Download Transcription in Eukaryotes I and II

Transcription in Eukaryotes I and II
Jörg Bungert, PhD
352-273-8098
[email protected]
Objectives
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Know the basal promoter elements and the basal transcription factors.
Know the RNA polymerase II CTD phosphorylation cycle.
Understand reporter-gene assays.
Know and understand enhancer function.
Understand Chromosome Conformation Capture (3C).
Know the structure and function of the mediator.
Know histone modifications and how they impact gene expression.
Know the different chromatin modifying activities (HAT, HDAC, HMT,
chromatin remodeling complexes).
Know what a DNAseI hypersensitive (HS) site is.
Know RNA polymerase I and III transcription complex formation.
Reading: Lodish 6th edition, chapter 7 (pp. 276-317).
Transcription in Eukaryotes - Overview
Eukaryotic RNA Polymerases
1.
Pol I - Transcribes rRNA genes (28S, 18S, 5.8S RNA genes); accounts for 80-90% of total
cellular RNA mass
2.
Pol II – Transcribes protein-encoding mRNA’s, and several non-protein encoding genes;
consists of 12 subunits that are conserved among diverse eukaryotic organisms
3.
Pol III - Transcribes tRNA, 5S RNA, 7S RNA (component of signal recognition particle genes,
and other small RNA-encoding genes (including genes encoding RNA’s involved in splicing;
tRNA and 5S genes have internal promoter (i.e., within body of gene)
4.
Each RNA Pol can be distinguished by differences in sensitivity to α-amanitin
5.
Each RNA Pol has multiple subunits, is similar from yeast to mammals, and is more complex
than the E. coli RNA polymerase
6.
Some subunits of each RNA Pol share some sequence similarity to E. coli RNA pol, and all
three eukaryotic RNA Polymerases share come common subunits
7.
The C-terminal end of the largest subunit of pol II subunit contains a repeated 7 a.a. motif
1. This repeated region is termed the C-terminal domain (CTD)
2. Active transcription is correlated with phosphorylation of CTD
3. CTD is associated with several RNA processing factors
8.
All three RNA Pols use TBP (TATA binding protein) in assembly of pre-initiation complex
9.
New report (2005) of previously unknown mitochondrial RNA pol (spRNAP-IV) transcribing
some nuclear mRNA-encoding genes with promoters different from those using pol II
Subunit Structure of RNA Polymerases
The TATA-Box
Basal Promoter Elements in Higher Eukaryotes
Housekeeping, constitutively
expressed, genes are transcribed from
multiple start sites. Tissue-specific
and regulated genes are transcribed
from a single start site. The
mechanism of dispersed transcription
initiation is not well characterized.
Focused transcription initiation takes
place at genes harboring specific basal
promoter elements (e.g. TATA, INR,
DPE). TATA and DPE are present in
about 15 to 20% of genes transcribed
by Pol II, the INR is found in about
50% of the genes.
Juven-Gershon and Tjian, Developmental Biology, 2009
RNA Polymerase II Transcription
The pre-initiation complex (PIC):
PIC assembles at the basal promoter region of a gene
PIC consists of RNA pol II, 5 general transcription factors (GTF’s), and promoter DNA
GTF’s = TFII D, TFIIB, TFIIF, TFIIE, TFIIH (and TFIIA?)
(e.g., HeLa cells)
Identified by in vitro
transcription assay (Run
Off) using as a DNA
template the adenovirus
major late promoter
(Ad2MLP).
E-Box
TATA-INR-DPE
Steps in Transcription Complex Formation
IIA
TBP binds to the
minor groove
and bends the DNA.
Bending may facilitate
melting of the DNA
or assembly of the
transcription complex.
The binding of TBP is
facilitated by TFIIA
Steps in Transcription Complex Formation
TFIIB bridges the TBP-TATA
box complex with the RNA
polymerase.
Steps in Transcription Complex Formation
TFIIF interacts with the RNA
Polymerase and is required
for preventing unspecific
initiation at non-promoter
sites as well as for efficient
transcription elongation.
Steps in Transcription Complex Formation
TFIIE stimulates the
helicase and ATPase
activities of TFIIH
Steps in Transcription Complex Formation
TFIIH is a multi-protein
complex that contains
kinase, helicase, and
ATPase activity. It is
required for melting
the DNA during initiation
and for the phosphorylation
of serine 5 of the CTD.
Steps in Transcription Complex Formation
“closed” complex
“open” complex
TBP/TATA box complex and TFIID
TFII D composed of::
•TBP – TATA-Binding Protein
•TAF’s – TBP-associated factors
Also, evidence for:
- Cell type-specific TBP’s
- TAF complexes w/out TBP that can functionally
replace TFIID
Structure of the Preinitiation Complex
TBP/TFIIA/TATA-Box
TBP/TFIIA/TFIIB/TATA-Box
Structure of the Elongating RNA Polymerase
Cheung et al., EMBO J. 2011
Phosphorylation of RNA Pol II CTD Correlates with
Gene Activation
CTD Heptapeptide: Thr Ser Pro Thr Ser Pro Ser (yeast, 26X)
Tyr Ser Pro Thr Ser Pro Ser (mammals 52X)
Red:
Antibody for
phosphorylated CTD
Green: Antibody for unphosphorylated CTD
74/EF/75b: ecdysone induced
high level transcription in
“puff” regions.
Transcription Elongation Factors
Transcriptional pause is a frequent event
during elongation caused by slight
misalignment of the RNA 3’OH and the
active site. It is self-reversible and
regulated by numerous cellular factors.
Transcriptionally arrested complexes, in
which the RNA 3’OH and active site are
irreversibly misaligned, resume elongation
after Pol II mediated transcript cleavage
and realignment of the active site and the
RNA 3’OH end, processes that are
stimulated by TFIIS.
Modification of RNA Pol II CTD During
Transcription Elongation
CTD phosphorylation cycle: TFIIH subunit Kin 28 phosphorylates Ser 5. Transcription
elongation complex (TEC) is arrested at checkpoint for pre-mRNA capping. P-TEFb
(Bur1/2 or Ctk1 in yeast) then phosphorylates Ser 2, which allows further elongation.
Specific phosphatases (possibly Ssu72 and FCP1, which are required for Pol II
Recycling) dephosphorylate the CTD at Ser 5 and Ser2.
Activation of Transcription
Tissue-specific Gene Expression
Simple gene expression array analysis.
cDNAs are spotted onto nitrocellulose
or nylon membranes and hybridized to
radio-labeled RNA from liver, kidney, or brain.
For identifying tissue-specific DNA control elements
the genomic regions of the liver-specific genes
can be isolated from genomic libraries. The
upstream promoter regions can then be ligated in
front of reporter genes and analyzed for tissuespecific expression.
Identification of Regulatory DNA Elements by
Progressive Deletions and Reporter Gene Assays
Assume TTR is a liver specific gene identified in
the previous experiment. The upstream regulatory
region of this gene was cloned in front of a reporter gene
(Reporter Gene: e.g. LacZ, luciferase, GFP).
The reporter gene constructs were transiently
transfected into a liver cell line.
The data demonstrate that a tissue-specific
cis-regulatory DNA element, required for activated
expression of the reporter gene is located between
the 5’end of fragment 2 and the 5’end of fragment 3.
In addition, a basal promoter element is located
between the 5’end of fragment 4 and the
5’ end of fragment 5.
The regulatory DNA element that is located
between the 5’end of fragment 2 and the 5’end of
fragment 3 can be further analyzed for the
presence of transcription factor binding sites.
A.
B.
C.
D.
In silico analysis for known factors using databases.
Electrophoretic Mobility Shift Assays (EMSA).
DNA Footprinting.
Chromatin Immunoprecipitation.
Modular Structure of Transcription factors
-Plus: Nuclear Localization Sequences (NLS), Protein/Protein
Interaction Domains, Sites of Modification.
Identification of Transcription Regulators by
Reporter-Gene Assays
The previous experiment identified
a cis-regulatory element of the
TTR gene that mediates high-level
expression in a liver cell line. To
identify a transcription factor that
binds to the cis-regulatory element
(X) and activates transcription, cotransfection experiments can be
performed. Plasmid 1 carries the
gene encoding the transcription
factor of interest, while plasmid 2
carries regulatory sequence X
linked to a reporter gene. A control
experiment would be performed
with plasmid 2 lacking sequence X.
Identification of Transcription Regulators by
Reporter-Gene Assays
Identification of DNA binding and Transcription
Regulation Domains
Genome Wide Analysis of Protein Chromatin Interactions Using
ChIP-seq or ChIP-chip
Farnham P.J., Nature Rev. Genet., 2009
Hormone mediated transfer of GR to the Nucleus
Enhancer and Promoter of a Liver-Specific Gene
Cooperative Binding of Transcription Factors to DNA
Enhanceosome
•Enhancer can be located upstream or downstream of genes (or in introns)
•Enhancer act in an orientation independent manner.
•Many Enhancer elements act in a tissue-specific manner.
•The size of enhancers is 200 to 400bps.
•They act by a tracking, looping, or linking mechanism.
Activation of the stripe 2 enhancer by a gradient of transcription
factors in the developing Drosophila embryo
The drosophila eve gene is regulated by
many enhancer elements that confer
Expression of the eve gene in specific
stripes of the developing embryo. The
stripe 2 enhancer is composed of binding
sites for activating proteins (Bicoid: Bcd,
Hunchback, Hb) and repressing proteins
(Kreisler, Kr, and Giant, Gnt). During
early embryonic development, a gradient of
transcription factors is set up in the
multi-nucleated embryo (syncytium). At
this stage, the concentration of bicoid and
hunchback is high in stripe 2, whereas the
concentration of kreisler and giant
is low.
Ptashne and Gann, 2002
Possible Mechanisms of Enhancer Function
Dean, A., Trends in Genetics, 2006
Transcription and Transcription Factories
Model of dynamic associations
of genes with transcription factories.
Chromatin loops (black) extruding
from chromosome territories (gray).
Transcribed genes (white) in RNA
Pol II factories (black circles).
Potentiated genes (free loops) that
are not associated with Pol II factories
are temporarily not transcribed and can
migrate to a limited number of preassembled Pol II factories (dotted arrows)
Szentimayr and Sawadogo, Nucl. Acids Res.. 2002
Osborne et al., Nat. Genet., 2004
Chromosome conformation capture
Dekker, Nat. Methods 2006
Active genes dynamically colocalize to shared sites
of ongoing transcription
Actively transcribed genes associate
with RNA Pol II foci. (a) RNA immunoFISH of Hbb-b1 transcription (red) with
RNA Pol II staining (green) in erythroid
cells. (b) DNA immuno staining of
Eraf (red) and RNA Poll II (green).
(c) Comparison of the percentage
of alleles exhibiting a gene transcription
signal by RNA FISH (black), with the
Percentage of loci that overlap with an
RNA Pol II focus by DNA FISH.
3C showing interactions between
b -globin LCR and the transcribed
Eraf and Uros genes in erythroid cells.
Calr is a ligation and PCR control.
E: erythroid
B: brain cells
nuclei were fixed for 5
Minutes (in a) or for 10 min (in b)
Osborne et al., Nat. Genet., 2004
Transcription Factories
Model of a transcription factory (diameter
70 nm) containing 8 polymerases (green
crescents). Genes are reeled through these
factories (one polymerase per gene).
Heat shock gene activation and formation
of a transcription factory that facilitates
reinitiation.
Sutherland, H., and W.A. Bickmore, Nat. Rev. Genet., 2009
Activation of Transcription via Mediator
Structure of Mediator
Transcription Repressors Recruit Chromatin Modifying
Protein Complexes
Silencing of Chromatin Domains by
Polycomb Repressor Complexes
The PRC complex mediates the silencing of genomic loci. It is recruited to specific sites in chromatin by
the PRC responsive elements (PREs) and proteins that interact with PREs. There are two PRC complexes.
PRC2 methylates H3K27 and represses gene expression. PRC1 recognizes methylated H3K27 and
methylates CpGs. This leads to a stably repressed chromatin configuration that can be transmitted to
daughter cells after mitosis.
Transcription Activators Recruit Chromatin Modifying
Protein Complexes
Histone Modifications In Euchromatin and Heterochromatin
“Histone Code”
A histone mark
associated with
sites of Pol II
recruitment is
trimethylated
H3K4. This
modification has
been shown to
assist in the
recruitment of
TFIID.
Dimethylated H3K4
is associated with
active chromatin
domains
H3K27 and H3K9
trimethylation is
associated with
repressed
chromatin.
H3K36 methylation
is associated with
transcription
elongation.
Histone modifications
Histone acetylation
• Occurs on lysine side chains of N-terminal tails of histones
• Changes charge of histone tails – makes them less basic (neutralizes positive charge)
• May weaken histone-DNA interactions and “open” the nucleosome
• May alter histone-histone interactions
• May alter interactions between histones and regulatory proteins
• May facilitate binding of regulatory proteins to cis-acting elements in DNA
• Effects may be on individual nucleosomes and/or higher order chromatin structure
• Makes histone tails more -helical
• Catalyzed by histone acetyltransferases (HAT’s)
• Reversible by histone deacetylases (HDAC’s)
Histone methylation
• Does not alter net charge of histone tails
• Mono-, di-, tri-methylation of lysine side chain
• Catalyzed by histone methyltransferases (HMT’s)
• Methylation of histone tails thought to be stable
• Histone demethylase isolated in 2005 – LSD1, lysine-specific demethylase, removes a
methyl group from one particular residue (H3, K4), probably others
ATP-Dependent Chromatin Remodeling Complexes
Steps in Gene Regulation
Genes are normally embedded in inaccessible
chromatin. The first step in the activation of
gene expression is often the recruitment of
chromatin remodeling complexes (Swi/SNF) by
pioneer transcription factors that recognize
their respective binding site in the context of
nucleosomes or higher order structure. In the
example shown here, the yeast DNA binding
factor SWI5 recruits the SWI/SNF complex to
its binding site in chromatin. In an ATP
hydrolysis dependent manner Swi/SNF
mobilizes nucleosomes, rendering a short
segment of chromatin accessible (nuclease
sensitive). Subsequently, SWI5 and other
DNA binding proteins recruit histone
acetyltransferases (HATs) like yeast GCN5.
Steps in Gene Regulation
HATs acetylate H3 and H4 N-terminal tails and
provide docking sites for other co-regulatory
protein complexes. For example, acetylated
histones recruit additional HATs that acetylate
neighboring nucleosomes leading to the
spreading of accessible chromatin.
Steps in Gene Regulation
The partial opening of chromatin structure allows other DNA binding transcription
factors to access their binding sites.
Steps in Gene Regulation
The binding of additional transcription factors is followed by
recruitment of mediator…
Steps in Gene Regulation
… and the transcription preinitiation complex. This leads to a segment of DNA that is completely devoid of a
nucleosome, thus forming a DNAseI hypersensitive site. The example shown above is just one way genes can
be activated.
In other cases, like heat shock genes, the transcription complex is already bound at the promoter but stalled.
Gene activation leads to a change in the transcription complex that renders it elongation competent.
Recent evidence suggests that highly expressed genes are recruited to transcription factories in the nucleus.
Transcription factories are domains in the nucleus that contain multiple aggregated transcription complexes.
Active Genes are Associated with Accessible Chromatin
Mapping DNase I Hypersensitive Sites
[ DNase I ] 0
1) Treat chromatin/cells with DNase I
2) Purify genomic DNA
3) Digest DNA with
restriction enzyme (BamHI)
4) Fractionate DNA on agarose gel
5) Blot gel to membrane (Southern blot)
6) Hybridize blot with probe
7) Expose gel to film for autoradiogram
- 15 kb
DHS1
- 3.1 kb
DHS2
- 1.5 kb
Southern blot
DHS1 DHS2
B
B
Exon 1
Probe
1.5kb
3.1kb
15kb
RNA Polymerase I Transcription
RNA Polymerase III Transcription
U6 Gene