Download HPV DNA PARTITIONING DURING MITOSIS AS FOLLOWED

HPV DNA PARTITIONING DURING MITOSIS
AS FOLLOWED BY FLUORESCENCE MICROSCOPY
by
ROBERT J. CARTER
LOUISE T. CHOW, COMMITTEE CHAIR
WILLIAM J. BRITT
THOMAS R. BROKER
IGOR CHESNOKOV
N. PATRICK HIGGINS
KENT T. KEYSER
TIM M. TOWNES
A DISSERTATION
Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
BIRMINGHAM, ALABAMA
2010
Copyright by
Robert J. Carter
2010
HPV DNA PARTITIONING DURING MITOSIS
AS FOLLOWED BY FLUORESCENCE MICROSCOPY
Robert J. Carter
Department of Biochemistry and Molecular Genetics
ABSTRACT
Human papillomaviruses (HPVs) are small, double-stranded deoxyribonucleic
acid (DNA) tumor viruses capable of establishing persistent infections in the epithelia.
After infecting actively-dividing basal cells, the papillomavirus (PV) genome is maintained as extrachromosomal nuclear plasmids. It is largely unknown how the viral genome is maintained in actively-dividing cells. Our lab demonstrated that several HPV
genotypes appear to employ a distinct strategy to facilitate partitioning of HPV DNA into
daughter cells during mitosis. Association of the HPV-11 origin of replication (ori)binding protein E2 with the mitotic apparatus via cellular adapter proteins is thought to
mediate equal partitioning of HPV genomes into daughter cells. The focus of this dissertation project was to devise and test a technique allowing direct visualization of replicated HPV DNA during mitosis.
First, I developed a tracking plasmid system for observing newly-replicated HPV
ori-containing plasmids during mitosis. Certain DNA-binding proteins only bind to a
unique DNA sequence. I incorporated DNA binding-site arrays into HPV ori-containing
plasmids, expressed DNA-binding domain-GFP fusion proteins, and visualized HPV
tracking plasmids using high-resolution, high-sensitivity microscopy. Usage of Gal4
DNA-binding site (Gal4-DBD)- and TetR GFP fusion proteins each failed. Alternatively,
rtTA3-eGFP-nls facilitated efficient, doxycycline(dox)-inducible, direct, in situ fluorescent detection of replicated HPV ori-containing plasmid without side-effects. TetO ar-
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rays I successfully-utilized were smaller than those previously-reported by others. With
my 48-copy, tandem TetO array, plasmid was only detectable after exposure of rtTA3eGFP-nls to dox. To our knowledge this is the first time that DNA has been induciblylabeled by fluorescence.
Secondly, I report that we were able to follow the fate of HPV plasmids during
mitosis using imagery. A short, pre-treamtent with dox prior to fixation allowed us to
visualize replicated plasmids as small, discrete foci with relatively-uniform intensities.
Control TetO array-only control plasmids mostly coalesced to form a small number of
large, bright aggregates. We suggest that replicated HPV DNA can persist in dividing
cells rather than being sequestered and degraded as was the case with control nonreplicated control plasmids.
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ACKNOWLEDGMENTS
I thank my mentors, Drs. Louise T. Chow and Thomas R. Broker, for allowing me
to conduct my research in their lab with their guidance and support. I believe firmly that
my dissertation work could not have been completed in any other laboratory in the world.
Their expertise, advice, patience, and criticism were instrumental in shaping who I have
become as a researcher.
I thank Tim Townes and Pat Higgins for their tremendous support and encouragement, without which I probably would have left science. Dr. Townes went in with our
lab to acquire the DeltaVision Spectris on which I completed much of my dissertation
work. My family and I appreciated Dr. Higgins’ scientific exactness, perfect integrity
and constructive feedback given to me at pivotal moments during my dissertation work.
He requested and shared contructs from David Sherratt at Oxford University that were
vital my completing my thesis project.
Peter Prevelige and Tim Townes provided substantial financial support that enabled our lab to remain entirely-intact, even during the leanest of economic times.
I appreciate those in the Broker/Chow laboratory who grew to become close
friends over the years. In particular, Wayne Wang and his wife have been close friends
and helpful for many years. Also, Biing-Yuan Lin and Nicholas Genovese were there to
encourage me to press on when I was ready to throw the towel in and walk away during
my most difficult times here at UAB. I thank Brian Van Tine for mentoring me as his
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rotation student and teaching me so much of what I know about microscopy. He was instrumental in my attending the advanced microscopy course at Cold Spring Harbor Laboratories in 2002. I also must mention Sanjib Banerjee, Lucas Yu, and Luan Dao and
thank them for conversations and life lessons that have shaped me to become who I am
today.
The training I received from Jason Swedlow, Abby Dernburg, and John Murray
and their assistants while at Cold Spring Harbor Laboratories has impacted every aspect
of my research.
Last but definitely not least, my family deserves special mention. They tolerated
the chaotic, demanding lifestyle that comes with having a scientist as a husband and father. My wife Aimee and children Ethan and Elise have been sources of smiles each time
I return home.
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TABLE OF CONTENTS
Page
ABSTRACT....................................................................................................................... iii
ACKNOWLEDGMENTS ...................................................................................................v
LIST OF FIGURES ........................................................................................................... ix
LIST OF ABBREVIATIONS..............................................................................................x
INTRODUCTION ...............................................................................................................1
HPV pathobiology and life cycle.............................................................................1
HPV genome structure and functions......................................................................4
Normal morphology of the epidermis and the impact of HPV infection ................9
Multiple roles for the upstream regulatory region (URR).....................................11
Control of gene expression by elements within Papillomavirus URR’s ........12
The URR contains the papillomavirus origin of replication (ori) ..................13
Viral genes and proteins ........................................................................................15
E1 ...................................................................................................................15
E2 ...................................................................................................................16
E1^E4 .............................................................................................................17
E5 ...................................................................................................................18
E6 ...................................................................................................................19
E7 ...................................................................................................................21
L1 and L2 .......................................................................................................22
The partitioning of papillomavirus DNA into daughter cells requires the URR and
E2 proteins.............................................................................................................22
Cell division: the mitotic apparatus and chromosome segregation .......................24
HPV DNA replication and early region message splicing ....................................25
Experimental aims and questions addressed in this project ..................................27
EXPERIMENTAL WORK................................................................................................28
Tracking DNA inside mammalian cells using Gal4-DBD, TetR, and rtTA3 GFP
fusion proteins................................................................................................28
Pre-requisites and experimental constraints to carefully consider during development and testing of HPV tracking plasmid systems.......................................32
Expression of the HPV E1, E2, and E4 proteins impacts cell cycle progression and can be cytotoxic........................................................................32
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peBFP2-11-E1E2 plasmid supports efficient ori-specific plasmid replication ..........................................................................................................33
Tight association of a GFP fusion protein in a large array to an HPV ori
plasmid can impede ori replication.........................................................37
Tracking plasmid detection sensitivity during mitosis erquires optimized expression of the eBFP2-11-E1E2 operon and rtTA3-eGFP-nls ...............40
Clear visualization of HPV DNA localization is obtained when rtTA3-eGFP-nls
binds to arrays of tetO sites embedded in tracking plasmids ........................43
The cellular distribution pattern of the 48-copy TetO array-containing plasmid ..........................................................................................................43
Plasmid does not associate with transiently-expressed rtTA3-eGFP-nls to
form foci until cells are treated with doxycycline ..................................47
The 11-URR/TetO array-containing tracking plasmid is efficiently-targeted
to microtubules during mitosis ...............................................................51
DISCUSSION....................................................................................................................57
Possibilities for future research .............................................................................59
Additional HPV tracking experiments of interest .........................................59
An alternative to ChIP to pull down discrete chromatin sequence................61
Super-resolution fixed- and live-cell imaging on an OMX microscope .......66
MATERIALS AND METHODS.......................................................................................66
GENERAL LIST OF REFERENCES ...............................................................................79
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LIST OF FIGURES
Figure
Page
1 Differentiation profile of normal epithelium ...................................................................3
2 HPV-11 genome organization and transcripts produced by the E1E2 operon ................7
3 HPV-11 URR features ...................................................................................................13
4 The replication efficiency of HPV-11 ori plasmids in the presence or absence of TetR
and doxycycline ............................................................................................................35
5 The replication efficiency of HPV-11 URR-containing plasmids in the presence or absence of rtTA3-eGFP-nls and doxycycline ..................................................................39
6 The distribution pattern of the negative control, 48-copy TetO array plasmid in doxycycline (dox)-treated cells ............................................................................................45
7 The inability to detect replicated 11-URR/48-copy TetO array plasmid in 293 cells not
treated with doxycycline (dox) .....................................................................................49
8 The distribution pattern of replicated 11-URR/48-copy TetO array tracking plasmid in
doxycycline (dox)-treated 293 cells..............................................................................55
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LIST OF ABBREVIATIONS
ATP
adenosine triphosphate
ATPase
adenosine triphosphatase
BFP
blue fluorescent protein
Bp
base pair
BPV
bovine papillomavirus
BS
binding site
C
carboxyl (terminus of a protein)
Cdk
cyclin-dependent kinase
cDNA
complementary deoxyribonucleic acid
Cy-3
cyanine 3
DAPI
4’,6-diamidino-2-phenylindole
DNA
deoxyribonucleic acid
E2BS
E2-binding site
EBNA1
Epstein Barr nucleic antigen-1
EBV
Epstein Barr virus
FK
foreskin keratinocyte
G1
gap 1 phase of the cell cycle
G2
gap 2 phase of the cell cycle
Gal4DBD
Gal4 deoxyribonucleic acid binding domain
GFP
green fluorescent protein
x
LIST OF ABBREVIATIONS (Continued)
H
hinge domain of a protein such as HPV E2
HAT
histone acetyltranferase
HDAC
histone deacetylase
HPV
human papillomavirus
HR
high-risk, referring to the neoplastic potential of a papillomavirus
Hsp
heat shock protein
IRES
internal ribosome entry site
Kb
kilo base (1000 nucleotides, a standard for representing the length of a
segment of DNA)
Kbp
kilo base pair
kDa
kilo Dalton, a unit of mass of a macromolecule
LR
low-risk, a non-oncogenic papillomavirus
M
mitosis
MAP
microtubule-associated protein (not to be confused with mitogen-activated
protein kinase, MAPK)
MARS
matrix attachment regions
MCM
minichromosome maintenance
mRNA
messenger ribonucleic acid
MTOC
microtubule-organizing center
N
amino (terminus of a protein)
NCR
non-coding region, also called the upstream regulatory region (URR) of a
papillomavirus
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LIST OF ABBREVIATIONS (Continued)
NLS
nuclear localization sequence
ORC
origin recognition complex
ORF
open reading frame
Ori
origin of replication sequence
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PFA
paraformaldehyde
PHK
primary human keratinocyte
PML
promyelocytic leukemia
pRb
retinoblastoma protein, p105
Puro
puromycin resistance gene
PV
papillomavirus
PVTD
papillomavirus transcription domain
RNA
ribonucleic acid
RNase
ribonuclease
rt
room-temperature
rtTA3
reverse tetracycline-inducible transcriptional activator protein
S
synthesis phase of the cell cycle
SSC
sodium chloride, sodium citrate
SV40
simian virus 40
TetR
tetracycline-responsive repressor protein
URR
upstream regulatory region (also see NCR)
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1
INTRODUCTION
HPV pathobiology and life cycle
Papillomaviruses are non-enveloped, icosahedral DNA tumor viruses with circular, double-stranded genomes of approximately 8 kb (Figure 2). Each PV is host speciesspecific with infectivity limited to either mucosal or cutaneous epithelium at specific
body sites. As of 2010, more than 150 different HPV types have been recovered from
infected human tissue and molecularly characterized (de Villiers et al. 2004). HPV infections are often sub-clinical. Active infections cause epithelial hyperproliferation, with
viral lesions being referred to as warts, papillomas, condylomas, or low grade dysplasias,
depending on the body site(s) being affected. Active infections usually regress but may
reactivate at a later time. At a low frequency, infections by the “high-risk” oncogenic
viruses such as HPV-16, HPV-18 and closely related types can progress to cause highgrade dysplastic lesions and carcinomas. Studies of men with penile cancer and women
with cervical cancer frequently reveal that high-risk HPVs are a necessary underlying
cause. Cervical cancer is the most prevalent cancer affecting women under the age of 35
(zur Hausen 2002). Globally, cervical cancer remains the second most common malignancy amongst women. It is projected that over 500,000 new cases of cervical cancer
will occur each year, and worldwide, more than 275,000 women will likely die of cervical cancer annually (zur Hausen 2000; zur Hausen 2002; Ellenson and Wu 2004). Studies of individuals within this same risk group show that HIV infection and AIDS in-
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creases the rate of HPV disease progression (Aoki and Tosato 2004). The immunosuppressed condition of individuals co-infected with HPV and HIV may wholly account
for the elevated pathogenicity of HPV oncoproteins. Contrary to the disease caused by
the so-called high-risk HPV strains, low-risk HPVs, such as HPV-6 and HPV-11, can
cause benign condylomas of the anogenital tract as well as recurrent laryngeal papillomatosis. Although benign in nature, patients with laryngeal papillomas can develop lifethreatening airway blockages unless surgically removed.
The papillomavirus life cycle can be sub-divided into two or possibly three
phases. The first is the initial infection of basal cells and the establishment of the infection. The second phase is the maintenance of the viral genome in the long-living basal or
parabasal cycling cells. The third phase involves high-level amplification of the viral genome; it occurs only in a subset of post-mitotic, differentiated cells comprising a
squamous epithelium. The capsid proteins are expressed and the progeny virions assembled in the uppermost several layers of cells, while maturation of virus particles takes
place in the squames or cornified envelopes of dessicated cells, from which the virions
are released upon mechanical disruption (Figure 1). Monolayer cell cultures are insufficient for studying most aspects of HPV biology because of the absence of cellular differentiation. To overcome this obstacle, a method was devised to produce differentiated
squamous epithelial tissue in vitro. By culturing primary human keratinocytes (PHKs)
atop collagen beds at the air-media interface, the cells divide and differentiate as they are
pushed upward and further away from the liquid medium interface. This system allows
production of organotypic epithelial tissue that is almost indistinguishable from clinical
specimens (Dollard et al. 1992). The differentiated strata are labeled alongside an image
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of a cross-section from an organotypic raft culture (Figure 1, left-hand column). The viral gene expression profile and certain viral processes such as virion assembly and release
are restricted to discrete layers of cells within the stratified squamous epithelium (Figure
1, right-hand column). With the organotypic raft culture system, the complete virus lifecycle from infection to the shedding of progeny virions can now be reproduced and studied in vitro (Chow and Broker 2006; Wang et al. 2009).
Figure 1: Differentiation profile of normal epithelium. Viruses infect basal and parabasal cells where low-level maintenance of the viral genome proceeds. After expression of
the E6 and E7 oncoproteins in the spinous layer has artificially induced and sustained reentry into S-phase, high-level amplification of the viral genome commences. Expression
of capsid genes, virion assembly, and the sloughing off of infectious virus particles are
restricted to the upper layers of the stratified squamous epithelium.
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HPV genome structure and functions
The HPV genome can be divided into three regions: the upstream regulatory region (URR), the early (E) genes, and the late (L) genes. All open reading frames (ORFs)
are encoded by the same DNA strand of the viral genome. All mRNA transcription proceeds in the same direction. Utilization of several promoters, two polyA sites and alternative mRNA splicing together provide access to each of the various ORFs. The following is a brief summary of cis elements identified in the URR and proteins encoded and
their known function(s) during the viral life cycle. Subsequent sections contain more detailed discussion of the URR, viral genes and proteins.
URR: Comprising approximately 700 bp of the PV genome, it contains transcriptional
regulatory elements, promoters and the viral origin of replication (ori). The ori is
comprised of multiple tandem binding sites for the viral E1 and E2 proteins necessary for the initiation and elongation of viral DNA replication.
E1:
E1 is the replicative DNA helicase required for initiation and elongation during
HPV genome replication. E1 is responsible for recruitment of the host DNA replication machinery to the origin, making the successful replication of the viral genome possible. The extremely-rare E1 transcript is derived from an unspliced
transcript spanning the multi-cistronic E1E2 operon that comprises much of the E
region.
E2:
A multi-function protein, E2 is the ori-recognition and binding protein required
for recruiting E1 to the ori to nucleate the assembly of the pre-initiation replication complex.
E2 is also a context-dependent positive as well as negative tran-
scription factor and is now also thought to be responsible for segregation of ori-
5
containing plasmids. The E2 mRNA arises from alternative E1 intragenic splicing
of transcripts spanning the E region. E2 message is more abundant than E1 message but is still present at very low levels.
E4:
The cytoplasmic E4 protein binds to the network of cytokeratin intermediate filaments. The spliced E1^E4 mRNA is the most abundant viral mRNA and originates from primary transcripts that span the much or the entire E region. Its role
in viral DNA amplification and in other stages of the viral life cycle is controversial and remains to be resolved.
E5:
E5 is a small trans-membrane protein primarily found in the endoplasmic reticulum and Golgi membranes that enhances growth factor receptor signal transduction, but its role in the viral life cycle is not particularly understood.
E6:
The high-risk HPV E6 protein causes the degradation of p53 protein and a number of other host proteins. The HPV E6 proteins from both the high- and low-risk
genotypes can inactivate the p53 transcription activities. As well, there are many
other host protein binding partners. The exact roles of E6 in the viral life cycle are
yet to be elucidated.
E7:
The high-risk HPV E7 protein causes the degradation of the pRB family of proteins. In contrast, the low-risk HPV E7 only destabilizes p130, a pRB-related protein which prevents the differentiated cells from reentering the cell cycle, but it
does not cause turnover of pRB. Thus, this E7 activity promotes S-phase re-entry
by the differentiated spinous cells, creating a milieu conducive to viral DNA am-
6
plification. This restricts high-copy viral genome amplification to the upper strata
of the squamous epithelium.
In addition, E5, E6, and E7 proteins all contribute to the down-regulation of the host immune surveillance and defensive responses.
L1 and L2: Major and minor capsid proteins, respectively.
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Figure 2: HPV-11 genome organization and transcripts produced by the E1E2 operon.
The HPV genome is roughly 8 Kb in length and possesses 8 open reading frames. The
major promoters are found within the URR or within the coding region. The main transcripts produced by the E1E2 operon portion of the genome include E1, E1^E2, and
E1^E4, and these encode the E1, E2, and E1^E4 proteins, respectively. The eBFP2-11E1E2 operon construct I generated results in blue-tagged E1 and E1^E4 proteins but native E2 protein.
The PV URR (approximately 500-700 bp in length) contains promoters as well as
numerous elements that bind host transcription factors (Bernard 2002). It also contains
the viral origin of replication (ori), which consists of several binding sites for viral E1 and
E2 proteins (Chow and Broker 2006). Early region genes include: E1, E2, E4, E5, E6,
and E7. The encoded proteins are thought to regulate viral transcription and DNA replication, but the actual roles of E4, E5 and E6 protein are not completely understood. The
late region encodes the L1 and L2 capsid proteins. The L1 or L1 plus L2 proteins can
self-assemble into empty particles (virus–like particles), but DNA packaging into virions
8
requires both L1 and L2. In benign lesions, HPVs typically persist in host cells as extrachromosomal nuclear plasmids. In cancers, the viral DNA is often integrated into host
chromosomes in the E1 or E2 ORF, disrupting the expression of all downstream genes.
Transcriptional initiation from alternate promoters coupled with alternative RNA
splicing and utilization of one of two polyadenylation sites result in more peptides than
predicted from genes. While most major PV messages and peptides have been at least
partially characterized, little is known about the significance of minor transcripts and the
peptides that they potentially encode (reviewed by (Chow and Broker 2006)).
HPVs have evolved to take advantage of biological processes unique to differentiated squamous epithelial tissues. Submerged cultures of undifferentiated, immortalized
or transformed epithelial cell lines do not support viral reproduction. Investigators have
learned to isolate neonatal foreskin primary human keratinocytes (PHKs) and grow these
cells into differentiated organotypic raft cultures that are almost completely indistinguishable from native skin (Chow and Broker 1997). Prior to inducing PHK differentiation into raft cultures, one or more HPV genes can be introduced via retrovirus-mediated
gene transfer to investigate the viral protein functions. Alternatively, full-length HPV genomic DNA can be excised from recombinant plasmids and transfected into PHKs. Raft
cultures of cells immortalized by transfected high risk HPV DNA support the production
of a small number of virus particles. More recently, a new strategy that enables high-titer
production of HPV-18 virus in PHKs was devised. PHKs are transfected with HPV-18
whole-genomic plasmids that also contain an antibiotic selection marker flanked by loxP
sites. Following selection of the transfected monolayer PHKs for drug-resistance, Cre
recombinase is expressed. The selection marker excises and results in a plasmid contain-
9
ing the intact HPV-18 genome and one loxP site. These cells, when grown as raft cultures, recapitulate the life cycle of HPV-18 and generate high-titer, infectious virus particles (Chow et al. 2009; Wang et al. 2009). This efficient, reproducible system makes it
possible to perform mutagenic studies of individual viral genes in the context of the
whole HPV in the squamous epithelium, independent of the immortalization functions.
The results show for the first time that E6 protein is essential for viral DNA amplification
(Chow et al. 2009; Wang et al. 2009). Using this system, the role E5 plays during the
virus life cycle is now being elucidated in our lab. Understanding the intricacies of the
HPV infection program will require this new method for culturing, generating and passaging HPV virus particles, with and without modifications to the viral genome. This
will be crucial to attaining a better understanding of virus-host interactions. The regulation of virus gene expression and viral genome amplification by the URR is important
during the HPV infectious cycle and is crucial for appropriate differentiation-dependent
expression of the different HPV proteins throughout the stratified epithelium.
Normal morphology of the epidermis and the impact of HPV infection
In uninfected skin (Figure 1), the basal cells are usually relatively quiescent
whereas the parabasal cells divide daily. The upwardly-displaced daughters of the parabasal cells exit the cell cycle and begin to differentiate, generating several layers of
spinous cells. In the cutaneous skin, a layer or two of granular cells develop above the
spinous cells and are further differentiated into layers of cornified envelopes. This differentiation program takes place over a period of 2-3 weeks in vivo but is achievable in 9
days to 2 weeks in raft cultures after lifting the assembly to the media-air interface. In
10
the absence of HPV, the spinous and granular cells are post-mitotic, having exited the cell
cycle never to re-enter it.
In HPV-infected tissues, when basal cells enter S-phase, low-level replication of
viral DNA proceeds to keep pace with replication of the host genome. Thus, low level
production of the E1 and E2 messages and protein products is necessary during this phase
of the virus lifecycle. Mutational analyses suggest that the E6 and E7 proteins, but not
the E4 and E5 proteins, are necessary for stable maintenance of the viral genome in transfected keratinocytes. Upon differentiation, HPV E7 begins to be expressed in the lowerto mid-spinous layers. Following retrovirus-mediated gene transfer and ectopic expression, the E7 oncoprotein overrides the cellular homeostasis conferred by p130 and promotes unscheduled S-phase re-entry in post-mitotic, differentiated primary keratinocytes
in organotypic cultures. Thus, the E7 protein conditions the differentiated cells so that the
HPV genome can amplify to a high copy-number (Cheng et al. 1995; Banerjee et al.
2006; Genovese et al. 2008). The function of E6 is not known, as E6 is neither necessary
nor sufficient to promote S phase reentry by the differentiated keratinocytes in raft cultures. However, constitutive expression of E6 and E7 in the PHK raft cultures result in a
tissue morphology resembling high-grade dysplasia (Halbert et al. 1992). Thus, E6 and
E7 are likely to contribute to the increased thickness of the parabasal strata in productively-infected lesions.
An E6-mutant genome cannot immortalize PHKs and thus could not be studied in
organotypic raft cultures until recently. Our lab has now established the ability to recapitulate the HPV life cycle in a manner that can ultimately yield high-titer production
of infectious virions in organotypic cultures of primary human keratinocytes (Chow et al.
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2009; Wang et al. 2009). This capability has bolstered the toolbox for studying HPV
immensely. Our research group has begun to examine the roles of E6 and E5 in the context of an intact HPV genome in the organotypic cultures.
Multiple roles for the papillomavirus upstream regulatory region (URR)
Within each known type of papillomavirus (PV), approximately 500-700 bp of
contiguous genomic DNA is commonly referred to as the upstream regulatory region
(URR), long control region (LCR) or non-coding region (NCR). The URR immediately
precedes the E6 gene and has no open reading frame. The E6-proximal portion of the
URR contains one of the major transcription promoters overlapping the PV origin of replication (ori). The transcription profiles in PV-infected squamous epithelial cells change
dramatically as these cells undergo differentiation. High-level viral gene expression and
viral genome amplification occur mainly in the middle to upper strata of the squamous
epithelium, whereas virion packaging occurs only in the superficial cells. These observations are significant because almost every aspect of the viral life cycle is partially or totally shielded from immune surveillance.
Recognition sites for many transcription and replication factors have been identified in the URR. Understanding how individual elements within the URR contribute to
the PV life cycle remains a formidable task to this day (Chow and Broker 1997; Parker et
al. 1997; Ozbun and Meyers 1998; Zhao et al. 1999a; Zhao et al. 1999b; Sen et al. 2002;
Bromberg-White and Meyers 2003; McLaughlin-Drubin et al. 2004; McLaughlin-Drubin
and Meyers 2004; Sen et al. 2004). I will describe briefly (1) the elements that control
12
viral gene expression and (2) the elements that constitute the papillomavirus origin of
replication (ori), which also functions to ensure that viral genome copies are partitioned
equally between daughter cells in the dividing basal and parabasal cells.
Control of gene expression by elements within papillomavirus URR’s
Numerous regulatory elements in the URR are vital to the differentiationdependent life cycle of PVs.
Just upstream of the E6 gene is the E6 promoter, generally
referred to as P1 or more specifically as P94, P95, or P97 in HPV types -11, -18, or -16,
respectively, according to the map location of the first transcribed nucleotide in the RNA
transcript. The enhancer region includes binding sites for host transcription factors such
as YY-1, NF-1, AP1, Oct-1, KRF, AP2, GRE, and Sp1 (Figure 3) (Bernard 2002). Varied functionality of these protein binding sites has been observed and is attributable to the
sequence context and the HPV type. Placement of the reporter gene lacZ under the differentiation-dependent control of the E6 promoter limits β-galactosidase expression
mainly to the spinous cells of the differentiated strata. Mutation, deletion, or replacement
of known URR promoter or enhancer elements often has a negative impact on promoter
activity (Parker et al. 1997; Zhao et al. 1997; Zhao et al. 1999a; Zhao et al. 1999b; Sen et
al. 2002; Sen et al. 2004).
The URR sequence immediately upstream of the E6 promoter includes several E2
binding sites (E2BSs) (Figure 3). The E2 protein was originally thought to modulate the
expression of the E6 and E7 oncoproteins, as E2 binding to the E6 promoter-proximal
binding site can prevent assembly of the pre-initiation complex onto the E6 promoter
which is situated a few bp downstream (Hou et al. 2000) and can prevent the binding of
13
SP1, which is also critical for HPV E6 promoter activity (Dong et al. 1994). However,
promoter repression by E2 was only observed in cell lines harboring integrated but not
extrachromosomal HPV plasmid DNA (Bechtold et al. 2003). In vivo, there is some
doubt regarding the ability of E2 to regulate viral promoters in cells containing extrachromosomal plasmid DNA (Bechtold et al. 2003). Rather, the primary function of E2
is to initiate viral DNA replication.
HPV-11 URR Features
93
Figure 3: HPV-11 URR features. List of host protein binding sites (top list) and viral
protein binding sites (bottom list). (adapted from (Zhao et al. 1999a), (Hou et al. 2000).
The URR Contains the Papillomavirus Origin of DNA Replication (Ori)
Several E2BSs flanking a series of overlapping E1 binding sites (E1BSs) comprise the minimal PV origin of replication (ori). Mutations in the E2BSs are deleterious
to viral DNA replication (Chiang et al. 1992c; Kuo et al. 1994; Stubenrauch et al. 1998a;
Stubenrauch et al. 1998b). The HPV E1 DNA helicase and E2 proteins interact directly,
and the role of the E2 protein is to recruit the E1 protein to the ori. Both will be discussed in detail in later sections. The host cell supplies all additional replication en-
14
zymes, protein factors, and deoxynucleoside triphosphate substrates. Thus, PV DNA replication proceeds only when the differentiated cells re-enter the cell cycle. Recent work
in our lab has demonstrated that viral DNA amplification occurs in G2 phase following
the completion of the host DNA replication in S phase (Wang et al. 2009). Our lab believes that E2-E2BS interactions are also instrumental for faithful partitioning of the viral
genome during mitosis in the basal/parabasal cell layers, thereby facilitating persistent
HPV infections.
15
Viral Genes and Proteins
E1: Replicative DNA helicase
The PV E1 phosphoprotein (68kDa MW) is an ATPase with DNA helicase activity. It binds to the ori, albeit with low specificity and low affinity. It is recruited to the
ori via an interaction with the E2 protein which binds to E2BSs with high affinity and
specificity. Indeed, transient replication of PV ori-containing plasmids in transfected cells
depends upon the presence of E2 (Ustav and Stenlund 1991; Chiang et al. 1992a). The E1
protein is necessary for the initiation of viral genome replication (Sun et al. 1990; Blitz
and Laimins 1991; Ustav and Stenlund 1991; Yang et al. 1991; Chiang et al. 1992a; Seo
et al. 1993; Yang et al. 1993; Kuo et al. 1994; MacPherson et al. 1994; Liu et al. 1995;
Raj and Stanley 1995; Sanders and Stenlund 1998). E1 oligomerizes into hexameric or
dihexameric rings, and intermediates of assembly are thought to be dimers or trimers (Liu
et al. 1998; Sedman and Stenlund 1998; Conger et al. 1999; Fouts et al. 1999; Titolo et al.
2000).
The ori-binding activity of E1 is enhanced through interactions with chaperone proteins such as Hsp40 and Hsp70 and, in the presence of Hsp40, ori-bound E1 protein is a
dihexamer (Liu et al. 1998; Fouts et al. 1999; Titolo et al. 2000) which functions as a potent bidirectional helicase in the presence of RPA (replication protein A) and topoisomerase I (Lin et al. 2002). The ori-bound E1 protein recruits DNA polymerase-α/primase
and RPA to the viral ori to initiate replication (Park et al. 1994; Masterson et al. 1998;
Conger et al. 1999; Han et al. 1999; Amin et al. 2000). Although assembly of the preinitiation complex onto the viral ori is inherently linked to the presence of E2 protein, the
16
formation of the E1 hexamer and PV DNA replication cannot actually commence until
E2 becomes dissociated from the PV ori sequence (Lusky et al. 1993; Yang et al. 1993;
MacPherson et al. 1994; Lin et al. 2002; Abbate et al. 2004).
E1 is transcribed from a differentiation-dependent promoter (E1 or P3 promoter)
located immediately upstream of the E1 ORF overlapping the E7 gene. The great majority of this transcript is alternatively-spliced to encode E2 or E4 protein. Only rare
unspliced mRNAs encode the E1 protein. In the high-risk HPV, E1 and E2 proteins can
also be translated from transcripts initiated from the E6 (P1) promoter.
E2: transcription factor; viral DNA replication factor; viral persistence factor
The 43kDa E2 and amino-terminus-truncated E2-related proteins were first identified as factors that bound with specificity to conserved 12-nt elements within the URR to
regulate expression of the E6 promoter (Spalholz et al. 1985; Androphy et al. 1987; Hirochika et al. 1987; Chin et al. 1988; Giri and Yaniv 1988; Gius et al. 1988; Hirochika et al.
1988; Chin et al. 1989; Ham et al. 1991; Chiang et al. 1992a; Dong et al. 1994). It was
subsequently shown that E2 is essential for initiation of viral DNA replication (Ustav and
Stenlund 1991; Chiang et al. 1992a; Chiang et al. 1992c; Liu et al. 1995). Recently, work
from our lab showed that E2 tethers the HPV ori-DNA to the mitotic apparatus to facilitate partitioning of the viral genome into daughter cells and thereby to ensure viral persistence, and it has now been shown that E2 interacts with several proteins of the mitotic
apparatus(Van Tine et al. 2004; Parish et al. 2006).
E2 can be functionally sub-divided into three distinct regions: the amino-terminus
(N), the hinge (H), and the carboxyl-terminus (C). HPV E2N is a transcriptional trans-
17
acting domain and associates with mitotic spindles. E2H associates with nuclear matrix
proteins. E2C is responsible for DNA sequence-specific binding and protein dimerization
and interacts very strongly with the mitotic apparatus; specifically, the E2C spindlelocalization domain has been mapped to a short stretch of peptides near the H domain
(Dao et al. 2006). Alternate promoter utilization and mRNA splicing result in multiple
E2-related proteins, most prominently the E8^E2C protein (also simply called E2C) expressed from a promoter within the E1 ORF and containing a different N terminal domain
of about 12 amino acids encoded by the E8 ORF.
Spliced transcripts that originate from the E1 promoter and remove much of the
E1 ORF can encode the full-length E2 protein. Using alternative mRNA splicing or an
alternative promoter, other minor E2-related proteins such as E1M^E2C of unknown
function are expressed. The utilization of P1 promoter coupled with RNA splicing can
also produce a transcript with potential to encode E2, as reported for some of the HR
HPV types.
E1^E4: Cytoskeletal binding protein.
The highly-abundant E1^E4 peptide of about 85 residues is encoded by a spliced
mRNA derived from a primary transcript which serves as the mRNA for the E1 protein.
Splicing links the initiation codon and approximately four following triplets to a site near
the beginning of the E4 ORF (Nasseri et al. 1987). Various properties have been attributed to the E1^E4 peptide, although the role of this small peptide during the PV life cycle
is not well defined. The E4 protein binds to the network of cytokeratin intermediate
filaments and causes the collapse of this cytoskeletal framework in transfected cell lines,
18
a phenomenon not seen in patient specimens or in productive raft cultures (Doorbar et al.
1991; Pray and Laimins 1995; Wang et al. 2004; Doorbar 2005). Many PV E4 proteins
contain a domain capable of blocking cell cycle progression during G2-phase when overexpressed alone in cycling cell lines by sequestration of cyclin B/cdk1 (Davy et al. 2002).
Whether this property of E4 is relevant to viral DNA amplification in differentiated epithelium is still a matter of debate. Other reports describe E1^E4 interactions with the mitochondria (Raj et al. 2004). The E1^E4 protein undergoes proteolytic cleavage and release of the amino terminal domain, followed by a conformational change that enables
some of the interactions with host proteins.
The unspliced E1 mRNA and the alternatively-spliced E2 mRNA are each of lowabundance relative to the E1^E4 mRNA. Thus, alternate splicing to create the E1^E4
message can regulate the expression of the HPV E1 and E2 replication proteins and replication efficiency (Deng et al. 2003), and such a role in balancing production of the replication proteins may indeed be achieved by the E1^E4 mRNA splicing.
E5: Enhancement of growth factor receptor signal transduction (Crusius et al. 1999;
Leykauf et al. 2004; Longworth and Laimins 2004)
HPV E5 is a small peptide which primarily localizes to the endoplasmic reticulum
(ER) membranes (Conrad et al. 1993; Sparkowski et al. 1995; Disbrow et al. 2003;
Krawczyk et al. 2008). HPV E5 does not interact with or activate EGFR, and its activation of receptor tyrosine kinases (identity unknown) is ligand-dependent. The MAP
kinase signaling cascade can lead to the transformation of NIH 3T3 cells. E5 also enhances the abilities of the E6 and E7 proteins to immortalize cells (Crusius et al. 2000;
Suprynowicz et al. 2005). E5 is believed to play a role during the establishment of infec-
19
tions by inducing basal and suprabasal cells to cycle more readily, thus expanding the
infected cell population. Its role in viral DNA amplification in differentiated cells is being
investigated in our lab using the newly-established PHK raft culture system. .
E6: Inactivation or degradation of host proteins, including p53, many PDZ containing
proteins, RasGap protein, and TIP60, and up-regulation of hTERT expression.
E6 is one of two human papillomavirus oncoproteins. The 151-residue
protein folds to form two zinc finger domains. This oncoprotein is sufficient to immortalize mammary epithelial cells (Liu et al. 1995) but not dermal or cervical keratinocytes
(Münger and Howley 2002). E6 protein can inactivate the p53 tumor suppressor protein,
which normally functions to halt cell-cycle progression when DNA damage is present. In
cells where p53 detects DNA damage but the cell does not repair it, p53 triggers caspasemediated apoptosis (Kastan et al. 1995). The high-risk HPV E6 protein can inactivate
p53 in different ways. Firstly, E6 can target p53 for degradation via a complex between
E6 and E6-AP which functions as a p53-specific ubiquitin ligase (Scheffner et al. 1993).
Secondly, E6 efficiently inhibits the binding of p53 to its target sequences (Lechner and
Laimins 1994; Thomas et al. 1995; Thomas et al. 1999). Thirdly, E6 binds to chromatinbound complexes of p53-p300 to prevent acetylation of p53 target genes, and this further
silences transcription of genes that would otherwise be activated during a p53-mediated
DNA damage response (Thomas and Chiang 2005). To the contrary, E6 proteins from
low-risk PV types do not destabilize p53, but it retains the ability to inactivate p53 transcription activation function (Thomas and Chiang 2005). Fourth, the C-terminus in highrisk E6 oncoproteins contains a PDZ ligand that interacts with PDZ-domain containing
20
proteins and causes them to be polyubiquitinated (Nakagawa and Huibregtse 2000; Pim
et al. 2000; Grm and Banks 2004; Hengstermann et al. 2005; Matsumoto et al. 2006;
Handa et al. 2007; Shai et al. 2007). Moreover, the N-terminus of E6 destabilizes Tatinteracting protein 60 kDa (TIP60) (Jha et al. 2010). A catalytic sub-unit of the nucleosomal acetyltransferase of H4 (NuA4) complex, TIP60 associates with the HPV major early promoter, acetylates histone H4, and results in the recruitment of Brd4 and repression of E6 expression. By destabilizing TIP60, the HPV E6 promoter and other cellular promoters are de-repressed. During a DNA damage response, TIP60-induced histone modifications can promote p53-mediated apoptosis. By destabilizing TIP60 both
low- and high-risk E6 proteins enhance cellular proliferation and survival (Jha et al.
2010). E6 proteins of the high-risk HPV types can up-regulate the transcription of
hTERT, a component of the telomerase complex, thus providing a critical contribution to
immortalization of cells (Kiyono et al. 1998; Oh et al. 2001; Lee et al. 2002; Gewin et al.
2004; Horner et al. 2004; Jeong Seo et al. 2004; Plug-DeMaggio et al. 2004).
The E6 coding region in low-risk HPV types is unspliced, but all high-risk types
contain alternative intragenic and intergenic splices, one of the key distinguishing hallmarks of these HPVs. The splice leads to a peptide that contains the amino terminus of
E6, but terminates shortly after the splice acceptor to create one of several possible forms
of E6* peptide, depending on the HR HPV genotype. The E6* peptide can counteract
some of the functions of the full length E6 protein. The splice itself balances the production of E6 vs. E6*, and it also affects the level of translation from the downstream E7
ORF by creating an untranslated spacer between the end of E6* and E7 (Schneider-
21
Gadicke and Schwarz 1986; Bedell et al. 1989; Hummel et al. 1992; Woodworth et al.
1992).
E7: Inactivation of pRB family of proteins (facilitates unscheduled S-phase re-entry)
E7 is a major viral oncoprotein. E7 proteins expressed by high-risk PVs bind to
and inactivate the retinoblastoma protein (pRB) which controls the G1-S transition in
submerged, proliferating cells. Thus, over-expression of the HR HPV E6 and E7 genes
can immortalize PHKs in vitro (Woodworth et al. 1992; Steenbergen et al. 1996) and initiate viral carcinogenesis in vitro. Cervical carcinoma cell lines such as HeLa, Caski, and
SiHa rely upon the continuous expression of this viral oncoprotein as well as E6 for proliferation (Schneider-Gadicke and Schwarz 1986; Tsunokawa et al. 1986) as ectopic expression of E2 to suppress the E6 promoter in these cell lines represses E6/E7 transcription and cause the cells to senesce (Dowhanick et al. 1995; Francis et al. 2000; Nishimura
et al. 2000; Wells et al. 2000; DeFilippis et al. 2003; Wells et al. 2003). In contrast, E7
proteins from low-risk PVs have a much lower affinity for pRB. However, E7 of both
HR and LR HPVs can induce unscheduled S-phase reentry in terminally-differentiated
spinous cells (Gage et al. 1990; Cheng et al. 1995; Banerjee et al. 2006) because the target of E7 in the differentiated keratinocytes is the pRB-related p130 protein which normally maintains the homeostasis of the differentiated cells (Sdek et al. 2006; Genovese et
al. 2008).
22
L1 and L2: Major and minor capsid proteins, respectively
L1 and L2 are the major and minor papillomavirus capsid proteins, respectively.
Expression of these proteins can be observed in cells of the upper spinous layers where
encapsidation of the viral genome takes place. The appropriate differentiation-dependent
expression of L1 and L2 in the context of the full HPV genome can result in extremelystable, highly-infectious virions. Papillomavirus capsids are non-enveloped, icosahedral,
and seen as 43-56 nm in diameter, depending on the staining method used for electron
microscopy. Mature virus particles are released as corneocytes are sloughed off. During
the next round of infection, the low-pH, reducing atmosphere in the endosome causes the
virus coat to break down, thereby permitting release of the viral genome into the invaded
cell.
When expressed alone in eukaryotic cells, L1 self-assembles into virus-like particles (VLPs) suitable for use in vaccines. Immunization with non-infectious VLPs stimulates a high-titer antibody response that is protective against immunological challenge by
native, infectious virions (Kirnbauer et al. 1992; Howley 1996; Schiller and Davies 2004;
Chow and Broker 2006).
The Partitioning of papillomavirus DNA into daughter cells requires the URR and
E2 protein
HPV DNA normally replicates and persists extrachromosomally as plasmid DNA
without ever becoming integrated into host chromosomes during the virus life cycle. On
very rare occasions, HPV DNA will erroneously integrate into a host cell chromosome,
23
primarily found in HR-HPV caused cancers. It has been proposed that integration of viral DNA disrupts the autoregulation of the E6 promoter and leads to upregulated expression of E6 and E7 oncoproteins, as a result of capturing a stable host polyA sequence located downstream of the integration site. Many aspects of the mechanisms of replication
of the PV genome are able to be studied accurately in monolayer cells or in cell-free systems because PV DNA replication can occur when E1 and E2 are supplied in trans from
expression vectors (Chiang et al. 1991; Ustav and Stenlund 1991; Yang et al. 1991;
Chiang et al. 1992b; Kuo et al. 1994). Monolayer cells are also suitable while studying
segregation of HPV DNA into daughter cells during mitosis. Recent findings from our
lab identified novel interactions between the HPV-11 E2 protein and the mitotic spindles,
and these interactions have been proposed to ensure faithful partitioning of HPV ori DNA
into daughter cells during cell division (Van Tine et al. 2004; Dao et al. 2006). The ability of HPV E2 proteins to tether viral ori-containing DNA to the mitotic spindle apparatus
could have serious biological ramifications in a cell containing integrated HPV DNA.
The HPV ori could function as an additional centromere. The ability of E2 to bind to an
integrated viral ori while simultaneously interacting with the mitotic spindles could induce a DNA breakage-fusion-bridge cycle causing genomic instability, and this genomic
instability would not end until E2 expression is disrupted (Dao et al. 2006). This could
explain why E2 is no longer expressed in all transformed cells lines that harbor integrated
HPV DNA. The E2 proteins from multiple PV types have been found to interact with
specific host proteins, and these interactions are thought to tether PV DNA to mitotic
chromosomes to enable viral DNA partitioning (You et al. 2004; Baxter et al. 2005;
24
Brannon et al. 2005; McPhillips et al. 2005; Zheng et al. 2005; McPhillips et al. 2006;
Oliveira et al. 2006; Parish et al. 2006; Yu et al. 2007; Cardenas-Mora et al. 2008).
Cell division: the mitotic apparatus and chromosome segregation
Mitotic cell division in mammalian cells requires a vast array of intricate, interdependent events and checkpoints to ensure that chromosomes segregate equally to yield
sister cells with identical diploid genomes (Decordier et al. 2008). Chromosomal migrations are carefully controlled by bipolar spindles of microtubules nucleated by a pair of
centrosomes. Daughter strands of a replicated chromosome known as sister chromatids
pair up and become physically joined together early during mitosis at a chromosomal locus known as the centromere. During mitosis, a complex scaffold of proteins assembles
onto each centromere to form a structure known as a kinetochore. Kinetochores mark the
major contact site between mitotic spindle microtubules and chromosomes (Burke and
Stukenberg 2008). Regulation of the microtubule dynamics by centrosomes at the cell
poles and kinetochores at microtubule-kinetochore interfaces ensures that chromosomes
will traffic and segregate appropriately during cell division (Weaver and Cleveland 2006;
Morrison 2007; Musacchio and Salmon 2007; Burke and Stukenberg 2008; Decordier et
al. 2008).
Prior to sister chromatid separation and chromosome segregation, paired sister
chromatids move toward a metaphase plate and remain there until anaphase promoting
complex (APC) inhibition is relieved (Burke and Stukenberg 2008). A cohesin ring
keeps one sister kinetochore attached to the other until entry into anaphase (Decordier et
25
al. 2008). For the mitotic checkpoint signal at pro-metaphase to be relieved, each kinetochore must exist in a spindle-attached state, and sister kinetochores must be bi-oriented
with one kinetochore being drawn by the spindle toward one centrosome while the sister
is drawn to the opposite pole. Bi-orientation of sister chromatids causes the mitotic
checkpoint apparatus to “sense” tension between kinetochore pairs. A single pair of sister kinetochores that are improperly-attached or wrongly-oriented is sufficient to prevent
entry into anaphase. Proteins involved in maintenance and/or relief of the mitotic checkpoint prior to anaphase include Mad1, Mad2, CenpE, CenpF, BubR1, AuroraB, INCENP
and Survivin, just to name a few (Brown et al. 1996; Yao et al. 2000; Mao et al. 2003;
Mao et al. 2005). Once all sister chromatids are bi-oriented, APC inhibition is relieved,
the cohesin ring complex is cleaved, sister chromatids begin segregating toward opposite
spindle poles, cytokinesis occurs, and daughter cells almost always have inherited identical cellular DNA content (Burke and Stukenberg 2008; Decordier et al. 2008).
HPV DNA replication and early region message splicing
Replication of the HPV genome is one portion of the virus life cycle that can be
re-capitulated fairly well in monolayer cells. For HPV genome replication to initiate successfully and then be completed, the E1 and E2 proteins must associate with each other
and also their respective recognition sites in the papillomavirus (PV) URR. The normal
relative abundance of E1 and E2 is dictated by alternative splicing of their common primary transcript as the E1E2 operon is transcribed. The E1 and E2 messages are minor
RNA species though. The relative amount of a third product of the same primary tran-
26
script that is translated as the E1^E4 protein is much more abundant than the E1 and
E1^E2 messages (Figure 2). Expression of E1 and E2 from separate plasmids prevents
E1^E4 production and maximizes ori plasmid replication efficiency. Although transfection of an 11-E1E2 operon plasmid cuts expression of E1 and E2 drastically, the relative
ratios of these replication proteins remain wild-type, and ori plasmid replication is still
very strong. Use of an 11-E1E2 plasmid for replication studies supports robust replication of ori plasmids at levels similar to when separate 11-E1 and 11-E2 plasmids are
used. E1, E2, and E1^E4 each cause cytotoxicity in transfected cells. Attempts by others
in our lab to make inducible E1-expressing cells have failed. Even the slightest induction
of E1 expression caused all cells to die. Inducible E2 cell lines have been generated but
also exhibit substantial signs of cytotoxicity. The only way to be certain that the relative
abundance of E1 and E2 is held constant in replication studies is to utilize the E1E2 operon. Utilization of the E1E2 operon leads to the production of E1^E4 which is undesirable and also cytotoxic, but the ability to normalize the ratio E1 expression ratio to that of
E2 is very helpful.
27
Experimental aims and questions addressed in this project
The goals of the present work are:
1) Develop plasmids that can be tracked by direct, in situ immunofluorescence imaging.
2) Visualize the partitioning of HPV ori-containing plasmids in dividing cells.
3) Determine whether the E2BSs which facilitate efficient PV DNA replication must
also interact with E2 during mitosis to ensure that HPV genomes segregate into
daughter cells evenly.
28
EXPERIMENTAL WORK
Tracking DNA inside mammalian cells using Gal4-DBD, TetR, and rtTA3 GFP fusion proteins
Hybridization conditions for visualizing discrete nucleic acid targets by fluorescence in situ hybridization (FISH) often disturb or destroy other cellular features of interest. As the Kd of the lac repressor-lac operator complex is 10-13M, integrated tandem arrays of at least 256 lac operator sites have permitted non-destructive, direct, fluorescent
in situ visualization of target DNA in both fixed and living cells using chimeric fusions
between the repressor and a tracer such as green fluorescent protein (GFP) (Miller and
Reznikoff 1980; Robinett et al. 1996; Tsukamoto et al. 2000). These techniques can also
yield better resolution than can FISH. More recently, it was reported that single membrane-bound protein molecules could be visualized in bacteria by fluorescence (Yu et al.
2006). However, the signal-to-noise ratio for most experimental purposes requires binding site arrays that are quite large. For in situ DNA localization experiments in bacteria
involving detection of one or more discrete genetic loci, it has been the convention to use
~240 tandem copies of the operator (Lau et al. 2003; Wang et al. 2008a; Wang et al.
2008b). In yeast, in situ localization of DNA has relied upon as few as 112 copies of the
lac operator (Michaelis et al. 1997). In mammalian eukaryotic cells, where cell size and
volume are greater, refractive scattering of fluorescent signal has challenged investigators. In some instances, researchers relied upon clonal cell lines where multiples of the
256-copy lac operator array are stably integrated at a single genetic locus. With a plas-
29
mid containing 256 lac operators, cell lines have been isolated and tested with singlelocus insertions of either 10, 50, or 1000 copies of a lac operator-containing plasmid
(Tsukamoto et al. 2000; Janicki et al. 2004). The net effects were cell lines containing a
chromosomal insertion at a single locus of either 2.56*103, 1.28*104, or 2.56*105 lac operator sites. This is not feasible for studies where extrachromosomal, replicating HPV
ori-containing plasmids are to be followed. Since limiting any HPV tracking plasmid to
~8kb in size is preferable, ~40-50 copies of an appropriate operator site would be ideal.
Researchers have used either the Lac Repressor (LacI) or the TetR proteins fused
to either CFP, GFP, or YFP fluorescent proteins for visualizing DNA (Robinett et al.
1996; Michaelis et al. 1997; Tsukamoto et al. 2000; Ono et al. 2004; Ebersbach et al.
2005; Higuchi and Uhlmann 2005; Fiebig et al. 2006; Yu et al. 2006; Wang et al. 2008a;
Wang et al. 2008b). Our lab has previously used the yeast Gal4 DNA-binding domain
(Gal4-DBD)-GFP fusion protein to observe HPV ori-containing plasmid which also contains 40 copies of a Gal4 binding site (Gal4BS) array (Van Tine et al. 2004). Using the
high-sensitivity, high-resolution DeltaVision Spectris microscope system, I found that the
Gal4-DBD/Gal4BS system has a serious drawback and can produce artifacts. In the absence of a Gal4BS array, Gal4-DBD-GFP was able to form very fine foci around the kinetochores and associate with cellular chromatin during mitosis. In light of this observation, I abandoned the Gal4-DBD/Gal4BS system for my further studies of HPV DNA
replication and segregation. The tracking protein needs to bind reversibly to a very specific DNA sequence to allow ori-specific replication and only be induced to bind its recognition sequence until just prior to fixing cells for use in microscopy. These types of in
situ DNA detection experiments remain technically and experimentally challenging. The
30
lac repressor has constitutively-high affinities for its operator nucleotide sequence, and
this is inappropriate for our experimental purposes.
Researchers have turned to the family of tetracycline(tet)/doxycycline(dox) inducible proteins. Among those proteins that can inducibly bind to or release from the tet operator are TetR, tTA, and rtTA. The usefulness of the Tet repressor-GFP fusion protein
(TetR-GFP) and the tetracycline operator (tetO) array have been described and discussed
in depth. Under opposite conditions, VP16 fusion proteins known as tTA and rtTA proteins are each capable of binding to promoter-proximal tetO sites to activate transcription.
TetR and tTA exhibit high affinity for tetO sites only in the absence of doxycyclin (dox)
while rtTA only binds tetO sites efficiently after exposure to dox (Gossen and Bujard
1992; Michaelis et al. 1997). Compared to rtTA in mammalian cells, a variant known as
rtTA3 with the S12G, F86Y and A209T substitutions exhibited 5-fold better activity and
25-fold greater sensitivity, in response to the addition of dox (Das et al. 2004).
For any tagging protein to be useful for visualizing HPV DNA replication and/or
segregation, rtTA3 and TetR must not interfere with the replication competency of the
URR-TetO plasmid when the wild-type relative amounts of E1 and E2 are expressed via
co-transfected HPV-11-E1E2 operon-containing plasmid. Expression of neither TetR nor
rtTA appeared toxic to the cells. Neither dox-induced nor the uninduced TetR and rtTA
proteins interfered with the replication efficiency of the control plasmid containing only
the HPV 11-URR, as assessed by Southern blot hybridization. In contrast, when either
TetR or rtTA was allowed to bind to the tetO sites in the URR-containing plasmid for the
entirety of the experiment, replication was severely depressed.
31
To study the segregation of replicated HPV plasmid DNA, I tested TetR and
rtTA. Doxycycline was used as the inducer drug in lieu of tetracycline because it is water-soluble and more stable. In their high-affinity conformation, both TetR and rtTA3 are
capable of binding to the same TetO DNA recognition sequence. The tetO DNA sequence recognized by TetR and rtTA3 is as follows:
+9..+8.......+6......+4........+2........0........-2........-4.........-6.........-8...-9
5’- T C C C T A T
C A G T G A T A G A
3’- A G G G A T A G T C A C
(Gossen and Bujard 1992)
T A T C
T
G
A -3’
C
T -5’
32
Pre-requisites and experimental constraints to carefully consider during development and testing of HPV tracking plasmid systems
Expression of the HPV E1, E2, and E4 proteins impacts cell cycle progression and
can be cytotoxic
The abundance of E1 protein relative to that of E2 is normally dictated by alternative intragenic and intergenic RNA splicing from primary transcripts spanning multiple
cistrons. When ectopically-expressed, the use of the HPV-11 E1E2 operon allows both
HPV replication proteins to be expressed from a single plasmid. In addition, the E1^E4
mRNA and protein are the predominant products. Using the more-stable blue fluorescent
protein variant referred to as eBFP2, I generated the eBFP2-11-E1E2 expression plasmid
which encodes predomantly the eBFP2-E1^E4 protein along with eBFP2-E1 and the native E2 protein (Ai et al. 2007). I was able to identify the cells successfully transfected
with the plasmid expressing the abundant cytoplasmic eBFP2-E1^E4 protein.
To assess whether the eBFP2-tagged 11-E1E2 operon can support the replication
of an HPV ori-containing plasmid, I conducted transient replication assays in 293 cells.
If positive, the eBFP2-11-E1E2 operon would be useful for visually verifying that E1 and
E2 are both expressed in cells being imaged. If E1 and E2 expression from the same
plasmid is feasible, the total number of plasmids being transfected during experiments
can also be reduced. For all HPV ori plasmid replication assays being presented, low molecular weight plasmid was isolated 60 hours post-electroporation. Since each transfected
plasmid originated and was purified from DNA methylase-positive bacterial cell strains,
all plasmids were methylated at the time of transfection and therefore susceptible to di-
33
gestion by DpnI, whereas ori DNA newly-replicated in 293 cells will not be methylated
and will be resistant to Dpn1 digestion. To assess ori replication in 293 cells, half the
plasmid purified from each transfection was digested by a single restriction enzyme (oddnumbered lanes) while the remaining half was simultaneously-treated with DpnI endonuclease (even-numbered lanes). All non-replicated plasmid will be susceptible to degradation by DpnI. By running non-DpnI-treated (odd-numbered lanes) and DpnI-treated
samplesfrom the same experiment in adjacent lanes, bands representing non-replicated
and replicated plasmid DNA will be readily-visible upon Southern blot hybridization.
Whenever high-level replication of the plasmid is observed, the sum total intensity of ori
band(s) should be high and almost identical in both the DpnI and non-DpnI-treated lanes.
This assay is highly-sensitive and can show whether expressed E1 and E2 proteins are
functional and at adequate levels. This assay can also show whether ori plasmid replication is affected by the expression of fluorescent fusion proteins like TetR-eGFPnls or
rtTA3-eGFP-nls or the treatment with a drug such as doxycyclin (dox). With the TetO
array-containing HPV ori plasmid, we can also determine whether ori plasmids can replicate while TetR-eGFP-NLS is bound to the plasmid via a tetO array.
peBFP2-11-E1E2 plasmid supports efficient ori-specific plasmid replication
Fig. 4a shows that peBFP2-11 E1E2 supported replication of a minimal ori plasmid
(spanning HPV-11 nts 7730-99) as well as when the HPV-11 eGFP-E1 and native E2
proteins were expressed from separate vectors. Expression of the low levels eBFP1-E1
and the native E2 replication proteins at their natural relative ratio (lanes 3,4) was almost
34
as good as when the proteins were individually over-expressed to high levels at nonnative ratios (lanes 1,2).
To assess whether the peBFP2-11-E1E2 would be suitable for experimental studies of HPV DNA replication and segregation, it was co-transfected with a plasmid which
contained the HPV-11 URR (nts 7070-99) spanning the full ori. Fig. 4b showed peBFP211-E1E2 was able to support the replication of plasmid containing only the HPV URR(lanes 1-6).
35
Figure 4: The replication efficiency of HPV-11 ori plasmids in the presence or absence
of TetR and doxycycline. a) 293 cells were transiently-transfected with a minimal HPV11 ori plasmid. Additionally, plasmids encoding HPV-11 GFP-E1 or native E2 proteins
(lanes 1, 2) or, alternatively, the eBFP2-11-E1E2 plasmid were co-transfected (lanes 3,
4). Robust ori replication was observed under both conditions b) 293 cells were transiently transfected with HPV-11 ori containing plasmid with (lanes 7-12) or without
(lanes 1-6) an array of 48 copies of TetO sequence. Additional cells were transientlytransfected with a non-ori-containing plasmid with an array of 48 copies of TetO sequence (lanes 13-18). In each experiment, the eBFP2-11-E1E2 expression plasmid was
also co-transfected. As indicated (+ or -), in some experiments, the expression vector of
TetR-eGFP-nls was co-transfected. Cells were cultured in DMEM either in the presence
or absence of 0.5 μg/ml doxycycline, also indicated (+ or -). Low-molecular weight
36
plasmid DNA was purified 60 hrs post-transfection, digested with ApaLI alone (oddnumbered lanes) or along with DpnI (even-numbered lanes), and analyzed by Southern
blot hybridization. HindIII-cut, gel-purified 11-URR/eBFP2-11-E1E2/48-copy tetO array
plasmid served as the template to generate the radio-probe. A single asterisk points to the
linearized DNA band which corresponds to the ori plasmid (lanes 1-6), the 48-copy TetO
array plasmid (lanes 13-18) or the plasmid with both the ori and the TetO array (lanes 712). A double-asterisk marks the open-circle DNA band of these same plasmids due to
incomplete ApaLI digestion.
37
Tight association of a GFP fusion protein in a large array to an HPV ori plasmid
can impede ori replication.
Whether TetR-eGFP-nls can alter the ability of HPV-11 URR-only plasmids to
replicate efficiently was next assessed by Southern (Figure 4b) using low molecular
weight plasmid purified from 293 cells 60 hours post-electroporation. Compared with
baseline replication levels (Figure 4b, lanes 1,2), URR-containing plasmid replication
was unaffected by TetR-eGFP-nls expression either alone (Figure 4b: lanes 3,4) or together with dox treatment (Figure 4b, lanes 5,6). Replication of the plasmid containing
both the HPV URR and the TetO array sequence (Figure 4b, lanes 7,8) was almost identical to the replication of the simple URR-containing plasmid (Figure 4b, lanes 1-6). Replication of the plasmid containing both the ori and TetO array was inhibited substantially
when TetR-eGFP-nls was expressed (Figure 4b, lanes 9,10). Inhibition of replication by
TetR-eGFP-nls was reversed by culturing cells in media containing doxycycline (dox)
through the entire incubation period to reduce the affinity of TetR for the TetO array
(Figure 4b, lanes 11,12).
Observing replicated, 11-URR / 48-copy tetO array plasmid in situ by fluorescence microscopy would be impossible unless dox can be removed to allow the binding
of GFP-TetR to tetO sites. However, restoring the affinity of TetR-eGFP-nls for TetO
sites by rinsing dox out of cells proved unfeasible due to the tight binding of TetR to dox.
Due to this limitation and drawback of TetR-eGFP-nls, a different strategy for fluorescently-tagging our tracking plasmids would be essential.
38
To image HPV ori-containing tracking plasmid DNA, I felt that rtTA, which only
binds to TetO sites in the presence of dox, might be better-suited. An rtTA variant known
as rtTA3 has lower-level background affinity for TetO sites in the absence of dox. I generated an rtTA3-eGFP-nls expression plasmid and tested its impact on replication of HPV
ori plasmids in 293 cells (Figure 5). Compared with baseline replication levels (Figure 5
lanes 1,2), URR-only plasmid replication was unaffected by rtTA3-eGFP-nls expression
either alone (Figure 5, lanes 3,4) or together with doxycycline (dox) treatment (Figure 5,
lanes 5,6). Replication of HPV URR / 48-copy TetO array plasmid (Figure 5, lanes 7,8)
was very similar to replication of URR-only plasmid (Figure 5, lanes 1-6). Expressing
rtTA3-eGFP-nls did not impair replication of the 11-URR/48-copy TetO array plasmid
(Figure 5, lanes 9,10), unless dox had also been added to cell culture media throughout
the incubation period (Figure 5, lanes 11,12). No replication of the TetO array-only
plasmid was detected (Figure 5, lanes 13-18).
39
Figure 5: The replication efficiency of HPV-11 URR-containing plasmids in the presence or absence of rtTA3-eGFP-nls and doxycycline. 293 cells were transiently transfected with HPV-11 URR-containing plasmid with (lanes 7-12) or without (lanes 1-6) an
array of 48 copies of TetO sequence. Additional cells were transfected with 48-copy
TetO array-only plasmid lacking the ori (lanes 13-18). In each experiment, the eBFP211-E1E2 expression plasmid was also co-transfected. As indicated (+ or -), in some experiments, the expression vector of rtTA3-eGFP-nls was co-transfected. Cells were cultured in DMEM in the presence or absence of 0.5 μg/ml doxycycline(dox), also indicated
(+ or -). Low-molecular weight plasmid DNA was purified 60 hrs post-electroporation
and analyzed by Southern blot hybridization. All purified plasmid was digested with
ApaLI. HindIII-cut, gel-purified 11-URR/eBFP2-11-E1E2/48-copy tetO array plasmid
was used as template to generate radiolabelled probe. A single asterisk points to the linearized DNA band that corresponds to the URR-only plasmid (lanes 1-6), the 48-copy
TetO array-only plasmid (lanes 13-18) or the plasmid with both the URR and the TetO
array (lanes 7-12). A double-asterisk marks the open-circle DNA band of these same
plasmids due to incomplete digestion by the ApaLI.
40
Tracking plasmid detection sensitivity during mitosis requires optimized expression
of the eBFP2-11-E1E2 operon and rtTA3-eGFP-nls
To reduce the overall number of different plasmids that had to be electroporated
for each experiment, I attempted to consolidate plasmids by driving the expression of either eBFP2-11-E1E2 or rtTA3-eGFP-nls from the HPV-11-URR / 48-copy TetO array
plasmid. In theory, the 11-URR / eBFP2-11-E1E2 / 48-copy TetO array plasmid should
have been able to function as an autonomously-replicating tracking plasmid. Also in theory, the 11-URR / rtTA3-eGFP-nls / 48-copy TetO array plasmid should have been able
to function as a self-detecting tracking plasmid. Being able to use either of these strategies would have meant that a maximum of two plasmids would need to be electroporated
for each experiment. This would be, in theory, much superior to utilizing up to three
plasmids for each transfection. The two-plasmid approach proved, however, to be problematic.
If my replicon were to be useful, I needed to be able to have eBFP2-11-E1E2 operon expression reach ideal levels at the same time that the rtTA3-eGFP-nls expression
was optimal. Since the tracking plasmid is replicating exponentially, the gene copy number of the eBFP2-11-E1E2 operon is also rising exponentially. This causes a rapid spike
in E1, E2, and E1^E4 expression levels during the middle and later parts of the experiment. At the same time, the plasmid copy number of the rtTA3 expression plasmids
holds steady or is falling. This means that optimal expression levels of the HPV replication proteins and the GFP plasmid-tagging protein could not be attained simultaneously.
The expression levels of the E1E2 proteins varied from one cell to another depending on
the extent of plasmid replication, and cytotoxicity was observed frequently. When E1,
41
E2, and E1^E4 were being expressed, cells on average were expressing extremely-high
levels of the proteins. Replication was extensive, but almost no mitotic cells could be
found, probably because of the ability of the E1^E4 protein to sequester cyclin B/cdk1 in
the cytoplasm (Knight et al. 2004; Knight et al. 2006). Thus, the replicon was unusable
and expression of the E1E2 operon needed to be driven from a distinct plasmid at an optimized amount.
I did not attempt to use rtTA3-eGFP-nls in a self-detecting tracking plasmid because I anticipated difficulty titrating rtTA3-eGFP-nls expression levels, based on my
experience with TetR. Overall, this approach would lead to the production of too much
of the GFP fusion protein. Just as carefully-controlled levels of eBFP2-11-E1E2 are central to minimizing cytotoxicity, carefully-titrated expression levels of rtTA3-eGFP-nls are
vital for maximizing tracking plasmid detection sensitivity.
When rtTA3-eGFP-nls was expressed from one dedicated plasmid and the
eBFP2-11-E1E2 operon from another, I was able to achieve satisfactory titration of
rtTA3-eGFP-nls, E1, E2, and E1^E4 expression plasmids for transfection. All imagery
presented hereafter was acquired by using this approach.
I observed less cell-to-cell variation in the amount of rtTA3 being expressed.
During the titration experiments, cells expressing different amounts of the GFP fusion
protein were imaged, but the best sensitivity and resolution were obtained in cells where
a moderate amount of rtTA3-eGFP-nls protein was expressed. Regardless of how wellor poorly-titrated rtTA3-eGFP-nls expression was, big, bright green spots of replicated
DNA were easily-detected in the nuclei of non-dividing cells. It was in mitotic cells that
the benefit of carefully-titrating rtTA3-eGFP-nls expression became evident. During mi-
42
tosis, there are hundreds or thousands of dim, discrete spots. Over-expression of rtTA3eGFP-nls masks detection and often prevents resolution of the small, dim foci that each
might represent as few as one copy of replicated HPV tracking plasmid. The only way to
better-normalize cell-to-cell expression levels of rtTA3-eGFP-nls would be to generate
stable cell lines. I have generated stable, clonal 293-rtTA3-eGFP-nls cell lines that express the fusion protein to different levels but have yet to explore their utility. (I thought
you had to supplement the cell line with additional expression vector. I did with the
pooled TetR-eGFP cells that were non-clonal, but with the clonally-selected rtTA-eGFPnls cell lines I did not have to)
By optimizing peBFP2-11-E1E2 used in the transfection, the mitotic index was
greatly-improved relative to early pilot experiments where E1 and E2 had been expressed
from two separate plasmids, and I was able to image large numbers of mitotic cells with
ease.
As already stated, I attempted to use both TetR-GFPnls and rtTA-GFPnls for imaging purposes to follow the HPV tracking and control plasmids. Attempts to image
plasmids with TetR-GFPnls were entirely unsuccessful. Due to the tightness with which
TetR binds to dox, it was unfeasible to wash the drug out of treated cells. Even after
changing the DMEM multiple times at long intervals, only dim spots in interphase cell
nuclei were apparent, and no discernible foci were detectible in mitotic cells. TetR-GFP
is unsuitable for use in experiments where URR-TetO plasmid needed to be visualized
inside cells capable of supporting HPV DNA replication.
43
Clear visualization of HPV DNA localization is obtained when rtTA3-eGFP-nls
binds to arrays of tetO sites embedded in tracking plasmids.
The cellular distribution pattern of the 48-copy TetO array-containing plasmid
Make it clear that all experiments were conducted by transfection or cotransfection of one
or more plasmids. (as opposed to a cell line which already expresses rtTA3-eGFP-nls
The TetO array negative control plasmid (pTetO) contains 48 tetO sites. When
this plasmid was transfected into 293 cells together with the rtTA3-eGFP-nls expression
plasmid, treatment with doxycycline (dox) for four hours prior to fixing the cells allows
this control plasmid to be visualized as bright spots. Depending on spot intensities and
sizes, each spot from experimental (Figure 8) and control cells (Figure 6) can be grouped
into one of three categories. In dox-treated cells where TetO array-containing plasmid
either with (Figure 8) or without (Figure 6) the HPV-11 URR has been transfected, spots
are usually: 1) small & dim, 2) larger & brighter, or 3) humongous & extremely bright.
The pTetO control plasmid was not generally seen as small, dim spots. When I fixed
these control cells 36 hours post-electroporation, bright or dim small spots were not seen
in the vast majority of the nuclei. Instead, almost all pTetO plasmids were detected in the
cytoplasm as multiple clusters that were large and extremely-bright (Figure 6, interphase
#2 panels). In interphase cells fixed 60 hours post-electroporation, there was at most one
or two cytoplasmic cluster(s) of control plasmid (data not shown). In cells where the 11URR/TetO array plasmid (p11-URR-TetO) and the rtTA3-eGFP-nls expression plasmid
were co-transfected without the eBFP2-11-E1E2 operon expression plasmid, the fate of
44
the plasmid was identical to the TetO array-only control plasmid (data not shown).
The
same is also true when the TetO array-only control plasmid cells were also co-transfected
with rtTA3-eGFP-nls expression plasmid and the eBFP2-11-E1E2 operon plasmid (data
not shown).
We believe the vast majority of TetO array-containing plasmids in these control
experiments have coalesced to form clusters due in part to recombination or that they
were sequestered to certain domains in interphase cells where they are degraded or lost
during subsequent rounds of the cell cycle. A portion of the cytoplasmic plasmid aggregate clusters were seen co-localizing with microtubules (Figure 6, interphase cell #2, column A).
45
Figure 6: The distribution pattern of the negative control, 48-copy TetO array plasmid in
doxycycline (dox)-treated 293 cells. 293 cells were transiently transfected with rtTA3eGFP-nls expression plasmid and 48-copy TetO array plasmid.
36 hours post-
electroporation, cells were fixed after being treated with 2 μg/mL dox. Fixed cells were
stained and then imaged using z-stepping. β-tubulin (Columns B & D; pseudo-colored
46
red) and chromatin (Columns C &D; pseudo-colored blue) were stained using Cy3conjugated monoclonal antibody and DAPI, respectively. The TetO array plasmid is
visible as green foci. To maximize the signal-to-noise ratio, background, non-specific
signal was subtracted from the red, green, and blue image channels. rtTA3-eGFP-nls not
associating with a TetO site on the negative control plasmid resulted in a haze in each
image. The hazy rtTA3-eGFP-nls signal was subtracted from the green channel, while
ensuring that all green foci remained intact. This maximized the visibility and contrast of
green foci. The background-subtracted image file of each cell was then used to generate
the maximum-intensity projections seen here in the figure panels.
The TetO array plas-
mid produces bright green foci (Columns A-D). Image analysis software calculated
whether TetO array plasmid foci and β-tubulin did or did not co-localize. Partial or total
colocalization of green foci with β-tubulin is displayed as magenta or white (Column A).
Magenta represents the majority of plasmid-microtubule co-localization events while
white indicates where the brightest plasmids are co-localized with microtubules. Each
scale bar represents 5μm.
47
Plasmid does not associate with transiently-expressed rtTA3-eGFP-nls to form foci
until cells are treated with doxycycline.
Whenever rtTA3-eGFP-nls was expressed from transiently-transfected plasmid in
293 cells in the absence of TetO sites, no foci were observed in any cells, whether or not
they were mitotic. The GFP fusion protein was evenly-diffused inside the nuclei of interphase cells and throughout mitotic cells, regardless of whether the cells were treated
with doxycycline (dox) (data not shown). No foci were detected in rtTA3-eGFP-nlsexpressing, dox-treated 293 cells where the URR-only negative control and eBFP2-11E1E2 expression plasmids were co-transfected. Observations were virtually-identical
when the TetO array-only negative control and eBFP2-11-E1E2 plasmids had been cotransfected and the cells were fixed without having been exposed to dox (data not
shown). (you need to shown one such control image, no dox, no foci. The control cells I
show in Figure 7 are cells with 11-URR-TetO plasmid that have not been exposed to dox.
As this is actually a more-stringent control, is Figure 7 sufficient or should I also show
that this is the case with the TetO-only plasmid?) Figure 7 shows cells transientlytransfected with the p11-URR-TetO, peBFP2-11-E1E2, and rtTA3-eGFP that have not
been exposed to dox. For rtTA3-eGFP-nls to bind to any TetO array-containing plasmid
and yield detectable green foci, dox was absolutely essential. For the purpose of presentation, I did not subtract all rtTA3-GFP haze from the control cells in Figure 7 because
little or no GFP signal would remain to be shown. In Figures 6 and 8, rtTA3GFP haze
was subtracted as thoroughly as possible. Aside from that, all imaging conditions, image
processing and data analysis were standardized were held constant from control to ex-
48
perimental cells ensure that my interpretations of all results were completely unbiased.
Each and every positive mitotic cell I encountered was imaged. When I combine the controls I have shown (Figure 7) and/or discussed in this section with controls shown (Figure
6) and/or discussed in the previous section, I can comfortably place a high degree of confidence in the experimental results to be discussed next.
49
Figure 7: The inability to detect replicated 11-URR / 48-copy tetO array plasmid in 293
cells not treated with doxycycline (dox). 11-URR/48-copy TetO array plasmid was electroporated into 293 cells together with the rtTA3-eGFP-nls and eBFP2-11-E1E2 expression plasmids. Cells were fixed 36 hours post-electroporation. Cells were stained for β-
50
tubulin (Columns B & D; pseudo-colored red) and chromatin (Columns C &D; pseudocolored blue) using Cy3-conjugated monoclonal antibody and DAPI, respectively.
rtTA3-eGFP-nls is shown in green. Background signal in the red and blue channels was
subtracted with the same stringency as with cells in figures 4 and 6. No foci were detectable in these cells untreated with dox. Rather, the rtTA3-eGFP-nls signal was diffused
throughout the nuclei in interphase cells or the cell in mitotic cells in these negative control cells. This signal was not subtracted. Only extracellular rtTA3-eGFP-nls true background signal was subtracted. Image analysis software determined where green haze and
β-tubulin did or did not co-localize. Partial or total co-localization of the green rtTA3eGFP-nls haze with β-tubulin is displayed as magenta (Column A). Scale bars in each
image represent 5μm
51
The 11-URR/TetO array-containing tracking plasmid efficiently-targeted to microtubules during mitosis.
The experimental 11-URR/48-copy TetO array tracking plasmid was cotransfected together with the peBFP2-11-E1E2 and prtTA3-eGFP-nls expression plasmids, and the 293 cells were treated with doxycycline(dox) prior to fixation. Very different results (Figure 8) were observed when compared to controls (Figure 6). In Figure 8,
the vast majority of green foci were similar in intensity. Some tracking plasmid clusters
or aggregates were observed, but they were smaller and dimmer than the ultra-bright,
large aggregates seen in Figure 6. A breakdown of what I observed during different
phases of the cell cycle follows.
The p11-URR-TetO tracking plasmid showed a highly-dynamic localization pattern in mitotic cells. In interphase cells (Figure 8, row #1), p11-URR-TetO replicated
efficiently. I observed multiple bright foci that varied in size from small to extremelylarge. Each focus was equivalent to multiple copies of tracking plasmids. I suggest that
they are replication centers, as no or few such foci were observed in the control experiments described above.
At the nuclear periphery, almost every replication centers
showed co-localization with a single microtubule at a single, discrete point (Figure 8,
Column A, row #1). At prophase (Figure 8, row #2), most of the large replication centers
with dense amounts of p11-URR-TetO had dissociated to become very small and discrete
spots with relatively uniform brightness. The couple larger clusters of plasmids may
have represented replication centers that had yet to disperse. This same pattern was observed at pro-metaphase (Figure 8, Prometaphase cell, Columns A and B).
52
During metaphase, maximum co-localization of the 11-URR-TetO tracking plasmids with mitotic microtubules was observed (Figure 8, Metaphase cell, Columns A, B).
Additionally, the large clusters of un-dispersed, replicated DNA visible at pro- and prometaphase had dissipated and only discrete, tiny green foci were observed.
At anaphase, a substantial fraction of the tracking plasmids co-localized with the
mitotic spindle fibers (Figure 8, Anaphase cell, Columns A and B). Most notably, the 11URR-TetO plasmids co-localized with the central spindle fibers as well as microtubules
proximal to the centrosomes. Some of the plasmids co-localized with the central spindle
fibers were slightly larger than green foci observed at metaphase. During telophase and
into cytokinesis (Figure 8, telophase and telophase/cytokinesis cells, Columns A and B),
much of the tracking plasmids still co-localized with microtubules. A greater number of
medium-sized spots were observed than was the case during anaphase.
At the completion of cytokinesis, the majority of the ori plasmids had successfully
migrated back into the nuclei of each daughter cell. We observed equal partitioning of the
11-URR-TetO tracking plasmid in this and the other mitotic cells shown in Figure 8.
Plasmids remaining at the cleavage furrow and midbody were likely associated with E2
(Dao et al., 2006) and might have eventually migrated back into the nucleus. Alternatively, they might be lost upon mitosis.
When I began to study the localization pattern of p11URR-TetO (Figure 8, Column A, rows 1-8) and pTetO (Figure 6, Column A, rows 1-8) relative to microtubules in
both interphase and mitotic cells, a technical challenge emerged that required utilization
of more intensive image processing and analysis tools. Each image panel in figures 6-8 is
a maximum intensity projection. With maximum-intensity projections, a simple overlay
53
of the red (β-tubulin) and green (rtTA3-eGFP-nls) yields yellow when there is colocalization, but it is challenging to distinguish red and various shades of yellow accurately (Column B, Figures 6 and 8). Another issue with maximum-intensity projections
where only the tubulin (red) and rtTA3-eGFP-nls (green) are shown is even more serious
(Figures 6-8, Column B). Yellow does not necessarily mean that the red and green are
co-localized when looking at a particular X-Y coordinate in a maximum-intensity projection. As a hypothetical example of this, non-spindle-associated green foci of either
pTetO (Figure 6) or p11URR-TetO sometimes show up as yellow if there is a microtubule in any separate z-plane that is either above or below that particular green spot.
When the maximum-intensity projections of the red and green channels are generated, the
x-y coordinate marking the plasmid will appear yellow even though the plasmid is not colocalized with a microtubule. To solve this problem, the co-localization analysis tool in
SoftWoRx was utilized to identify co-localization and produce a visual rendering of
whether microtubules associate with pTetO and p11URR-TetO (Column A, Figures 6 and
8 respectively). The co-localization data was assigned by me to the blue channel. When
I overlaid blue co-localization image data with the green channel, I was left with colocalization spots that were different shades of turquoise. As with viewing different
shades of yellow, an image displaying greens and turquoises would have been less than
optimal.
To circumvent this problem, I duplicated the blue co-localization channel inside
the red image channel to yield magenta spots wherever green foci and β-tubulin were colocalized. Co-localization spots that are magenta are easy on the eyes and print extremely well. Overlaying the magenta atop the green image channel yields can yield an
54
image with mixtures of different-colored spots that are green (not co-localized with βtubulin), magenta (dim green foci co-localized with β-tubulin), and/or white (bright-green
foci co-localized with β-tubulin).
In control images where dox was not added, p11URR-TetO is invisible, and the
free rtTA3-eGFP-nls is evenly dispersed throughout the nuclei of interphase cells and the
entirety of mitotic cells (Figure 7). The magenta in these control cells does not depict
plasmid co-localization (Figure 7, Column A). Rather, the magenta renders each instance
when the free rtTA3-eGFP-nls is co-localized with the β-tubulin (Figure 7, Column A).
In figures 6 and 8, non-plasmid-associated rtTA3-eGFP-nls signal was subtracted as
thoroughly as possible. Residual green signal is overwhelmingly-specific and is comprised of the green plasmid foci, clusters, and aggregates plus whatever tiny bit of residual rtTA3-eGFP-nls remained after the stringent subtraction. By this approach, I was
able to study potential co-localization of pTetO and p11URR-TetO accurately, and subsequently, I was able to present the co-localization data clearly for all viewers to see
(Figures 6-8, Column A).
55
Figure 8: The distribution pattern of replicated 11-URR/48-copy TetO array tracking
plasmid in doxycycline (dox)-treated 293 cells. 11-URR/48-copy TetO array plasmid
56
was electroporated into 293 cells together with the rtTA3-eGFP-nls and eBFP2-11-E1E2
expression plasmids. 36 hours post-electroporation, cells were fixed following a 4-hour
treatment with 2 μg/mL dox. Fixed cells were stained and then imaged using z-stepping.
β-tubulin (Columns B & D; pseudo-colored red) and chromatin (Columns C &D; pseudocolored blue) were stained using Cy3-conjugated monoclonal antibody and DAPI, respectively. The 11-URR/TetO array plasmid is visible as green foci. Image processing
was as described in the legend to Figure 4. The 11-URR/TetO array plasmid produces
distinct, green foci (Columns A-D). Shown in column A, image analysis software calculated whether TetO array plasmid foci and β-tubulin did or did not co-localize. Magenta
(column A) represents the majority of plasmid-microtubule co-localization events while
white indicates where the brightest plasmids are co-localized with microtubules. Each
scale bar represents 5μm.
57
DISCUSSION
I have developed a system to visualize replicated HPV-11 ori plasmids and have
followed the plasmid distribution through the cell cycle. My conclusion was that the ori
tracking plasmid colocalized with mitotic spindles, enabling equal partitioning into
daughter cells. This conclusion is in agreement with previous reports from our lab (Van
Tine et al. 2004; Dao et al. 2006). In those reports, either Gal4-eGFP and native HPV-11
E2 protein bound to the HPV-11 ori plasmid or GFP-HPV-11 E2 alone was used as reporter, not replicating tracking plasmids. From these three studies, it is also apparent that
the binding of 11-E2 to p11URR-TetO (or any other URR-containing tracking plasmids I
characterized) is necessary to allow segregation of the tracking plasmid during mitosis.
We suggest that this association with mitotic spindles provides a mechanism by which
viral DNA can persist in dividing cells. It should also be mentioned that there is evidence
that the E2 proteins of other HPV types, primarily those of the beta HPVs, associate with
mitotic chromosomes, rather than the mitotic spindles (Skiadopoulos and McBride 1998;
McBride et al. 2004; You et al. 2004; Baxter et al. 2005; Brannon et al. 2005; McPhillips
et al. 2005; Zheng et al. 2005; McPhillips et al. 2006; Oliveira et al. 2006; Parish et al.
2006; Yu et al. 2007; Cardenas-Mora et al. 2008)
I note that the transient replication system used in my study is not comparable to
viral DNA replication which occurs in the basal cells in organotypic raft cultures. Rather,
it is equivalent to viral DNA amplification in post-mitotic differentiated cells. In the longliving basal cells where HPV DNA must partition properly to persist, there are somewhere between 50 and 200 copies of HPV genomic DNA per cells. The levels of viral E1
58
and E2 proteins expressed from the homologous promoter would be much reduced relative to the ectopically expressed viral protein in 293 cells. We theorize that the host protein to which E2 interact to effect the attachment of ori tracking plasmid to microtubules
can be overwhelmed by the high levels of the E2 protein. Although ChlR1 and MKlp2
have both been shown to associate with E2 proteins of multiple PV types previously, it is
unclear how many molecules of each of these proteins are present per mitotic cell. Their
possible saturation by the over-expressed E2 may account for some or all of the nonmicrotubule-associated HPV tracking plasmids as well as those found in the cleavage furrow and midbody (Parish et al. 2006; Yu et al. 2007). These plasmids might be lost upon
cytokinesis. Despite this caveat, our observations of the p11-URR-TetO tracking plasmid
in the transient replication system do provide relevant insights (Figure 8). Our new tracking plasmid system is however not perfect. Exposure times for capturing the green channel were 5 seconds, and hence, I was not able to perform live cell imaging.
My new tracking plasmid system is a significant improvement over previouslyreported systems that utilize TetR and Lac Repressor instead of rtTA3. In addition to being one of only a few people who were able to use this strategy to label DNA directly in
mammalian cells, I have reduced the number of TetO sites required substantially. With
this inducible, rtTA3-eGFP-nls-mediated DNA tagging system, the DNA that is to be observed can remain unbound and undisturbed by rtTA3-eGFP-nls until the addition of dox
four hours prior to fixing the cells. As a result, negative impact to DNA replication or
transcription can be minimized. While researchers can theoretically rinse dox off of TetR
to restore its affinity for TetO sites near the endpoint of the experiment, my experience is
that this strategy was impractical. The drug associates with TetR and rtTA3 too tightly to
59
be removed. With Lac Repressor, the protein always exhibits a high affinity for LacO
sequences. As such, Lac Repressor could not be used for my experimental purposes as
the bound Lac Repressor would have prevented the tracking plasmid from replication.
I have optimized the rtTA3-eGFP-nls system to tag TetO sites a great deal. To
my knowledge, my results show the first time that a TetO-containing DNA sequence has
been fluorescently-tagged by rtTA3 following the addition of dox inducer. This is a
novel, valuable approach whereby we can now study HPV persistence and DNA segregation. Additionally, I should be able to adapt this model system with relative ease in order
to address biological questions in other model systems and organisms. My thesis work
with the rtTA3-eGFP-nls and TetO constructs has advanced the ability that researchers
have to study discrete DNA elements by fluorescence microscopy a great deal.
Possibilities for future research
Additional HPV tracking experiments of interest
My new HPV ori tracking plasmid system is a powerful tool that will certainly
yield more-detailed findings in the future. For instance, co-staining with an antibody to
PML, a major component of ND10 bodies, can help localize the replication centers in interphase cells. Previously, our lab has reported that a fraction of the E2 protein colocalizes with ND10 bodies (Swindle et al. 1999). Using GFP-E1, the replication centers were
found to be near PML-positive sites (J-H Yu, L.D. Dao, T.R. Broker and L.T. Chow, personal communication). The tracking plasmids will allow us to identify the replication
centers relative to host nuclear domains and will yield new insights into how HPV plas-
60
mid DNA is dispersed at and around sites of active replication prior to, after, and during
HPV DNA replication.
Another line of investigation would be to introduce the tracking plasmid and the
prtTA3-GeGFP-NLS into primary human keratinocytes that already harbor the HPV-18
genomic plasmid (Wang et al. 2009). Because of the highly conserved nature of the viral
ori sequence, the HPV-18 E1 and E2 should be able to replicate the tracking plasmid
which contains the HPV-11 ori. Additionally, the ability to introduce genomic HPV-11
DNA by the same strategy used with HPV-18 is being developed. The advantage of this
experiment will be the low copy numbers of the replicating tracking plasmid supported
by the low levels of viral replication proteins expressed from the HPV-18 plasmid. This
way, the partitioning of the tracking plasmid will reflect the environment in the basal
cells more closely than is possible in 293 cells where the tracking plasmid amplifies to
extremely high copy numbers.
Either in 293 cells or in HPV-18 containing PHKs, staining with additional mitotic marker proteins was performed previously with the Gal4DBD-GFP tracking plasmid
system (data not shown). These host marker protein can be used for further characterization of the mitotic localization pattern of the new 11-URR-TetO tracking plasmid system.
Markers I can already stain for include Bub1, AuroraB, AuroraA, CenpE, CenpF, Crm1,
and multiple microtubule-associated motor proteins. Additionally, the sera from human
Crest schleroderma patients (an autoimmune disease) can be used to stain kinetochores.
Together, staining with these antibodies will hopefully produce a more-detailed understanding of the dynamic localization of HPV DNA during mitosis.
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Since we now know that E2 must bind to 11-URR plasmids to allow colocalization of tracking plasmids with the mitotic apparatus, it would be useful to perform
mutagenesis and truncation mapping of the URR to determine the precise region(s)
and/or elements that are sufficient to facilitate association of HPV plasmids with the mitotic apparatus. I have generated clones incorporating a wide range of URR truncations
and/or modifications while I was working with the Gal4DBD-GFP tracking plasmid system. These truncated and/or mutated URR fragments can easily be transferred into my
new TetO tracking plasmid system to replace the wild-type 11-URR. These experiments
would narrow down the elements within the URR critical for HPV DNA partitioning during mitosis.
The use of transiently-transfected plasmid to express rtTA3-eGFP-nls has proved
technically-challenging. A significant portion of cells either express too much or too little rtTA3-GFP, and these cells cannot yield useful imaging data. I have generated clonal
cell lines that stably-express rtTA3-eGFP-nls at low and moderate levels. It remains to
be seen whether these stable 293 cell lines will be more advantageous than transient expression of the rtTA3-eGFP protein.
If one of the clonal cell lines expresses rtTA3-
eGFP-nls at optimal levels, we might be able to image p11-URR-TetO in almost all cells
positive for both the tracking plasmid and the E1E2 operon, and this would represent an
enormous improvement over present and past strategies where two and three-plasmid cotransfections are required.
An alternative to ChIP to pull down discrete chromatin sequence
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The most commonly-practiced approach for studying protein-chromatin relies
upon pull-downs with antibody specific to a protein of interest. There are inherent problems with this strategy though. First, proteins rarely associate with the locus of interest
exclusively. Theoretically, antibody pull-downs will isolate chromatin sequences anywhere in the genome that the pull-down protein is bound. Especially while studying
some rare events such as developmental gene switching and differentiation, relying on a
native, cellular protein to pull down a specific chromatin fragment of interest can result in
authentic background signal. That authentic background can drown out rare events to the
point where drawing conclusions is impossible. In this instance, high-resolution chromatin mapping would be more appropriate, but currently, this is difficult and tedious. My
work with TetR, rtTA3, and my new TetO-based tracking plasmid system has given me
some ideas of how to isolate chromatin at a single locus efficiently and with a low
amount background. The presence of other cellular proteins of interest on the pull-down
chromatin can then be subsequently analyzed.
By knocking TetO arrays in at genetic loci of interest via recombineering
(Copeland et al. 2001; Court et al. 2002; Sawitzke et al. 2007; Bamps and Hope 2008),
cross-linked, purified chromatin from a specific genomic locus should be able to be studied efficiently. Genomic DNA would be sonicated to yield chromatin fragments of the
appropriate average size. Unlike in bacteria, TetR-GFP-nls can be expressed in mammalian cells without toxicity, and the fusion protein could be purified in its active form from
crude cellular lysate. Whether it is best to express TetR alone, as a GFP, or as a GFP-nls
fusion protein remains to be determined. GFP and the NLS might expendable if a good
enough antibody to TetR is available. It might be advantageous to eliminate the NLS al-
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together to reduce background affinity for the TetO sites. Alternatively, use of antibody
raised against GFP might allow TetR more steric flexibility and enhance the ability of
TetR to bind TetO sites. This non-enriched, TetR-eGFP-nls-containing lysate could then
be added to sonicated chromatin isolated from the cells to be studied. Biotin-conjugated
anti-GFP monoclonal antibody could be incubated with the TetR-GFP-nls-exposed
chromatin. This mixture could then be incubated with Streptavidin resin to pull down all
TetO array-containing chromatin fragments. In theory, the resin should only associate
with TetO array-containing chromatin fragments, mediated by biotinylated anti-GFP
monoclonal antibody. After appropriate washing to remove unbound chromatin and cellular proteins, the TetO array-containing chromatin fragments could be released from the
resin through the addition of doxycyclin (dox). While better than standard ChIP, an ever
more favorable strategy is described next.
I propose a simpler approach whereby a TetR fusion protein could be expressed,
bound to biotinylated microbeads, and used to affinity purify TetO array-containing
chromatin fragments directly without any need for antibody. A fungal, avidin-like protein, tamavidin 2 was recently used for affinity purification purposes, but unlike streptavidin, high levels of this avidin homologue can be expressed and purified in soluble form
from bacteria (Takakura et al.). Mammalian cell lines such as Cos7’s can express fusion
proteins in abundance whenever bacterial expression would be toxic as is the case with
TetR. It remains to be determined whether the fusion protein could be expressed and purified using insect cells. To re-state it again, it remains to be determined whether TetR
should be expressed with or without being fused to GFP. I propose that TetR-Tamavidin
fusion protein could be stably- or transiently-expresed in Cos7 cells. Crude Cos7 lysate
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with soluble TetR-eGFP-nls-avidin could be incubated with biotinylated microbeads, and
unbound cellular material could be washed from the resin. Next, unenriched, sonicated
chromatin fragments with and without the TetO array-tagged locus of interest would be
incubated with theTetR-eGFP-nls-tamavidin-bound biotinylated microbeads. The microbeads could in theory pull down all TetO array-containing chromatin fragments along
with any proteins that were crosslinked to the chromatin. After washing, the only chromatin and protein remaining on the resin should be the TetO-containing chromatin fragments. Addition of dox should allow all chromatin and protein to be released into solution gently, and the TetR-eGFP-nls-tamavidin should all remain in the insoluble, beadcontaining fraction. This strategy should allow for the efficient, relatively-simple purification of specific chromatin fragments and any interacting cellular protein complexes.
To my knowledge, this strategy for high-resolution chromatin purification or a similar
technique has not yet been reported in the literature.
The ability to purify and characterize affinity-enriched, chromatin fragments from
genetic loci where a TetO array had been incorporated via homologous recombination
would be a significant technological advance. Targeted knock-ins of modified DNA sequences without the need for antibiotic selection has become simpler and more efficient
thanks to techniques like recombineering. The same biochemical toolset used to analyze
chromatin isolated using standard ChIP protocols could be utilized, including coimmunopreciptations, quantitative PCR, blue-native gel electrophoresis, mass spectrometry, Westerns, and gel-shift assays. Since, this strategy requires no tagging and/or overexpressing of cellular and/or exogenous proteins, all cellular protein interactions and expression profiles can remain undisturbed and completely wild-type. While not replacing
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the need for standard ChIP protocols, these novel approaches for purifying specific
chromatin fragments would reduce background signal and enable researchers to study
dynamic chromatin-protein interactions during gene activation, gene repression, and gene
silencing. This could produce a better understanding of how expression of individual
genes are regulated and enable better dissection of complex processes like cell maturation, differentiation, re-programming.
Apart from the chromatin pull-downs, visualization of TetO-tagged loci might be
feasible if the cells being studied can tolerate transient transfection and expression of the
rtTA3-eGFP-nls plasmid. To have the ability to resolve this, it might be necessary to incorporate a TetO array on each side of the genetic locus to be studied. This sort of experiment could provide a visual readout of whether the chromatin of interest is hetero- or
euchromatic. Euchromatin would result in either a single, bright spot or two smaller ones
of differing intensities. Heterochromatin would appear as one or two spots smaller than
spots produced by euchromatin at the same locus. The total GFP fluorescence of each
spot(s) should remain constant, regardless of whether it is hetero- or euchromatin. While
ChIP provides an average picture of what is occurring in a population of cells, direct in
situ visulazation of DNA by fluorescence microscopy could permit observation of some
dynamic processes in single cells. The ability to study specific loci by ChIP and fluorescence microscopy studies could provide new insights including the dynamics and regulation of single genes.
66
Super-resolution fixed- and live-cell imaging on an OMX microscope
With more than two-fold greater effective resolution than the standard DeltaVision Spectris, re-imaging the specimen presented earlier in my dissertation on the OMX
super-resolution microscope platform (Applied Precision, Inc.) should yield additional
details and insight regarding how HPV genomes partition into daughter cells during mitosis. The OMX system employs structured illumination in the X/Y and Z axes to determinee the precise location of fluorophores in three dimensions (Gustafsson et al. 2008;
Baddeley et al. 2010; Duleh and Welch 2010; Nakamura et al. 2010). The system is also
capable of collecting images more rapidly and with greater sensitivity than is the case
with the DeltaVision Spectris. This might make live-cell, time-lapse imaging a possibility with the HPV tracking plasmid system I developed. Ultimately, this work could result
in substantial improvement to our present understanding of how HPV DNA partitions
into daughter cells, and other more basic biological questions such as those described in
the preceding section might also be better-studied by using this more-advanced, higherresolution microscope platform.
Materials and Methods
Plasmids
The HPV-11 expression plasmids peGFP-11-E1E2, peGFP-11E1dm, pMT2-11E2 and the origin of replication-containing plasmid p7730-99 have been described previously (Kuo et al. 1994; Liu et al. 1995; Deng et al. 2003).
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1. pTetR-eGFP-N1 (lacks SV40 Tag nls; TetR ORF semi-codon-optimized; GFP kozak
and AUG deleted): the high-fidelity KOD Hot Start kit was used to generate this clone.
Frag-A (770bp) and Frag-B (660bp) PCR products were generated where pCDNA6/TR
and peGFP-N1-ΔkozakΔAUG were used as templates, respectively. The 3’ of Frag-A
and 5’ of Frag-B include 21nt of overlapping, identical sequence that was used to prime a
second round of PCR. The Frag-A and Frag-B PCR products were gel-purified (Qiagen
Inc.) and used for a second round of PCR where the sense-strand from Frag-A annealed
to the anti-sense strand from Frag-B to yield a 1.43 kbp fragment. The PCR reaction was
paused, and the forward primer used to amplify Frag-A and reverse primer used to amplify Frag-B were added to the PCR reactions. The Frag-A forward and reverse primers
(IDT Inc.) were ”FragA-FOR-pCDNA6TR-Nhe” (5’-ACC-GTT-GCT-AGC-GTT-TAAACT-TAA-GCT-TGG-TAC-CCG-3’) and “FragA-REV-pCDNA6TR” (5’-TGC-TCACCA-TGG-TGG-CGG-CCT-CGA-GCC-CTA-TAG-TGA-GTC-GTA-TTA-CAA-TTCTTT-GCC-3’), respectively. The Frag-A PCR product included an Nhe restriction site
(GCT-AGC), the subsequent UTR-rabbit β-globin intron#2-UTR from the pCDNA6/TR
vector, an XhoI cut site (CTC-GAG), a Kozak sequence (GCC-GCC-ACC), and the first
ten base-pairs of the TetR open reading frame (ORF) (ATG-GTG-AGC-A). The third
codon was changed from TCT to AGC to improve protein expression in human cells.
The Frag-B forward and reverse primers were “For-XhoIkozakTetRGFP” (5’-CTCGAG-GCC-GCC-ACC-ATG-GTG-AGC-AGA-CTG-GAC-AAG-AGC-AAG-GTGATT-AAC-AGC-GCC-CTG-GAG-CTG-C-3’)
and
“FragB-Rev-TetRCTDHindBam”
(5’GAC-CGG-TGG-ATC-CAA-GCT-TTC-GGA-CCC-GCT-TTC-ACA-TTT-CAGCTG-TTT-TTC-CAG-TCC-GCA-TAT-GAT), respectively. The portions of the final
68
1.43 kbp PCR product that were contributed by the Frag-B PCR product were the XhoI
site (CTC-GAG), kozak sequence (GCC-GCC-ACC), TetR codon-optimization of the
third (TCT to AGC), sixth (GAT to GAC), seventh (AAA to AAG), eighth (AGT to
AGC), ninth (AAA to AAG), fourteenth (GCA to GCC), and fifteenth (TTA to CTG)
TetR codons in the 5’-ORF of TetR. The 3’-portion of the Frag-B PCR product resulted
in the codon-optimization of five additional triplets. With the sequence corresponding to
the L-E-K-Q-L stretch of amino acids, the Leucine (TTA to CTG), Glutamine (CAA to
CAG), and Leucine (CTT to CTG) codons were optimized. The two downstream Serine
codons in the TetR ORF were also optimized (AGT to AGC and TCT to TCC, respectively). The TetR ORF was followed immediately by G and A nucleotides, a HindIII site
(AAGCTT), a BamHI site (GGATCC), and lastly more of the spacer between the TetR
and GFP ORFs (ACC-GGT-C). In all, seven codons of the 5’ TetR ORF were optimized
(resulting in the first 17 codons being highly-optimized) and five codons in the 3’ portion
of the TetR ORF were optimized (resulting in the final 15 codons in the TetR ORF being
highly-optimized). Since I mutated the in the 5’-The gel-purified, full-length 1.43 kbp
PCR product was cut with NheI and BamHI. This fragment was ligated into the backbone of peGFP-N1-ΔkozakΔAUG between these same sites to yield a plasmid with a final overall size of 6.1kb.
2. pTetR-eGFP-nls (N1) version (the TetR ORF is semi-codon-optimized; between the
TetR and GFP ORFs, the kozak preceding GFP is wild-type; the GFP AUG is wild-type).
The NheI/AgeI restriction digest fragment from pTetR-eGFP was purified and ligated
into a modified version of the peGFP-N1 backbone where a linker (tcc-gga-ctc-aga-tct-
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cga-gct) and SV40 T-ag nuclear localization signal (cca-aaa-aag-aag-aga-aag-gta-gtc)
were fused to the carboxyl-terminus of GFP at an earlier time. The wild-type GFP kozak
sequence and start codon are intact.
3. prtTA3-eGFP-nls (start codon of GFP changed to ATC): the sequence between the
two XhoI sites in pTetR-eGFP-nls was excised and removed. The replacement fragment
with the rtTA3 ORF and a mutated GFP start codon (ATG to ATC) was commerciallysynthesized (IDT DNA Inc.) based on the rtTA3 ORF (Open Biosystems/Thermo Scientific Inc.). This ensured that rtTA3-eGFP-nls (but not free GFP) protein was solelyexpressed.
4.
pTetR-eGFP-nls-C1: The NheI-TetR-eGFP-nls-XbaI fragment was excised from
pTetR-eGFP-nls and ligated between the same sites in the peGFP-C1 backbone. This
yielded a plasmid with the peGFP-N1 multiple cloning site but the peGFP-C1 plasmid
backbone so that I could insert a TetO array into the MluI site at a later time.
5. pTetR-eGFP-nls-TetO: Using TetO clones and a strategy that were previouslydescribed (Lau et al. 2003), I generated and inserted a 48-copy TetO array into pTetReGFP-nls-C1 at the MluI site.
6. p11-URR-TetR-eGFP: The full-length, wild-type HPV-11-URR (nt7070 to 7933/1 to
99) was amplified by PCR from the previously-described p11Rc (Deng et al. 2003) using
recombinant Taq polymerase (Invitrogen) except that forward and reverse primers (IDT
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DNA Inc) incorporated AseI/AclI sites at the 5’ and BssHII/AseI sites were added onto
the 3’-end of the URR. The URR was inserted into the AseI site of pTetR-eGFP-nls-C1
in the forward orientation. The complete HPV-11 URR was PCR-amplified from p11Rc
to yield a 5’-AseI-AclI-11-URR-BssHII-AseI-3’ product that was ligated into the AseI
site of pTetR-eGFP-nls-C1.
7. p11-URR-TetR-eGFP-TetO (lacks an nls): An 5’-AseI-AclI-11-URR-BssHII-AseI-3’
fragment was used to insert the HPV-11-URR into pTetR-eGFP-C1. The 48-copy tetO
array was amplified by PCR and inserted into the MluI site of p11-URR-TetR-eGFP.
8. peGFP-N1-ΔkozakΔAUG – PCR mutagenesis was used to delete the kozak sequence
and start codon of GFP from the peGFP-N1 vector (Clontech).
9. p11-URR: The region between the BssHII and MluI sites was excised from p11URR-TetR-eGFP-TetO. The sticky ends were filled in and re-ligated to yield this plasmid.
10. pTetO: The URR, CMV promoter, TetR-GFP ORF and the other DNA between the
AseI and NotI sites of p11-URR-TetR-eGFP-TetO was excised and discarded. The
sticky ends that remained were filled in and re-ligated to yield this plasmid.
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11. p11-URR-TetO: The region between the BssHII and NotI sites in p11-URR-TetReGFP-TetO was excised. The sticky ends were filled in and re-ligated to yield this plasmid.
12.
peBFP2-11-E1E2: The AgeI-eBFP2-BglII fragment from the peBFP2-C1 (unpub-
lished) replaced the AgeI-eGFP-BglII fragment I removed from peGFP-11-E1E2.
13. p11-URR-eBFP2-11-E1E2: Using peBFP2-11-E1E2 as a backbone, the 11-URR was
inserted at the AseI site as done with other plasmids.
14. peBFP2-11E1E2-TetO: Using peBFP2-11-E1E2 as the backbone, the 48-copy TetO
array was inserted at the MluI site.
15.
p11-URR-eBFP2-11E1E2-TetO: Using p11-URR-eBFP-11-E1E2 as a backbone,
the 48-copy TetO array was amplified by PCR and inserted at the MluI site.
16. pBabePuro-TetR-eGFP-nls: The NheI/XbaI fragment from pTetR-eGFP-nls-N1 was
excised and the ends were filled in.
This was ligated with the filled-in ends of
BamHI/EcoRI-digested pBabePuro.
17. pBabePuro-rtTA3-eGFP-nls: The NheI/XbaI fragment from prtTA3-eGFP-nls was
purified and the ends were filled in. This fragment was ligated with the filled-in ends of
BamHI/EcoRI-digested pBabePuro.
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18. TetO array plasmids: pLau02(3 TetO sites), pLau04(6 TetO sites), pLau06(12 TetO
sites), and three plasmids with 24 TetO sites each (pLau10-1, pLau10-2, and pLau10-3)
were all generously shared with us by David Sherratt through Pat Higgins. Longer TetO
arrays were generated using these plasmids and a previously-published strategy (Lau et
al. 2003).
Cell culture for imaging
293 cells were maintained in phenol red-free Dulbeccos’s modified eagle medium
(Mediatech cat#17-205-CV) with 10% fetal bovine serum and 2 mM L-glutamine (Mediatech cat#25-005). Transfections were conducted by electroporation as described previously (Chiang et al. 1992a). For imaging purposes, electroporated cells were seeded onto
poly-L-lysine coated coverslips. Round, 40mm, #1 coverslips (Bioptechs Inc. cat#401313-0319-2)
were
acid-washed,
rinsed,
and
stored
in
70%
EtOH
(http://spectorlab.cshl.edu/acid_clean.html). Flamed coverslips were laid atop parallel
lengths of 1 mL plastic pipettes that had been glued to the bottoms of 60mm tissue culture dishes. Poly-L-lysine stock solution (Sigma-Aldrich cat# P8920) was diluted 1:10
with distilled water to yield a 0.01% w/v solution, and approximately 1.3mL of diluted
poly-L-lysine was pipetted onto each coverslip and allowed to stand there for 30 minutes.
The solution was aspirated off and coverlips were dried in a warm SpeedVac. Dried,
coated coverslips were double-UV-sterilized (BioRad GS Gene Linker) and stored until
use. Each coverslip was seeded with one-third the cells from an electroporation. As
such, each transfection could generate three identical coverslips.
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Non-clonal pools of TetR-eGFP-nls-293 cells were generated by infecting parental 293 cells with pBabePuro-TetR-eGFP-nls retrovirus produced in AM-12 cells and selected with 1 μg/mL puromycin (Sigma-Aldrich). For clonal rtTA3-eGFP-nls-293 cell
lines, pools of non-clonal cells were first generated using pBabePuro-rtTA3-eGFP-nls
retrovirus. Clusters of fluorescing rtTA3-eGFP-nls-293 cells were marked individually
and passaged using 6- and 8-mm cloning cylinders (Corning Inc cat#3166). Cell sorting
was used to sort the different populations of cells and generate a variety of different
clonal cell lines.
Cell fixation, cell staining, and indirect immunofluorescence
293 cells seeded on coverslips were placed in 60mm, treated tissue culture dishes
(Corning Inc.) and cultured for (36 - 60 hrs) at 370C in 5% CO2. Coverslips remained in
the same 60mm that they were cultured in until it was time to apply the antibody. The
cells were exposed to 2 μg/mL doxycycline for the four hours prior to harvest for imaging
as follows. Since the salts in DMEM caused the dox to precipitate out of solution when
dox stock solution was added directly to the culture dish and swirling the dishes to redissolve precipitated dox caused many cells to detach, I opted to prepare DMEM with
2μg/mL dox several hours in advance of it being used. To do this, I used 125-mL glass
bottles to prepare either 50- or 100-mL batches of induction media. The dox-containing
media was stored in 5% CO2 at 37C with the cap loose until the time when I added it to
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the coverslip-containing dishes
Media was vacuum-aspirated using long-tip Pasteur pi-
pettes, and 4-mL of dox-containing media was added to the treated cells. Mock-treated
cells received 4mL of fresh DMEM that lacked dox. All solutions used during fixation,
permeabilization, and the rinses were applied using 60-mL syringes (BD Inc) with 18-Ga
needles. Solutions were gently-applied to the edges of the 60mm tissue culture dishes to
keep from disturbing or detaching cells excessively. Solutions were removed gently by
pouring them off into a waste beaker. To preserve microtubules and spindle fibers, great
care was taken to ensure that cells never cooled prior to fixation. For fixation, the
DMEM was poured off and cells were fixed directly using freshly-prepared 4% paraformaldehyde in 1x-PBS (pH 7.2) that was maintained at 37C. I observed that fewer cells
detached when I added a small amount of PFA to the media prior to pouring it off and
putting the cells into pure, 4% PFA solution. After a few seconds and gentle mixing, the
dilute DMEM-PFA mixture was poured off and 3-mL of warm, 4% PFA-PBS was added.
Coverslip dishes were set atop a small stack of paper towels to allow the fixative to remain warm for longer. After eight minutes, the fixative was poured off and residual PFA
was aspirated using a syringe and needle. For five minutes each, three rinses at roomtemperature with 1x-PBS pH7.2 followed. Cells were permeabilized for 10 minutes using a 4oC solution of 1x-PBS/0.5%-Triton-X. As with fixation, I found that fewer cells
were lost due to detachment if I first added ~1-mL of the permeabilization buffer to the
PBS, prior to pouring the PBS off and adding pure permeabilization solution. After a few
seconds of gentle swirling, the dilute PBS-triton-X solution was poured off and replaced
with the pure 1x-PBS-0.5% Triton-X permeabilization buffer for ten minutes. Three
washes with 1x-PBS, five minutes each, at room-temperature followed. For the third
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PBS rinse, I used the same basic strategy as I did during fixation and permeabilization. I
poured all but about 0.5mL of the second PBS rinse buffer off, taking care to minimize
exposure of the cells to air and then added ~4mL of fresh 1x-PBS. A few seconds later, I
repeated this procedure and then let the cells sit for five minutes. To block and reduce
background staining, 400μL of a 50% goat serum/1x-PBS pH 7.2 blocking solution was
added to each dish (final [goat serum]=5% v/v). Immunostaining was performed using
60mm wide slides (Corning) where I had applied histology pen wax to all but a square,
~45mm portion of the slide surface. Coverslips were inverted and set atop a 400μL droplet of the staining antibody which I pre-diluted using 50% goat serum/PBS. Coverslips
were removed from their 60mm dish filled with the blocking solution by squeezing the
sides of the dish with my left hand. This allowed me to get underneath the coverslip with
forceps using my right hand. I only gripped the outermost ~3-4mm of coverslips to
minimize destruction of cells. Coverslips were lifted out of the dish gently. I held the
coverslips with my left hand by the very edge and blotted excess blocking solution from
the side of the coverslip that did not have cells growing on it. I grabbed the same 3-4mm
surface of the coverslip with the forceps and gently laid it atop the 400μL bead of diluted
antibody on the glass slide that was resting inside a standard slide storage box with wet
paper towels lining the bottom. Cells were incubated with antibody overnight. The following morning, staining boxes were removed from the cold box. Again, I used 60-mL
syringes to apply all wash solutions. Fresh 60-mm plates with 5mL of 1xPBS/0.1%
tween-20 (Sigma-Aldrich) were set up in advance prior to the first wash. To remove the
coverslips from the slides where they were being stained, I added ~1-1.5mL uniformly
around the edge of each coverslip. This caused the coverslips to float up enough so that I
76
could easily-insert a pair of forceps and grasp ~3-4mmof the surface of the coverslip.
The coverslips were gently lifted off of each slide, inverted, and promptly-added to one
of the 60mm dishes that already contained the PBS/0.1% tween-20 solution for the first
wash. Three 5-minute PBS/tween washes were then followed by three 5-minute 1x-PBS
rinses. Care was taken to ensure that the third PBS rinse was done incrementally, as was
done following permeabilization. DAPI was added to the third PBS rinse solution for 10
minutes to stain for chromatin. Two PBS rinses followed (each five minutes), and cells
were set atop kimwipes (cell-side up) to dry before being mounted with ~40μL of antifade prepared per David Spector’s (Cold Spring Harbor Laboratories) protocol. The recipe is available at: http://spectorlab.cshl.edu/acid_clean.html. In my hands this anti-fade
is better and easier to use than any commercially-available anti-fades I have tested. It
should also be noted that pure p-phenylenediamine (Sigma-Aldrich) is white. Crystals
usually arrive as a tan or brown color.
I rinsed large batches of the crystals multiple
times in non-pH-adjusted 10x-PBS buffer until all crystals were white and the supernatant was clear. It is my observation that 10x stocks of the pure crystals can be stored
stably for several years at -80C. When 1x stock of the crystals is exhausted I simply thaw
a 10x stock tube and dilute it 10-fold to refresh our supply of 1x-p-phenylenediamine solutions. 1x and 10x stocks of the crystals are both stored in non-pH-adjusted 10xPBS at 80C. Using David Spector’s anti-fade recipe, 1.5mL tubes of colorless anti-fade could be
stored under nitrogen at -20C inside a tightly-capped bottle for several months without
becoming noticeably-discolored. Discolored anti-fade (more than a pale yellow or orange color) will result in autofluorescent staining of chromatin.
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For all antibody staining I used the blocking, staining, and wash conditions described in the previous paragraph, and antibody dilutions were as follows. For staining
microtubules, Cy3-conjugated, anti-β-tubulin mouse monoclonal antibody (Sigma) was
used at a 1:50 dilution. Chromatin was stained using DAPI. Slides with coverslips of
cells were stored at -20oC until it was time to image. Though images were not presented
in the body of my dissertation, I have successfully stained 293 cells using anti-CREST
serum (Cortex Biochem) at 1:800, AIM-1 antibody (BD Transduction Labs.) at 1:100,
aurora-A Kinase antibody (BD Transduction Labs) at 1:200, CENP-F (anti-Mitosin) antibody (BD Transduction Labs) at 1:100, CENP-E (abCam) at 1:250, and Crm1 antibody
(Santacruz) at 1:100 dilutions with very low background noise.
Transient replication assays
For each electroporation, 5x106 293 cells re-suspended in 250μL DMEM that
contained BES and 50μg of boiled, snap-cooled salmon-sperm DNA (Invitrogen). Wherever required the following amounts of plasmid were transfected: 500ng of p7730-99
HPV-11 ori, 1μg peGFP-11-E1dm, 5μg pMT2-11-E1, 5μg pMT2-11-E2, 5μg p11-URR,
5μg pTetO, 5μg p11-URR-TetO, 5μg peBFP2-11-E1E2, 6μg pTetR-eGFP-nls, and 6μg
prtTA3-eGFP-nls plasmid were utilized. Cells were grown either in the presence or absence of 2 μg/mL doxycycline (Sigma-Aldrich cat#D9891) for the entire duration of the
experiment. The harvest of the low molecular weight DNA and Southern blot hybrization were performed as previously described (Chiang et al. 1992a) DpnI was acquired
from Promega. Apa I and HindIII were acquired from New England Biolabs Inc.
78
Derivation of 293 cell lines expressing TetR-eGPF or rtTA3-eGFP
pBabePuro-TetR-eGFP-nls and pBabePuro-intron-rtTA3-eGFP-nls were used to
generate retroviruses. The plasmids were electroporated into Bosc23 cells. The following day, sterile-filtered supernatant containing polybrene (Sigma) at a final concentration
of 5.2 μg/mL was dripped over Am12 cells (at 50-60% confluency). Media was changed
after 6 hours. 24 hours later, infected Am12 cells began 48-hour selection period utilizing 1 μg/mL puromycin. Upon removal of selection, Am12 cells were grown to nearconfluency and passaged. rtTA3-eGFP-nls and TetR-eGFP-nls virus-containing supernatant from the respective Am12 cells was sterile-filtered, mixed with polybrene, and
dripped onto 10 cm plates of 293 cells that had been seeded with either 1- or 1.2E6 293
HEK cells the prior day. 36-48 hours later, one-week selection with 1 or 1.25 μg/mL
puromycin was carried out. Media and antibiotic were changed every two days. Pools of
non-clonal 293 cells infected with the TetR-eGFP-nls retrovirus did not continue expressing the GFP fusion protein. As a result of this observation I utilized cloning cylinders
and FACS sorting to produce clonal 293 cell lines that stably-express rtTA3-eGFP-nls at
low to moderate levels.
79
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