to be responsible for the majority of
ERSE mediated transcription (Yoshida et al., 1998; Yamamoto et al., 2004).
2.2.4.2
ERSE II
Following the discovery of the ERSE, another ERSE element was discovered. The
sequence is ATTGG-N-CCACG, and was found by investigating the HERP promoter.
The non-conserved region in the middle is shorter, with only one nucleotide, and the
ATF6 binding site is inverted to the opposite strand. Like the classical ERSE, the ERSE-II
is bound by the transcription factors ATF6 and NF-Y. It initiates transcription using a
similar mechanism (Kokame et al., 2001).
XBP1 is also able to bind the ERSE-II element. Additionally, and unlike the
classical ERSE, removing XBP1 attenuates, but does not entirely eliminate, the
Misiewicz 36
transcriptional activity of the ERSE-II (Yamamoto et al., 2004).
2.2.4.3
ERSE-like
A third ERSE-type element identified is the so-called ERSE-like element. This element
contains the sequence CCACG-N9-ATTGG. This element is similar to the classical ERSE,
except in this case, the NF-Y binding site is on the opposite strand, and the orientation of
the element is reversed. Furthermore, this element is dependent on XBP1 binding, but
not ATF6. This element was confirmed to regulate the ER-related gene WFS1, though it is
thought to work for other genes as well (Kakiuchi et al., 2006).
2.2.4.4
UPRE
The Unfolded Protein Response Element (UPRE) is different than the ERSE. Its sequence
is TGACGTGG/A, and is bound by XBP1 (Yoshida et al., 2001; Yamamoto et al., 2004). It
is known to regulate the expression of the gene HERP. Initially, it was thought that ATF6
would have a high affinity for this element due to its similarity to the ERSE (on the
opposite strand), but that has not proven to be the case; XBP1 activates this element.
2.2.5
Exit from ER stress
Exiting from ER stress occurs in one of two ways. If ER homeostasis is restored, the
Misiewicz 37
transcription and translation of ER stress will subside, and BiP will re-associate with the
sensors ATF6, IRE1α and PERK. The alternative is apoptosis, which induced by the ER
stress-regulated genes CHOP (GADD153). GADD34 is an eIF2α phosphotase which
helps restore translation when the cell exits ER stress.
2.2.5.1
Restoration of ER homeostasis
Attenuation of ER stress signaling appears to indicate a return to ER homeostasis. With
proper BiP binding, ER stress sensors become inactivated. Importantly, it has been noted
that when IRE1α remains active during ER stress, cells avoid apoptosis and restore
homeostasis better. In contrast, prolonged activation of PERK due to ER stress appears to
have the opposite effect, preventing a return to normalcy, and inducing apoptosis.
However, the cell can avoid this outcome if IRE1α signaling is maintained rather than
PERK signal transduction (Lin et al., 2009).
2.2.5.2
Activation of apoptosis
Prolonged ER stress signaling eventually leads to apoptosis (Hetz, 2012). After long
periods of ER stress, the transmembrane sensor transmitting the ER stress signal shifts
from IRE1α to PERK. PERK causes transcription of ATF4, and ATF4 activates expression
of GADD153/CHOP (Oyadomari and Mori, 2004). Some evidence suggests CHOP
protein downregulates B-cell lymphoma 2 protein (Bcl-2) activity, which is a major
inhibitor of Bax oligomerization (McCullough et al., 2001). Following a CHOP-mediated
removal of Bax inhibition, cytochrome-c is released and caspase-dependent apoptosis is
activated. PERK appears to be activated later in ER stress, consistent with a mechanism
for to protect the organism from unhealthy cells if earlier protective responses fail to
restore cell homeostasis.
Misiewicz 38
2.2.6
ER Stress and Disease
Accumulating evidence is beginning to show that ER stress plays a role in many
diseases. ER stress is implicated in scrapie diseases, due to the high load of unfolded
scrapie proteins in cells. Accumulation of PrPSc has been observed in the ER, as well as
an increase in BiP levels in CJD patients (Hetz and Glimcher, 2009). Furthermore, PrPSc
from mouse brains has been shown to induce ER stress when injected in cell cultures
(Torres et al., 2010). Given that scrapie diseases are characterized by plaques of insoluble
proteins, the fact that these accumulate in the ER and impede function is not surprising.
However, in addition to scrapie diseases, ER stress has also been observed in
familial Alzheimer disease (AD) caused by mutations in the gene Presenilin 1 (Doyle et
al., 2011). An excess of Aβ protein and formation of plaques in familial AD brains has
been observed. Additionally, there is evidence that BiP levels are increased in sporadic
AD cases, suggesting distress in AD affected brains (Doyle et al., 2011). In addition to
AD, ER stress has been identified in Parkinson's Disease, which also is characterized by
protein aggregates in the brain, as well as amyotrophic lateral sclerosis (Doyle et al.,
2011).
Outside of neural tissue, ER stress has been implicated in Type 2 Diabetes, where
it has been observed that ER stress markers (BiP, XBP1, phospho-eIF2) are upregulated
in liver and adipose tissues, in conjunction with a decreased sensitivity to insulin
signaling (Ozcan and Tabas, 2012). ER stress signaling has also been observed in cancer,
where cells undergoing rapid division have a high demand for protein synthesis. ER
stress markers BiP and XBP1 have shown upregulation in cancer, and there is a positive
correlation between cancer severity and the levels of ER stress markers (Ozcan and
Tabas, 2012; Carrasco et al., 2007; Fernandez et al., 2000).
Due to the widespread nature of ER stress in disease, an understanding of its
mechanisms and systems of control can lead to better treatments for these diseases.
Misiewicz 39
3
Study of the Human Genome
The human genome is nearly 3 billion basepairs long and consists of 23 chromosomes.
The recent complete sequencing of the human genome in 2001 was a milestone of
human achievement, although now the process of interpreting these findings is the
major challenge facing scientists. Since the completion of this project, it has become clear
that the large degree of variability and phenotypes cannot be simply explained by
sequence variation alone, so the study of regulation of expression has become
increasingly important, and many new technologies to facilitate this has come into being
in recent years.
3.1
Sequencing
DNA sequencing has made truly incredible advances in the last 10 years. The cost of
sequencing (per nucleotide) has been decreasing as a super exponential function (Lee
and Tang, 2012). Fundamentally, the process of sequencing involves extracting DNA
from an organism and determining the sequences of bases by a chemical reaction that
provides a unique signal for each different nucleotide (Sanger et al., 1977). Originally,
this “Sanger Sequencing” was conducted using gel electrophoresis, and the sequence of
nucleotides could be determined by the position of bands on a very long gel.
Subsequently, improvements to classical Sanger sequencing (such as heavy use of
robotics and automation and sophisticated alignment algorithms) allowed publication of
the first draft of the human genome in 2001 (Lander et al., 2001). While improved Sanger
sequencing offered major improvements over older methods, they were still somewhat
slow due to the requirement that DNA be cloned in bacterial artificial chromosomes.
Recent advances in microarray technologies have allowed for sequencing of entire
genomes in very short times, by conducting millions of very short reads over the entire
sequence of the organism and aligning the fragments using sophisticated alignment
Misiewicz 40
algorithms (Lee and Tang, 2012).
3.1.1
The current state of the art of genome sequencing
Current next generation sequencing technologies are highly sophisticated and allow
many sequences to be determined in parallel (massively parallel sequencing). In modern
next generation sequencing methods, DNA is sheared into various small pieces. The
DNA is spotted onto a microwell plate, where it is immobilized, and reacted with Taq
DNA polymerase and DNA synthesis reagents in a microwell. Alternatively, the Taq may
be immobilized in the microwell, and the DNA is added. In either case, the end result is
the same: on a given plate, many thousands of sequences can be read simultaneously,
each spot on the plate represents a short DNA fragment. The actual sequence is read
using a pyrosequencing method: Each individual nucleotide is added sequentially by an
injection system to the entire plate, and a microscope with a digital camera reads the
plate to determine the sequence based on following the addition of a reactive nucleotide,
which produces detectable light.
Once a complete series of short reads has been recorded, they are aligned to a
reference genome and eventually a complete sequence can be obtained. Using these
techniques a genome can be sequenced in a short matter of time, however computing
complexity has dramatically increased. Another advantage to the massive parallel
approach is the ability to obtain several reads over several different regions of the
genome, which can help improve confidence values if a particular read did not work
well for some reason.
3.1.2
The Human Reference Genome
Misiewicz 41
These advances in sequencing has resulted in a concerted effort to increase coverage of
the human genome. Initially, the first draft sequence was only from a few individuals,
but this is problematic due to the number of polymorphisms that are present in a small
group (Church et al., 2011). As a result, teams around the world have sought to sequence
people from many different backgrounds and populations to gain an understanding of
the “reference” genome for humans, notwithstanding an individual's variation. This
project has been embodied as the Genome Reference Commission, which contains the
“correct” version of the human genome (Church et al., 2011). For the work presented in
this thesis, such curated reference versions were used for bioinformatic analysis.
3.2
Annotations and the definition of loci and their promoters
Being able to read the human genome is one matter. Interpreting it is another. The
process of understanding human genetics has often been compared to a book: first one
must determine the letters, then the punctuation, then the meaning of the words. While
large scale sequencing projects have solved the question of reading the letters, currently
deriving knowledge from this information (i.e. understanding the syntax and structure
of this language) is the challenge of our time (Lander et al., 2001). One of the ways this
must be done is genome annotation, the process of assigning features to locations in the
genome. A great number of cDNAs have already been deposited into nucleotide
databases such as Genbank (Sayers et al., 2012). These can simply be aligned to the
genome directly. However, there are a great number of genes which are not known, and
two methods are being used to locate novel genes. One method is exome sequencing,
where the entire RNA content of a cell is sequenced and aligned to the reference genome
(a complete reference genome sequence is required for these kinds of studies, which has
only recently been the case). This large-scale method is better than simply aligning
previously deposited mRNA sequences to the genome because during the process of
exome sequencing, new mRNAs can be discovered. However, the cell types being
Misiewicz 42
studied in exome sequencing may not express all genes or in levels that allow them to be
detected. Thus, the method has some bias. The other method of gene finding is
predictive: a statistical model based on formal computer science theory allows for
predictions of gene probability based on the emission of certain letters (i.e. TATA often
indicates a gene, followed by possible splicing sites, etc.). These methods allow for
computational prediction of genes which can be subsequently validated in exome
sequencing projects (Ng et al., 2009).
4
Computational pattern matching algorithms in biological sequences
Automated pattern matching in large data sets is one of the most useful applications in
modern computing. In the 1960s, the idea of using computers to search biological data
was first implemented, as previously tasks such as sequence alignments were done
manually. While fundamentally the problem is quite simple, searching for large and
complex patterns in complex datasets can quickly become computationally taxing. The
simplest algorithm is a naïve approach where each letter in the query sequence is
compared to the target sequence. Depending on the length of the query and the target
sequencings, the computation time and storage requirements can become impossible. In
order to overcome this limitation, algorithms such as Smith-Waterman (Smith and
Waterman, 1981) and the Basic Local Alignment and Search Tool (BLAST) (Altschul et
al., 1990) were developed. They each have optimizations in place to compute optimal or
near-optimal
alignments
between
query
and
target
sequences.
Although Smith-Waterman and BLAST work well for exact sequences, allowing
substitutions and deviations of letters in the sequence can increase complexity and
computational requirements. In order to conduct searches in targets allowing complex
patterns (for example, ACT[g or c]ATCGTA), other methods are needed.
Misiewicz 43
4.1
Regular expressions
Regular Expressions were first proposed by a pioneer of computer science, S. C. Kleene
(Kleene, 1951). Although at first his paper did not make a very big impact, in it he
defined a framework for representing a string of text as a series of “emission” events
froma finite state automaton (FSA) (Russell and Norvig, 2010). A FSA is an abstract
machine: given a set of arbitrary inputs (which could be a text string, sequence, pattern
or any sort of information) the FSA produces an output according to its structure and the
probability of certain outputs. Finite state automata can be useful if we want to
determine the parameters of a process (for example, the frequency of a certain nucleotide
being emitted in a sequence of nucleotides). To achieve this, we make the assumption
that a given sequence is output from a FSA, and then use this output to estimate of the
parameters of the FSA that must have produced it. Alternatively, one can define the
machine and its parameters ahead of time (for example: the FSA emits [A or B] then [C
or D]), and determine if a given input string could have been produced by this FSA.
The use of FSA to model emission probabilities allows the application of several
useful mathematical theorems which can dramatically optimize the speed of text
searching. More specifically, when using a FSA for pattern matching, the output to test
(i.e. the text to search) can be considered in an arbitrary order. Inputting a user-specified
FSA specification allows for implementation of efficient search algorithms (which
perform much more quickly than a naïve search of all possible strings that satisfy the
pattern). An algorithm implementing Regular Expression based search and pattern
matching was first written by the pioneering computer scientist Ken Thompson
(Thompson, 1968). His algorithm or derivatives of it have subsequently been
implemented in a great number of computer programs that contain abilities for
advanced text processing.
The approach of Thompson's regular expression evaluation algorithm provides
dramatic improvements over the naïve approach due to optimizations. It first generates
the FSA corresponding with the user inputted pattern. Next, this FSA is used to produce
a table of all possible characters at a given position in the query string, an operation
Misiewicz 44
which can be done very quickly. Then, these tables are used to search the target string,
by simply comparing each character of the target text to the tables. The naïve approach
of searching, comparing each position of the target and query texts of length n and m,
requires mn steps in the worst case. Using regular expressions, only n operations are
needed to search for a pattern of length m in n. Thus Regular Expression search is an
order of magnitude faster than naïve search and allows for great fexibility of patterns,
which would not be possible with naïve search due to the enormous cost in computation
time and storage space.
5
Methods of detecting gene expression levels
In order to study the transcriptional state of the cell, several methods have been
developed to assess mRNA identity and abundance. One is Northern Blotting (Alberts et
al., 2008), where an oligonucelotide probe is hybridized directly to the mRNAs
immobilized on a membrane (typically after transfer from a gel). This provides a very
sensitive method for determining RNA concentration and identity (since the probe is
made by the researcher). Alternatiely, reverse transcriptase (which makes cDNA from
RNA) be used to produce template DNA for polymerase chain reaction (PCR). DNA
from PCR can be run on a gel and used for semi-quantitative analysis. Once the image is
stored in a computer, pixel intensity values can be computed for the image and used to
compute the intensity of the band. The resultant measurement can be normalized
against controls to establish semi-quantitative fold induction values. While both of these
approaches are functional, there is a better more precise method.
5.1
Quantitative Real Time PCR
Although PCR on DNA samples can be quantitative, the method is only relative.
Misiewicz 45
Furthermore, agarose gel electrophoresis depends on a DNA staining agent to enable
detection and identification of products from the PCR reaction. However, the sensitivity
and linear response of the dye used (commonly Ethidium bromide) must be taken into
account. Furthermore, ethidium bromide has a comparatively low sensitivity compared
to newer dyes such as SYBR Green. This means that in order to identify the amplicon on
a gel, many cycles of amplification must take place. This leads to problems however,
since PCR is an exponential reaction, after a sufficient number of cycles, reactants can be
come depleted and amplification can plateau. Thus, with sufficient cycling, differences
in sample concentration can be masked because all of the reactions will have “caught
up” to each other. The solution to this problem was proposed by Morris and Morrion
(Morrison et al., 1998; Morris et al., 1996). Instead of detecting the double stranded
output of the PCR reaction after all thermal cycles have completed, a dye is placed in the
PCR reaction itself, and a fuorometer detects fuorescence above background levels
(meaning double stranded product has been produced) in the cycler. As soon as
fuorescence exceeds background levels, the cycle number is noted, and the fold
abundance can be determined by extrapolating from a logarithmic curve (Pfaff, 2001).
This method allows for very precise quantitation of PCR amplification, although primers
must be designed very carefully and well-characterized to prevent multiple products.
Unlike conventional RT-PCR, qPCR cannot be used to quantify multiple size amplicons
in the same reaction. Furthermore, complex equipment, expensive dyes and analysis are
required for interpretation of qPCR results.
6
Working hypothesis and research objectives
The culmination of previous evidence has lead to the theory that PrP is protective
against stresses and insults. This is supported by PrP's demonstrated anti-apoptotic
function (by inhibiting Bax), the vulnerability of Prnp knockout cells to insults, and PrP's
demonstrated
redox
activity.
Furthermore,
mutations
in
prion
that
cause
neurodegenerative disease inhibit this activity.
Misiewicz 46
In concert with PrP's protective role, preliminary evidence in our lab indicates
that PRNP and PrP are upregulated in response to ER stress in neurons and MCF-7 cells
(Jodoin et al. Unpublished). Jodoin et al. also identified two ERSEs in the PRNP
promoter as well as a novel ERSE which has similarity to the previously identified
ERSEs. This novel element, called ERSE-26, appears to be able to regulate PRNP
expression, based on a luciferase reporter construct and mutagenesis experiments. In the
same paper, Jodoin et al. showed with Chromatin Immunopreciption (ChIP) that the
PRNP promoter can bind both ATF6 and XBP1, but which of the three ERSEs binds
which transcription factor is not known.
Although the ERSE-26 is present and functional in the PRNP promoter, its
existence and functionality elsewhere in the genome remain to be seen. Additionally, the
activating transcription factor is not known. In order to address these questions, using
bioinformatic tools and the human reference genome (Church et al., 2011), I conducted a
genome wide scan of all promoters to identify other ERSE-26 elements. After finding
other gene promoters which contain the ERSE-26, I conducted RT-PCR and qPCR on
neurons and MCF-7 cells treated with the pharmacological ER stressors Brefeldin A
(BFA), Tunicamycin (TM) and Thapsigargin (Thps). These three stressors induced
expression of some ERSE-26 genes, confirming its existence and function elsewhere in
the genome. Next, I produced a luciferase reporter construct containing the ERSE-26,
which when co-transfected with either sXBP1 or ATF6 allowed for the identification of
the activating transcription factor. The factor sXBP1 was identified as the transcription
factor activating the ERSE-26. In order to confirm this, we transfected MCF7 cells with
sXBP1 to see whether or not ERSE-26 containing genes were induced or repressed.
The promoter of PRNP is poorly understood, and understanding the mechanisms
of PRNP expression and control will lead to a better understanding of its broader
function in the context of the cell. Furthermore, by understanding what is co-regulated
with PRNP, PrP's role in the cell can be better elucidated. Finally, the role of ER stress in
disease is currently being recognized in a wide array of diseases; having a better
understanding for these pro-survival responses can help improve treatments for these
diseases.
Misiewicz 47
Misiewicz 48
Chapter 2 – Manuscript
Preface
This chapter is the manuscript to be submitted to PloS Genetics: Michael Misiewicz, Julie
Jodoin, Derek Ruths, Andréa C. LeBlanc (2012). Identification of a Novel Endoplasmic
Reticulum Stress Response Element regulated by XBP1.
Author Contributions
Michael Misiewicz conduced all experiments and writing, with the exception of the
western blots in Figure 4D which were done by Julie Jodoin. Derek Ruths and Andrea
LeBlanc were supervisors. Human primary cultures were obtained from The University
of Washington, and I wish to thank Vikas Kushal for his assistance in culturing them.
Misiewicz 49
Identification of a novel Endoplasmic Reticulum Stress
Response Element regulated by XBP1
Misiewicz 50
Introduction
The endoplasmic reticulum (ER) is the organelle where all proteins secreted by the cell
are folded. In addition to helping route proteins to their proper destinations, the ER is
also critical in ensuring that proteins assume their proper confirmations. In order to
assure this, eukaryotes have evolved complex mechanisms for quality control in the ER
(Hetz, 2012).
PrP is a secreted 26 kDa glycosyl phosphatidylinositol (GPI) anchored glycoprotein
encoded by the PRNP gene (Westergard et al., 2007; Linden et al., 2008). PrP has been
linked to many different important cellular processes, and it is the causative agent of
several transmissible, familial and sporadic spongiform encephalopathies, for which it
was originally discovered and named (Prusiner, 1982). In addition to its original
discovery as the causative agent of scrapie disease, normal cellular prion (PrP C) is
known to be involved in copper metabolism, synapse function, cancer and oxidative
stress (Brown et al., 1997a; b; Wong et al., 2001; Stöckel et al., 1998; Haeberlé et al., 2000).
Additionally, PrP plays a cytoprotective role in neuronal cells (Kuwahara et al., 1999),
and can be retrotranslocated from the secretory pathway to the cytosol to exert prosurvival activity by inhibiting the pro-apoptotic protein Bax (Jodoin et al., 2007; Roucou
et al., 2003, 2005; Jodoin et al., 2009).
Prion diseases are characterized by aggregation of aberrantly folded PrP, which is able to
catalyze the conversion of properly folded PrP into mis-folded isoforms (Prusiner, 1982).
As a result of this process, the cell experiences ER stress and activates the Unfolded
Protein Response (UPR) (Hetz, 2012). The UPR is a well conserved process that cells use
to restore ER homeostasis or enter apoptosis. In higher eukaryotes, three sensors of ER
status exist. The Inositol Requiring Enzyme 1 (IRE1α) is a transmembrane ribonuclease
that splices and activates X-Box Binding protein (XBP1) mRNA when the foldase BiP
(Grp78, encoded by HSPA5) dissociates from IRE1α and preferentially attaches to misfolded client proteins. (Yoshida et al., 2001). Additionally, IRE1α is able to activate JNK
signalling through the adapter protein TRAF2 (Hetz and Glimcher, 2009). Concurrently,
the transmembrane protein Activating Transcription Factor 6 (ATF6α), which is
Misiewicz 51
normally bound by BiP, is released upon BiP’s preferential attachment to client proteins.
ATF6α translocates to the Golgi complex, where it is cleaved by transmembrane Site 1
and Site 2 proteases to form a highly active transcription factor (Haze et al., 1999). The
third branch of ER stress signal transduction is mediated by the transmembrane PKRlike ER Kinase (PERK), which upon BiP dissociation is able to phosphorylate eukaryotic
translation initiation factor 2 (eIF2α). Phosphorylation of eIF2 arrests translation of most
messages, except for several with upstream activating open reading frames, such as the
Activating Transcription Factor 4 (ATF4) mRNA (Hetz, 2012; Vattem and Wek, 2004).
Following ER signal transduction, spliced XBP1 (sXBP1), cleaved ATF6 (ΔATF6α) and
ATF4 translocate to the nucleus where they are able to activate the expression of ER
stress responsive genes. ΔATF6α, sXBP1 or ATF4 interact with several promoter DNA
motifs called the ER Stress responsive element (ERSE), resulting in elevated expression
of genes containing this element (Yoshida et al., 2000, 1998; Kakiuchi et al., 2006).
Originally identified upstream of HSPA5 and the ER-related genes HERP and PDI6, the
classical ERSE contains the consensus sequence CCAAT-n9-CCACG. It was shown in a
yeast one hybrid screen that this element is able to bind the transcription factors ΔATF6α
and XBP1 (Yoshida et al., 2000). Yoshida et al. showed that ΔATF6α binds to the CCACG
portion of the ERSE, while the general transcription factor NF-Y binds the CCAAT part
(Yoshida et al., 1998). sXBP1 is also able to bind CCACG (Yoshida et al., 1998; Lee et al.,
2002). Removing the CCACG portion or altering the variable region to 8 or 10
nucleotides reduces or eliminates the ER stress-mediated transcriptional response. The
ERSE is able to function in either a forward or reverse orientation. Subsequently, several
other variations on the ERSE have been identified, such as the ERSE-II, which contains
the sequence ATTGG-N-CCACG and binds either ΔATF6α or sXBP1, and the ERSE-like,
with an inverted NF-Y binding site (Yamamoto et al., 2004; Kakiuchi et al., 2006).
Previously, it has been shown that the PRNP promoter contains several ERSEs as well as
a novel ER stress response element, called the ERSE-26 (Jodoin et al., unpublished). This
element contains the same sequence as the classical ERSE, except the 9-nucleotide
variable region is replaced with a 26-nucleotide variable region (giving CCAAT-n26CCACG). Expression of this element in a luciferase reporter system showed induction
Misiewicz 52
when cells were subjected to ER stress, and mutagenesis removed this. Furthermore,
Jodoin et al. demonstrated that PRNP expression is correlated with cancer severity and
ER stress (as determined by BiP), suggesting that ERSE-26 may play a pro-survival role
in ER stressed cells.
Jodoin et al. demonstrated XBP1 and ATF6 binding to the PRNP promoter, but they did
not show which transcription factor activates the ERSE-26, and the existence and
functionality of the ERSE-26 elsewhere in the genome remains unconfirmed. In order to
gain insight into the broader cellular context of this novel element and to determine
whether it is specific to PRNP or not, we conducted a bioinformatic analysis of the
complete human reference genome (Church et al., 2011) using a Regular Expression
model of the ERSE-26. Following the identification of genes whose promoters contained
this element within -2,000 bp or +500 bp from the transcription start site, reverse
transcriptase PCR (RT-PCR) and quantitative RT-PCR (qPCR) were conducted to assess
the fold changes in ERSE-26 genes in cultured primary human fetal neurons and MCF-7
cells treated with pharmacological ER stress inducers. Additionally, we produced a
luciferase reporter construct to identify which transcription factors are responsible for
ERSE-26 activity. We identified that sXBP1 transactivates the ERSE-26 in a luciferase
reporter assay. Finally, an extensive literature search was conducted to identify which
pathways and processes are affected by ERSE-26 regulation. We identified ERSE-26
genes involved in the oxidative stress response, neurotransmitter metabolism, ER
homeostasis and mitochondrial function. These co-regulated genes could provide useful
future prospects for better elucidating PrP’s cellular function.
Misiewicz 53
Materials and Methods
Genome-wide search for genes bearing an ERSE-26 element
In order to find genes in the human genome with an ERSE-26 in their promoter, a
program was written in Python. The sequence of the human genome, version GRC37.1
from the Genome Reference Commission (Church et al., 2011) was scanned using regular
expressions representing the plus and minus strand versions of the ERSE-26. The region
from -2,000 bp upstream of the transcription start site (TSS) to +500 bp downstream of
the TSS was considered to be the promoter region of the gene. The Regular Expression
pattern used was “ccaat.{26}ccacg”, matching precisely (no deviations were permitted).
Both ERSE-26 elements in forward and reverse orientations were searched. Additionally,
promoters in the positive and negative strands of the genome were examined. Source
code is provided in a Git repository located at:
(https://www.github.com/networkdynamics/erse-like-promoters-analysis).
Cell culture conditions
Human primary neuron cultures, obtained from human fetal brains subject to McGill
University ethical approval, were cultured as previous described (LeBlanc, 1995). Briefy,
brains were minced and subject to trypsinization. Following trypsinization, cells were
plated on poly-lysine coated fasks and grown in the presence of 5-fuorodeoxyuridine,
1X Penicillin Streptomycin and minimum essential media (MEM) media supplemented
with 10% bovine calf serum in a 5% CO 2 environment at 37° C. MCF-7 cells were grown
in Roswell Park Memorial Institute (RPMI) media supplemented with 10% Fetal Bovine
Serum (FBS) in a 5% CO 2 environment at 37° C. HEK293 cells were grown in Dulbecco’s
Minimum Essential Media (DMEM) supplemented with 1.7 g/L NaHCO 3 and 10% fetal
bovine serum (FBS) in a 5% CO2 incubator at 37° C. N2a cells were grown in DMEM
supplemented with 1.7 g/L NaHCO3 and 10% FBS in a 5% CO2 environment at 37° C.
Pharmacological Induction of ER Stress and other cell treatments
Neurons were subjected to ER stress, under conditions experimentally optimized to
minimize cell death and maximize ER stress induction (Jodoin et al. unpublished). In
Misiewicz 54
neurons, ER stress was induced by the drugs Brefeldin A (5 µg/mL), Tunicamycin 0.5
µg/mL and Thapsigargin (3.25 µg/mL), all from Biomol. Drugs were dissolved in
dimethyl sulfoxide (DMSO), and cells were treated for 6h, followed by immediate total
RNA extraction. One neuron preparation was treated with ER stressors in the additional
presence of 1 µg/mL actinomycin D. MCF-7 cells were also treated under conditions
optimized to minimize cell death and maximize ER stress induction, as previously
determined by Jodoin et al (Jodoin et al. unpublished). Cells were treated for 18h with
all drugs at a concentration of 5 µg/mL, and then subject to total RNA extraction.
Total RNA extraction and Reverse-Transcriptase Polymerase Chain Reaction
Total RNA was extracted from neurons using Trizol (Invitrogen) as described by the
manufacturer’s protocol. For MCF-7 cells, the Chomczynski Guanidine Thiocyanate
method was used (Chomczynski and Sacchi, 2006). RNA was quantitated on an H4 plate
reader (Biotek, Winooski, VT) for neurons, or a Nanodrop ND-1000 spectrophotometer
(Thermo Scientific) otherwise. cDNA was prepared using avian myeloblastosis reverse
transcriptase (AMV-RT), following the manufacturer’s protocol (Roche). Briefy, 1µg
total RNA was used for cDNA synthesis by AMV-RT with poly-A primers, subjected to
the manufacturer’s recommended thermal cycle, and used as a template for subsequent
PCRs.
Primer Design and PCR of ERSE-26 genes
For PCRs, primers were designed using the NCBI PrimerBlast software available online
at http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Sayers et al., 2012). Primers were
designed to have an annealing temperature of 60° C. All oligonucleotides were ordered
from a commercial supplier (Invitrogen), and optimized with a gradient of annealing
temperatures. See Supplemental Table I for all primer sequences. During all subsequent
PCRs, the optimal annealing temperature of a gene’s primer pair was always used.
cDNA was diluted to 1:200 concentration and PCR was performed with Taq DNA
polymerase
(NEB),
following
the
manufacturer’s
protocol.
Three
biologically
independent neuron preparations were used, and two technical replicates per gene per
preparation were tested. Owing to differences in genetic backgrounds of the primary
cultures, expression levels of each gene were analyzed separately in each preparation.
Misiewicz 55
For MCF-7 cells, three independent experiments were conducted for all ER stress drug
treatments and XBP1 transfections.
Misiewicz 56
Supplemental Table I – Primers used in RT-PCR
Note: a blank value in “Optimal TM” indicates no amplification
Misiewicz 57
Quantitative PCR of ERSE-26 genes
Next, qPCR was conducted on cDNA from neurons and MCF-7 cells using SYBR Green
Taq Mastermix (Quanta Biosciences). For several ERSE-26 genes, new primers had to be
designed, and the additional qPCR validated primers were obtained from PrimerBank
(Wang et al., 2012b), a repository of primer sequences and synthesized by a commercial
supplier (Invitrogen). These additional primers can be found in the Supplemental Table
II. For neurons, two technical replicates for each of the three biologically distinct
preparations were conducted. For MCF-7 cells, two technical replicates per biological
replicate were conducted. Using ERSE-26 primers that produced a single unique
amplicon, standard curves and amplification constants were determined. An Applied
Biosystems 7500Fast qPCR apparatus (Invitrogen) was used, and the manufacturer’s
default thermal cycle was used for all experiments. Primers were verified for specificity
and performance for qPCR. All output was converted to fold-induction values compared
to control (DMSO) treatments normalized with loading controls (HPRT1), using Pfaff’s
method (Pfaff, 2001).
Supplemental Table II – Primers used in qRT-PCR
Note: these primers were designed specifically for qPCR.
Misiewicz 58
Agarose gel electrophoresis and quantification
TAE agarose gels were run under identical conditions (run for 1hr at 10 V/cm), using
ethidium bromide staining. After gels completed, images were digitized using the
Syngene GeneSnap software and gel documentation apparatus. After files were saved as
uncompressed TIFFs, the ImageJ program from the NIH (Rasband, 2011) was used to
quantitate gel bands. Band intensity was converted to relative fold change compared to
control lanes (DMSO). Fold induction values determined with ImageJ for each treatment
were averaged across all replications of PCRs. RT-PCR of HPRT1 was used as a loading
control, and relative values were normalized against the fold change in HPRT1 levels.
HPRT1 was chosen as the most stable amongst several other possible loading controls
tested (ACTB, GAPDH).
Western Blotting of PrP in ER stress treated neurons
Cells were lysed for 20 min on ice in 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100
(v/v), 0.5% sodium deoxycholate (NaDOC) (w/v), 50 mM Tris-HCl, pH 7.5 lysis buffer
containing fresh protease and phosphatase inhibitors (38µg/mL AEBSF, 0.5µg/mL
leupeptin, 0.1µg/mL pepstatin, 0.1µg/mL N-α-p-tosyl-L-lysine chloromethyl ketone
hydrochloride, 4 mM sodium orthovanadate, 20 mM sodium fuoride). After
centrifugation of the lysate at 11 500 x g for 10 min at 4˚C, the supernatant was collected
as the detergent soluble fraction and the detergent insoluble pellet was resuspended in 2
% SDS and sonicated for 30 sec. The protein concentration was determined with the BCA
Protein Assay Reagents (Fisher, Nepean, ON). One hundred µg of protein was
precipitated with 4 volumes of ice-cold methanol overnight at -20˚C, centrifuged at 11
500 x g for 15 min at 4˚C and the dried pellet solubilized in Laemmli sample buffer (2%
SDS (w/v), 5% β-mercaptoethanol, (v/v), 10% glycerol (v/v), 0.01% bromophenol blue
(w/v), 62.5 mM Tris-HCl, pH 6.8). The proteins were boiled for 3 min, separated in a
10% or 15% SDS-PAGE gel and transferred to PVDF membranes. PrP was detected with
the 3F4 monoclonal antibody recognizing human PrP (1/2000, anti-PrP109-112) or the
polyclonal R155 antiserum (1/500, anti-PrP36-56), which recognizes mouse and human
PrP and was produced in our laboratory. Antibodies specific for β-actin (1/5000, clone
AC-15, Sigma, Oakville, ON), mitochondrial Hsp70 (1/1000, clone JG1, Affinity
Misiewicz 59
BioReagents, Golden, CO), and BiP (GRP 78, 1/250, clone H-129, Santa Cruz
Biotechnology Inc), were used for western blot analyses. Immunoreactivity was revealed
with 1/5000 anti-mouse or rabbit IgG conjugated to horseradish peroxidase secondary
antibodies (GE Healthcare, Baie d’Urfe, QC) and chemiluminescence reagents from GE
Healthcare or Millipore (Billerica, MA, USA) and detected with the Molecular Dynamics
Storm 840 (GE Healthcare) or Kodak Biomax MR film (Carestream Health, Toronto, ON).
The level of PrP was calculated from the intensity of the immunoreactive bands
analyzed with the Image Quant TL software (GE Healthcare). The ratio of PrP over βactin was calculated.
Cloning of ERSE-26 fragment into pMetLuc2 vector
The pMetLuc2-Reporter construct (pML2, obtained from Clontech) is plasmid that
encodes a luciferase gene from the copepod Metrida Longa. This gene contains a
powerful endogenous signal peptide, causing the protein to be secreted into the growth
media. This allows for a no-lysis protocol for assaying luciferase activity in transfected
cells by simply reading the luciferase activity (when a substrate is added) of the media.
To clone the PRNP promoter fragment into pML2, primers were designed to amplify the
region from -391bp to -274bp, around the ERSE-26 (Mahal et al., 2001). The following
primers were used (restriction sites in bold): Forward 5’-GAG CTC TCT CCA TTA TGT
AAC GGG GA-3’, Reverse 3’-GCG AAT TCT CAG TTG ATA CCG CCT GCG G-5’.
Primers contained the restriction endonuclease sites for EcoRI and SacI. The PCR
product and pMetLuc2-reporter vector was digested using EcoRI and SacI, followed by
ligation with T4 DNA Ligase (Fermantas). DH5α E. coli were transformed using
standard protocols and DNA was prepared for transfection using the alkaline lysis
method (Birnboim and Doly, 1979). The resulting pML2-EL26 construct was sequenced.
Transfection of HEK293 cells and Luciferase assay
HEK293 cells were plated in 6 well plates at approximately 500,000 cells/well. Twenty
four hours after plating, using the polyethyleneimine (PEI) method (Boussif et al., 1995),
cells were transfected with pMetLuc2-Reporter-ERSE-26 and plasmids encoding
ΔATF6α (amino acids 1-373) or sXBP1 (Lee et al., 2002). Following 6h of transfection,
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media was changed, and cells were allowed to grow for 20h. After 20h, media was
collected. Luciferase assays were conducted on 50 µl of HEK293 growth media. Substrate
(Ready-to-Glow Secreted Luciferase substrate, Clontech) for the luciferase protein was
prepared according to the manufacturer’s protocols. Assays were conducted in a 96-well
white, opaque plate (Costar) and total luminescence was determined using an H4 plate
reader (Biotek, Winooski, VT). Three reads were conducted per well, and two separate
pMetLuc2-Reporter-ERSE-26 constructs were tested in three biologically independent
experiments each. As a control, total RNA was extracted as before to verify sXBP1 and
ATF6α transcripts in transfected cells.
Western Blotting of XBP1 and ATF6α in HEK293 cells
HEK293 cells were extracted with NP-40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl,
1% NP-40, 5 mM EDTA pH 8.0). Extracts were separated by 10% SDS-PAGE, transferred
using a Biorad Transblot Turbo apparatus to PVDF membrane, blocked 1h in 5% milk,
then incubated with 1:100 anti-XBP1 (SC7160, Santa Cruz) or 1:100 anti-ATF6 (IMG-273,
Imgenex) to confirm expression of transgenes. Membranes were detected using antimouse IgG horseradish peroxidase at 1:5000 in 5% milk (Jackson) and visualized with
ECL Prime (GE Healthcare) on Kodak BioMax MR Film.
Mutagenesis of pMetLuc2-ERSE-26 constructs
Following cloning of the PRNP promoter fragment into the pMetLuc2 vector, the ERSE26 was mutated by PCR with mutagenic oligos. Primers were designed to target the
second part of the ERSE-26, the putative XBP1/ATF6 binding site: CCACG was changed
to ATCTA. Two additional basepairs after the XBP1/ATF6 site were also changed from
TC to GA, yielding a final mutation of ATCTAGA. DNA was synthesized using Pfu
Turbo (Agilent Technologies) and subject to DpnI digestion to remove non-mutated
plasmid DNA. DNA was then transformed into DH5α E. coli using standard techniques
and prepared using the alkaline lysis method. Transfection in HEK293 cells was done as
before. The following primers were used for mutagenesis, with the mutation in bold,
and the former location of the CCACG site underlined: 5’-GAT TTT TAC AGT CAA
TGA GAT CTA GAA GGG AGC GAT GGC ACC C-3’ and 5’-GGG TGC CAT CGC TCC
CTT CTA GAT CTC ATT GAC TGT AAA AAT C-3’.
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Transfection of MCF-7 cells
MCF-7 cells from ATCC (Manasas, VA) were cultured as described above and
transfected with Lipofectamine 2000 following the manufacturer’s protocol (Invitrogen).
Cells were transfected with either pCGN-sXBP1 (Bommiasamy et al., 2009) or vehicle
(pCGN-EGFP). Total RNA was extracted using Chomczynski’s method, and cDNA was
synthesized using AMV-RT as before. Next, qPCR was conducted like in previous MCF7 experiments. Fold induction values were computed using Pfaff’s method as before.
Statistical analysis
For RT-PCR conducted on neuron preparations, statistics were conducted on the average
fold-induction values computed by densitometry. For each biologically independent
neuron preparation, a one way analysis of variance was conducted, followed by
Dunnet’s post hoc test. For qPCR on MCF-7 cells, an analysis of variance was conducted
on each of the genes tested in qPCR, followed by Dunnet’s post-hoc test.
For the luciferase assays, a one-way analysis of variance followed by Tukey’s Honest
Simple Difference post-hoc test was used to determine whether or not luminescence
values were statistically significant. For all statistics, a p-value less than 0.05 was taken
to be significant and all were computed using InStat version 3.1a (Graphpad Software).
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Results
XBP1 activates ERSE-26 in HEK293 and N2a cells
To determine if the ERSE-26 responds to ER stress-regulated transcription factors,
HEK293 cells were co-transfected with the pML2-ERSE-26 luciferase reporter construct
and pCGN-sXBP1 or pCGN-ATF6(1-373). Co-transfection of pML2-ERSE-26 and sXBP1
resulted in high luciferase activity (Fig. 3A), suggesting that sXBP1 is a transcription
factor that binds the ERSE-26. ΔATF6α did not activate ERSE-26-mediated transcription,
unlike its ability to transactivate the canonical ERSE. In cells transfected with pML2empty and sXBP1, some non-significant luciferase activity was detected. This may be
due to a low-affinity XBP1 site in the pML2 plasmid as identified by a computation
search of the Transfac database of promoter binding sites (results not shown).
Mutagenesis of the putative ATF6α/XBP1 binding site (CCACG) of the pML2-ERSE-26
abrogated transactivation by sXBP1 (Fig. 3B). As expected, pML2-luciferase under the
control of a CMV promoter showed very high luciferase activity, while mock, nontransfected or pML2-transfected cells showed no luciferase activity (Fig. 3A&B). RT-PCR
(Fig. 3C) and western blots (Fig. 3D) confirmed expression of constructs. Co-transfection
of pML2-ERSE-26 and sXBP1 in N2a cells gave identical results (Suppl. Fig. 1). Together,
these results indicate that sXBP1 is the transactivating factor for the ERSE-26.
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Figure 3 – ERSE-26 activity is XBP1 dependent. A.
Luciferase assay shows XBP1 induces luciferase activity in
reporter construct. *: p<0.001. B. Mutation of ATF6/XBP1
binding site removes this activity. *: p< 0.01 C. Ethidium
bromide agarose gel of DNA obtained by RT-PCR of
transfected cells, indicating sXBP1 and ATF6 expression in
transfected cells. D. Western Blot of transfected cells
showing
sXBP1
and
ATF6
expression,
confirming
transfection. In panels A and B, Error bars indicate SEM.
Supplemental Figure 1 – Activity of secreted luciferase in
N2a cells transfected with pML2-EL26 and sXBP1. Human
sXBP1 activates pML2-EL26 secreted luciferase activity in
mouse cells.
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ERSE-26 was found in the promoters of 38 genes
To assess if ERSE-26 regulates other genes, a Python program was written to search the
Genome Reference Consortium human genome (Build GRC37.1). In total, 38 genes
contained an ERSE-26 within -2000bp and +500bp from the transcription start site of the
gene (Table I). None of these genes also contained canonical ERSEs except C6orf20,
which contained an ERSE-II, and GADD45B and LRRC55, which contained XBP1
binding sites in their promoters. Twenty-six ERSE-26 matches were found in the reverse
(3’ to 5’) direction, and 12 were found in the forward direction. As a control for the
search program, the search was repeated with the canonical ERSE (CCAAT-.9-CCACG)
as a target pattern. Forty-five genes, including the previously known ERSE-containing
genes HSPA5 (Bip, Grp78), XBP1 and PDIA6 contained an ERSE within -2000bp and
+500bp from the transcription start site of the gene (Suppl. Table II, page 58). These
results show that the ERSE-26 is not unique to the PRNP promoter and suggest that ER
stress may regulate a wide variety of genes previously unsuspected to be involved in the
ER stress response.
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Table I
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Supplemental Table III
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ERSE-26 genes are upregulated in primary human neuron cultures treated with BFA, TM
or Thps
To confirm that the ERSE-26 containing promoters are trans-activated during ER stress,
primary human neurons were treated with BFA, TM, or Thps (Fig. 4). RT-PCR identified
that 20 of the 38 ERSE-26 genes were transcribed in primary human neuron cultures
(Fig. 4A). ER stress induction was confirmed in TM and Thps-treated neurons by the
presence of the shorter 257 bp sXBP1 amplicon. The lack of sXBP1 amplicon in the BFAtreated human neurons indicates differential regulation of the ER stress transduction
pathways. Relative to the DMSO control, each of the pharmacological inducers of ER
stress (BFA, TM, or Thps) induced expression of some of the ERSE-26 genes in at least
one neuron culture (Fig. 4A). Genetic variability may explain differential ER stress
responses in the primary human neuron preparations. Following densitometric analysis
(Fig. 4B), GADD45B showed a significant increase for all three stressors in preparation 1,
and a significant increase under TM and Thps treatment for preparation 2, but no
increases in preparation 3. Despite the variability in responses across preparations,
SESN2, NUDT9-T1 (transcript variant 1), GAD2, and ERLEC1 showed an upward trend.
In contrast, the genes USP4, YPEL3, LRRC55 and SMPD1 showed downward trends in
all cases. The genes GNAQ, KBTBD2 and NOL10 were mostly unchanged across all
preparations.
Further analyses by qPCR showed that BFA, TM, and Thps all significantly increased
HSPA5 (BiP) and sXBP1 mRNA levels, thus confirming the activation of the ER stress
response under these conditions (Fig. 4C). Furthermore, at least one of the ER stressors
increased PRNP, ERLEC1, GADD45B, SESN2, and SLC38A5 mRNA levels in primary
human neurons. Similar to previous observation, the response of some genes to ER
stress varied with the type of ER stress despite the fact that each stressor clearly induced
ER stress. Increased PrP and BiP accompanied ER stress increased mRNA levels (Fig.
4D).
Transcriptional inhibition with actinomycin D stunted the ER stress-mediated
upregulation of PrP.
Together, these results indicate that ERSE-26-containing genes can be differentially
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transactivated depending on the ER stress and on the genetic background of the
individual.
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Figure 4 – Induction of ER stress increases ERSE-26 gene
expression in cultured primary human neurons. Figure
represents three independent preparations, in duplicate. A.
Representative Gel electrophoresis of ERSE-26 genes
detected by RT-PCR in cultured primary human neurons,
indicating which ERSE-26 genes are detectable in neurons.
XBP1 and HPRT1 shown as controls. B. Panel B shows
each pharmacological treatment as a bar graph, and the
expression of each gene in each neuron preparation is
indicated by a different color bar within each graph. Upper
panel: Fold change of ERSE-26 genes following treatment
with BFA compared to DMSO (1 fold change). Middle
panel: Fold change of ERSE-26 genes following treatment
with TM. Bottom panel: Fold change of ERSE-26 genes
following treatment with Thps. *: p<0.05, **: p<0.005. Error
bars indicate SEM. Within each treatment, significance was
assessed by a one-way analysis of variance followed by
Dunnett’s post-hoc test. C. By qPCR, average fold
induction of several ERSE-26 genes and ER stress controls
averaged across two biological replicates and two technical
replicates. Error bars represent SEM. A one-way analysis of
variance followed by Dunnet’s post-hoc test were used to
assess significance. *: p < 0.05, **: p < 0.005. NT indicates
no treatment and -S indicates serum deprivation. D. In
cultured primary neurons, PrP increase following ER stress
treatment is transcriptionally dependent. The top two
panels show induction of PrP by western blot during ER
stress.
However,
when
the
transcription
blocker
Actinomycin is applied to the neurons, PrP levels do not
increase in ER stress.
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ERSE-26-containing genes are upregulated in ER stressed breast carcinoma MCF-7 cells.
To determine if the variability observed with different ER stressors is dependent on the
genetic background of the cells, we stressed MCF-7 cells with BFA, TM or Thps and
subjected the mRNA to qPCR. The results showed increased HSPA5 when cells were
treated with all three stressors and increased sXBP1 mRNAs when cells were treated
with BFA and Thps, confirming the ER stress response (Fig. 5A). PRNP, ERLEC1,
GADD45B, SESN2, and SLC38A5 showed a significant increase in mRNA when MCF-7
cells were treated with BFA. However, TM treatment caused increased expression only
for ERLEC1, and Thps treatment induced expression of ERLEC1 and GADD45B. To
assess if XBP1 alone can transactivate ERSE-26 containing genes in a cellular system,
MCF-7 cells were transfected with a vector expressing sXBP1 and expression levels was
monitored by qPCR. As expected, sXBP1 mRNA levels were increased 155 fold over
mock-transfected cells (Fig. 5B). The control EGFP vector induced a slightly higher level
as well but this was not significant. HSPA5 mRNA levels were induced approximately 3
fold with sXBP1, as expected for an ER stress regulated gene. However, only PRNP
showed increased mRNA levels with sXBP1 expression. These results suggest that
regulation of ERSE-26 gene expression required additional transactivating factors.
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Figure 5 – MCF-7 cells show induced ERSE-26 gene
expression following ER stress treatment or sXBP1
transfection A. Fold induction of ERSE-26 genes in MCF-7
cells treated with BFA, TM or Thps for 18 h as determined
by qPCR. Induction of HSPA5 and spliced XBP1 were used
as ER stress controls. A one way analysis of variance
followed by Dunnet’s post hoc were used to assess
significance, *: p<0.05, **: p< 0.005, ***: p< 0.0001. Data
represent three biologically independent experiments
performed in duplicate. B. ERSE-26 gene expression levels
in sXBP1 transfected cells compared to mock transfected
cells. **: p < 0.005. Data represent two biologically
independent experiments performed in duplicate.
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Discussion
Following the previous identification of the ERSE-26 (Jodoin et al. Manuscript
submitted), we set out to determine whether or not this element is unique to PRNP or
represents a new, novel mechanism for cells to respond to ER stress. Interestingly, during
the discovery and characterization of the ’classical’ ERSE in the late nineties and early
2000s, it was demonstrated that the ERSE is dependent on a precise spacing of nine
nucleotides between ATF6 and NF-Y sites (Yoshida et al., 2000). The authors
demonstrated this with luciferase reporter constructs with either eight or ten basepairs
added between binding sites, but larger numbers were not tested. Our results here
suggest that other ERSE configurations are possible, and that the ERSE-26 operates
through previously identified ER stress transcription factors. Previously known
functions of PrP appeared to be co-regulated with PRNP by the ERSE-26, and we also
identified putative new processes that are co-regulated with PRNP.
Our search program identified 38 genes in the human genome with the ERSE-26
in their promoter. Importantly, this indicates that the element exists elsewhere in the
genome and is not PRNP specific. In addition, most of the genes found in the search did
not contain other ERSEs and were not previously known to be ER stress regulated. These
results are a new set of ER stress regulated genes, previously unknown.
We restricted our search to exact matches only. Allowing for more sophisticated
searches, using position weight matrices or Hidden Markov Models might produce for
hits. Additionally, our definition of 'promoter' was rather arbitrary. A more nuanced
definition of the promoter region to search might expand hits. However, since we found
abundant genes using the aforementioned parameters, we did not adjust these values,
but it seems like a logical first step for followup experiments.
Next, we showed ERSE-26 gene induction in cells that are pharmacologically ER
stressed. The behavior of ERSE-26 genes in neuron cultures was inconsistent across
genetic backgrounds. All genes showed upregulation in at least one preparation, but it
was not repeated across all three for most genes. However, the genes GADD45B showed
consistent behavior across at least two treatments in two preparations, and SESN2
showed an upward trend in two treatments in two preparations. In qPCR experiments,
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which utilize a far more accurate detection method, GADD45B, and SESN2 showed
upregulation. Both are previously known as stress responsive genes: their status as
ERSE-26 genes might explain one way they are activated. In contrast to neurons, more
ERSE-26 gene tested in MCF-7 cells was upregulated, indicating that perhaps the ERSE26 is more or less functional in different cell types. PRNP, which is ERSE-26 regulated,
has been shown to be protective against apoptotic insults in breast cancers; perhaps
ERSE-26 regulation is the mechanism causing this (Diarra-Mehrpour et al., 2004).
Co-transfection of a luciferase reporter construct and the activated ER stress
transcription factors ATF6 and XBP1. We elected for co-transfection because the use of
the drugs BFA, TM or Thps would interfere with the secretory pathway in a variety of
ways, confusing the results. XBP1 is the transcription factor primarily responsible for
ERSE-26 activity, while the classical ERSE was found by Yoshida et al. to be mostly only
ATF6 activated. However, XBP1 has been shown to bind the ERSE in a yeast one-hybrid
screen, and yeast Hac1p (XBP1 homolog) binds the ATF6 promoter (Lee et al., 2002;
Yoshida et al., 1998). Subsequent to the discovery of the ERSE, additional ERSE-like
motifs have been found, and have been shown to be regulated by XBP1 and/or ATF6 to
differing degrees (Yamamoto et al., 2004). Thus, the ERSE-26 behaves similarly to other
ERSEs. Despite the evidence for XBP1, we cannot at this time rule out that ATF6 plays a
role in ERSE-26 activity. Possibly transfection with ATF6 requires co-transfection with
NF-Y, the obligate partner of ATF6 in the classical ERSE.
As further evidence for XBP1’s ability to induce ERSE-26 gene expression,
transfection of MCF-7 cells showed increased ERSE-26 genes. Thus, experimentally, the
ERSE-26 can be activated in two ways: either pharmacologically or genetically.
ER stress is implicated in a variety of diseases such as Alzheimer and cancer,
understanding how cells respond to ER stress is important to properly treat these
diseases. To gain insight into the transcriptional program regulating PRNP during ER
stress, we conducted an extensive literature search to identify functions of ERSE-26
genes (automated clustering did not give any significant results). Some potential novel
roles for PRNP in the cell were identified as well as confirmation for previously
published functions of PrP.
One notable functional group identified in this search was genes related to cell
adhesion and synapse function. GAD2, LRFN4, LRRC55, and SLC38A5 all play a role in
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this process. GAD2 is one of two terminal rate limiting enzymes that cleave glutamic
acid into γ-Aminobutyric acid (GABA) (Erlander et al., 1991). LRFN4, also known as
SALM3 is known to bind Post Synaptic Density 95 protein, and induces neurite
outgrowth (Seabold et al., 2008). LRRC55 was recently identified as a protein known to
enhance activity of calcium activated potassium channels (Yan and Aldrich, 2012).
Finally, SLC38A5, also known as SN2 or SNAT5, is an astrocyte expressed amino acid
transporter that permits astrocytes to release glutamic acid for neurons to cleave to
GABA (Cubelos et al., 2005). Both drugs and XBP1 transfection showed that LRFN4 and
KBTBD2 were very upregulated, as well as SLC38A5. GAD2 showed expression during
treatment with BFA. These findings give support to previously published evidence that
PrP plays a role in the synapse (Linden et al., 2008). Additionally, we have identified
biological processes and co-regulated genes that future studies can use to pinpoint the
role of PrP at the synapse.
Another notable genes in the results is GADD45B. It was originally discovered as
part of a family containing GADD153 (CHOP) (Zhan et al., 1994) and the prionassociated pro-survival EIF2a phosphatase GADD34 (Moreno et al., 2012). This protein is
a known activator of p38 MAPK signaling that is known to promote survival (Takekawa
and Saito, 1998; Takekawa et al., 2002; Engelmann et al., 2007). GADD45B’s upregulation
is consistent with PrP’s previously known pro-survival function (Kuwahara et al., 1999;
Roucou et al., 2005; Jodoin et al., 2007). Notably as well, p38 MAPK signaling has been
shown to promote changes to the cytoskeleton, which is consistent with our observation
that beta-actin levels changed dramatically during ER stress activation (Guay et al.,
1997). This gene was consistently upregulated in neurons treated with BFA, TM and
Thps, sometimes as much as 8 fold. It seems clear this gene is co-regulated with PrP, and
this further strengthens the case for normal PrP’s pro-survival role. In addition to the
known stress responder GADD45B, MYEOV2 (myeloma over-expressed 2) was highly
upregulated in neurons, although not in MCF-7. This protein, about which nearly
nothing is known, was identified as highly expressed gene in myelomas. Thus is seems
logical that this gene would be activated in a stress response, especially since it is known
that ER stress is a hallmark of cancer.
The final group of function that appeared was related to oxidative stress. The
genes NUDT9, TIMM44, SESN2 and GADD45B are known to be localized to the
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mitochondria or as responders to oxidative stress (Takekawa and Saito, 1998; Budanov et
al., 2010; Perraud et al., 2003). Although TIMM44 did not show induction following
XBP1 transfection or drugs in neurons, it showed activation in MCF-7 cells treated with
drugs. NUDT9, a gene whose function remains largely unknown, was strongly induced
by XBP1 transfection. Interestingly, both of these proteins are known to localize to the
mitochondria, and TIMM44 is known to interact with chaperones (Moro et al., 1999).
SESN2 and GADD45B (the latter is even used as a marker for p53 activity) are both
regulated by p53 in response to environmental stresses (Budanov, 2011; Takekawa and
Saito, 1998). Even more interesting, our results included a transcription factor FOXO4;
the FoxO family of transcription factors are known to regulate Sestrin family members
(Budanov et al., 2010). However, we were not able to detect FOXO4 expression in MCF-7
cells or neurons. Most importantly though, is that PRNP contains a p53 binding site
(Vincent et al., 2009). Thus all of these genes share a common regulator in addition to
their ERSE-26. This further bolsters the evidence for the cytoprotective role of PrP. PRNP
has been previously identified as a oxidative stress responsive gene (Shyu et al., 2004;
Wong et al., 2001; Qin et al., 2009); the evidence here bolsters its anti-oxidative stress
role.
There appear to be two functional groups of genes identified in this bioinformatic
survey. Genes that respond to oxidative stress and promote cell survival, and genes that
appear to play a role at the synapse and its function. It has been shown that cytosolic
PrP, retrotranslocated from the membrane is needed for PrP to exert its protective
functions. Furthermore, it is a well known fact that synapses are required to ensure
neuronal health. The genes identified and validated here seem to refect this two-sided
nature of physiological PrP, and its easy to imagine how scrapie diseases can disrupt
these processes. Although we have not shown that PrP interacts directly with anything
identified here, that seems to be a promising area of future study, given these genes’
implications in processes that PrP is involved in.
In conclusion, we have identified many new genes that contain a previously
described novel promoter element similar to the ERSE. We have showed that this
element appears to be responsible for transcriptional activity in a variety of
environments, and that genes regulated by it (and co-regulated with PrP) are involved in
previously identified cytoprotective roles in the cell (i.e. these genes were already shown
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to be protective). The results presented here can be used to help identify the exact
function of PrP, whose role in all these processes is known but remains unexplained.
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Chapter 3 – General Discussion
The function of PrP in the cell remains somewhat unclear to this day. PrP is implicated in
a variety of cellular functions, including metal ion binding, synapse localization, antioxidative stress response, and anti-Bax function (Westergard et al., 2007; Linden et al.,
2008). In particular, PrP's role as an anti-Bax agent is convincing; the particular isoform
of PrP has been identified, as well as the exact region of the protein that is needed, and
the role of disease mutations in this anti-Bax activity (Bounhar et al., 2001; Roucou et al.,
2003, 2005; Jodoin et al., 2007; Lin et al., 2008; Jodoin et al., 2009) . However, this remains
somewhat controversial in the field of prion research, as there are some investigators
who claim to have found a cytotoxic effect for cytosolic PrP. (Ma et al., 2002; Anantharam
et al., 2008).
Despite these conficting reports however, a clear picture of PrP C as a protective
gene is beginning to emerge. One area that, until now, had been poorly studied is the
regulation of PRNP in this context. Our lab recently characterized the expression of
PRNP as a responder to ER stress. Furthermore, in the same work, the association of PrP
with longevity in breast cancer tissue was established and correlated well with BiP
expression (Jodoin et al., unpublished). Importantly, it was shown that this ER stress
regulation of PRNP was due in part to a novel ERSE. Jodoin et al. demonstrated the
existence and function of this element in the PRNP promoter, but the current work
expands this into the rest of the genome. Furthermore, I've shown that genes regulated
by oxidative stress appear to be co-regulated with PRNP. While ER stress regulation is a
new role for PrP, regulation by oxidative stress is congruent with PrP's previously
identified function as an oxidative stress responder. The evidence presented here shows
that these two responses can be regulated by the same transcriptional element,
suggesting overlap between these two responses.
There are two important results of the genome-wide search and subsequent
search. One, the element is not unique to PRNP, and represents a novel mechanisms for
mediating ER stress transcription. This puts the ERSE-26 in the same category as the
ERSE, ERSE-II, ERSE-like, and UPRE. Furthermore, it is important to note that in this
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work, only exact matches were used for the search. More sophisticated models allowing
substitutions in the element can be produced, and the number of ERSE-26 genes will be
larger than what is reported here. Two, the ERSE-26 is a marker for ER stress mediated
genes, like the ERSE. A great deal of the genome remains very poorly understood; many
genes have been identified by exome sequencing or computational searches, however
nothing is known about them. By examining their promoters for certain regulatory
elements, much can be inferred about the function of these newly discovered genes.
Indeed, there are several genes like this in the ERSE-26, and given that I have shown this
element is a marker for a certain pattern of expression, already we know more about
them than we did before.
Understanding the nature of what genes are regulated alongside PRNP by the
ERSE-26 is also very relevant. The genes that contained an ERSE-26 were genes that have
been previously shown to be protective. The response to ER stress in cells is a deeply
conserved transcriptional program, going back as far as yeast. Studying these
transcriptional programs as a whole is informative because we can gain a much more
holistic understanding of how the cell functions. Thus, the genes found in this survey
would seem to corroborate the growing protective role for PrP, since the most
upregulated genes (GADD45B and SESN2) were previously known stress responders
and pro-survival genes. It seems unlikely that the literature suggesting PrP C can be toxic
is not correct, given all the evidence now assembled that indicates PrP is protective
against a variety of insults. Additionally, PrP C is highly and continuously expressed in
many different cell types, which does not make sense for a cytotoxic gene. Furthermore,
PrP's protective nature also suggests a mechanism by which scrapie can be exacerbated:
mis-folded PrP produces stress in cells, which upregulate PrP in response, which leads
to more substrate being available.
Having a more complete understanding of ER stress is very relevant to disease.
ER stress has been implicated in diseases that are of enormous challenge and burden to
healthcare systems around the world, namely Alzheimer Disease and cancer (Doyle et
al., 2011; Ozcan and Tabas, 2012). A more complete understanding of ER stress could
lead to invaluable treatments or remedies for these diseases which are the cause of
untold human suffering and burden. Thus, any better understanding of the regulation of
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ER stress and how cells cope with stresses is of high importance.
In a much broader sense, the methods presented in this paper are of great
importance. Biology is now entering the age of the large-scale experiment. Microarrays,
protein interaction assays, and genome sequencing projects are producing massive
amounts of data, more than can be analyzed by hand. This is a sharp break from the
past, where the methods available only allowed the study of a small number of
questions simultaneously. As a testament to the amount of data biologists are now
producing, one can simply look at the size of Genbank, which grew by nearly 20% last
year alone (containing 339 billion basepairs as of August 2011), and doubles every 18
months (Benson et al., 2012). And it is not simply biological data that is being produced:
all of humanity produced 1.8 zettabytes of information in 2010, or 1.8 trillion gigabytes,
representing 9-fold growth in five years. By 2015 nearly 8 zettabtyes are expected, which
will be a major challenge to computer scientists to understand, interpret and extract
knowledge from this information (Gantz and Reinsel, 2011)
However, there is a difference between information and knowledge, and one of
the major challenges of these large scale projects is interpreting the data. This is one of
the areas where bioinformatics can be of great use to biologists. As mentioned earlier,
“wet bench” methods have a restricted throughput, meaning that in order to gain
knowledge about how parts of a cell interact, specific topics of interest must be
identified for study. Using bioinformatics will allow scientists to identify research
questions and topics for time consuming wet bench research. The sequencing of the
human genome is one of the proudest moments for science, on par with the moon
landing or the construction of computers and the internet. Recently-released commercial
products and advances in sequencing technologies have put us on the cusp of the era of
the $1,000 genome, compared to billions in 2000. In a short matter of time, it seems that
every individual will have his genome sequenced as a routine matter during his life. The
need for scientists who have the tools to deal with this massive deluge of information
will only be increased in years to come – but having the ability to confirm findings in the
lab will be equally important. The work presented in this thesis represents a
combination of the old world and the new, which will be critically important in years to
come.
Misiewicz 83
Conclusion
The evidence presented here shows that PRNP can be expressed in response to
ER stress. This activity is regulated by the ERSE-26, a novel ERSE. It is functional and
extant elsewhere in the genome, and transactivated by XBP1.
The genes upregulated by the ERSE-26 are implicated in the response to
Oxidative Stress. This corroborates the previous evidence for PrP function. PRNP helps
contribute to the longevity of cells that express it, due to its protective activity, which is
why it is upregulated by the ERSE like 26 when cells are stressed. Furthermore, another
tissue that experiences large amounts of non-pathological ER stress is the immune
system, where BiP was first discovered. In my view, it is not a coincidence that these
tissues also express high levels of PrP. Taken together, the ERSE-26 mediated regulation
of PRNP corroborates growing evidence that PRNP is a cytoprotective gene, responds to
oxidative stress, and helps cells survive perturbations.
Misiewicz 84
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