Transcript Combinatorial Transcription: expression/regulation depends

Combinatorial Transcription:
expression/regulation depends on the
combination of elements in the promoter
Human Metallothionine promoter
GC box
MRE- metal response element
BLE- enhancer that responds to activator AP1
GRE- Glucocorticoid response element
The role of histone H1
5 mM NaCl
1 mM NaCl
H1 binds to the
nucleosome where the
DNA enters and exits
the core.
- H1
+ H1
H1 is needed to form the zig-zag structure.
What is the effect of histones on
transcription in vitro?
• Assemble core histones on a plasmid (1/200
bp), nucleosomes inhibit transcription by
blocking promoter binding sites.
• Addition of H1 further represses transcription (by
binding to the linker DNA), but this can be
overcome by activators such as Sp1.
• There are regulatory proteins, such as the
glucocorticoid-receptor complex, that can
remove histones from certain promoters.
2 Models for Transcriptional Activation
H1 (yellow) covers promoter,
remove it and bind activators
(factors).
Nucleosome covers promoter,
still repressed after H1 removed.
Remove nucleosome with
special factors.
In Vivo Studies
• Promoters of active genes are often
deficient in nucleosomes
SV40 virus
minichromosomes
with a nucleosomefree zone at its twin
promoters.
Can also be shown for
cellular genes by DNAase
digestion of chromatin –
promoter regions are
hypersensitive to
DNAase!
Function of Activation Domains
• Function in recruitment of components of the
pre-initiation complex in eukaryotes (the
RNAP holoenzyme is recruited in
prokaryotes)
• Act independent of DNA-binding domains
– Can make chimeric factors that function --- i.e.,
combine the DNA-binding domain from one factor and
activation domain from another and get the expected
activity
Activation from a Distance:
Enhancers
•
There are at least 3 possible models
Factor binding to the enhancer induces:
1. supercoiling
2. sliding
3. looping
Models for enhancer function
Basal factors
promoter
Enhancer with bound protein
RNAP II
E- enhancer
Psi40- rRNA promoter
Transcription of DNAs 1-5 was tested in Xenopus oocytes.
Results: good transcription from 2, 3, and 4 (also 2 >3 or 4) but not 5.
Conclusion: Enhancer does not have to be on same DNA molecule, but
must be close.
Rules out the sliding and supercoiling models.
Looping out by a
prokaryotic
enhancer binding
protein visualized by
electron microscopy.
NtrC – protein that
binds enhancer and
RNAP
σ 54 polymerase –
RNAP with the 54 kDa
sigma factor
Chromatin Insulators and
Boundary Elements
Confining Gene Expression
Some of the organisational properties of the
eukaryotic genome reside in the ability of
chromatin to establish autonomous units that
specify levels and patterns of gene expression.
i.e. enhancers act on a promoter in a specific domain, but are
unable to act on a promoter in a separate domain.
The candidates charged with the function of establishing and
delimiting domains of expression are boundary or insulator
elements. These set up independent territories of gene activity.
- Boundary or insulator elements have two characteristic effects
on gene expression:
1. They confer position independent transcription to transgenes
stably integrated in a chromosome.
2. They buffer a promoter from activation by enhancers when
located between the two.
Figure 3 Mechanism of insulator effect on enhancer function. (a) Diagram of two genes, X and Y, located within a chromosomal domain
defined by two insulator sequences (ins) and their associated proteins (ibp). Enhancers located between the two genes (en1and en2) can activate
transcription from the promoter of either gene. (b) If a boundary element such as the gypsy insulator (gyp) is inserted between the two
enhancers, a new chromosomal domain forms, leaving gene X in one domain and gene Y outside. One of the insulators forming the original
domain is now free to form other domains with alternative boundary elements (in this case containing genes Z1and Z2). Enhancer 1(en1) is
now unable to act on the promoter of gene Y because of the new location of the gypsy insulator. Nevertheless, this enhancer is still functional
and competent to activate transcription from the promoter of gene X, located within the same chromosomal domain.
Two models for insulators origin:
How do the insulators work?
A) Chromatin model (Barrier): insulators blocks the spreading of active
and inactive chromatin structures.
B) Decoy model: insulators form non-productive interactions with
enhancers, preventing them from interacting with their target promoter
The gypsy Retrotransposon of Drosophila
Yellow gene
Figure 1 Structure and function of the gypsy insulator. (a) Insulator sequences (ins) are composed of 12 copies of the binding site for the
su(Hw) protein (su), which interacts in turn with the mod(mdg4) protein (mo). The complex of both proteins binds to insulator sequences
and interferes with the function of enhancers present distally from the promoter with respect to the location of the insulator. Enhancers are
diagrammed as ellipsoid bodies on the DNA. In the case shown here, the enhancers that control expression of yellowin the wings (wng) and
body cuticle (bc) of the fly are affected (represented by an X over the enhancer), whereas those responsible for expression in the larval
tissues (lv), bristles (br) or tarsal claws (tc) can function normally. Exons of the yellowgene are indicated by open bars, and the intron by a
thin line. The direction of transcription is also indicated. (b) In a mod(mdg4) mutant, the protein product of this gene is missing, and only the
su(Hw) protein is bound to insulator sequences. In this case, repression of transcription is bi-directional, the insulator behaves as a silencer,
and none of the enhancers can act on the promoter.
One example of a specific insulator trought enhancer-promoter interaction!
Where is the insulator activity?
The core is not methylated (Fig5), but doesn’t work as a promoter (Fig6)
HpaII, Sma and Hae meth sensitive
MspI (isoesch of Hpa II) meth insensitive
INSULATORS: Common Features with Promoters, enhancer, but
can not stimulate transcription.
TRANSCRIPTIONAL ELONGATION
1. HIGH RESOLUTION STRUCTURE OF ELONGATING RNA POL II.
Two common things during transcriptional elongation:
1. Arrest (irreversible backsliding 7-14 nucleotides)
2. Pausing (back-tracking 2-4 nucleotides)
RNA pol II is a long time not synthesizing RNA.
2. THE SII FAMILY OF RNA POL II ELONGATION FACTORS
3. CTD PHOSPHORILATION AND TRANSCRIPTIONAL ELONGATION
Eukaryotic RNA polymerase II
Pol IIa
kinase + ATP
Pol IIo
phosphatase
CTD of large subunit of Pol II
P
P
P
P
P
CTD of large subunit of Pol II
P
CTD has repeat of (YSPTSPT)26-50
Phosphorylation of Pol IIa to make Pol IIo is needed to
release the polymerase from the initiation complex and
allow it to start elongation.
NELF and DSIF promote arrest of unphosphorylated RNA polymerase.
P-TEFb phosphorilates CTD and Spt4/5
Relieve of NELF and DSIF inactivation
4. TFIIF, ELL, AND ELONGIN FAMILIES OF TRANSCRIPTION ELONGATION
FACTORS
TFIIF: Increases the efficiency of transcriptional initiation by significantly reducing the
frequency at which RNA Pol II aborts transcription during synthesis of the first few nt.
Also stimulates then elongation
ELL: Important for elongation. Deletion of this gene provokes big changes in the
transcription of long genes, but has no effect on shorter ones.
ELONGIN: Involved in transcriptional elongation. Is a component of the proteasome.
How does RNA polymerase transcribe through
regions with histones/nucleosomes?
1. RNA Pol II can elongate through the nucleosomes
2. RNA Pol II transcription can “remodel” the nucleosomes
1. RNA Pol II can elongate through the nucleosomes
2. RNA Pol II transcription can “remodel” the nucleosomes
A. ELONGATOR COMPLEX:
Sequential histone acetylation by transcription factor-targeted histone
acetyltransferases and by a transcription-coupled histone
acetyltransferase. Polymerase II association with the promoter
precedes binding of the elongator, which requires phosphorylation of
the polymerase II CTD.
B. FACT:
The ebb and flow of histones. (A) The histone
chaperone activity of Spt6 helps to redeposit histones
on the DNA, thus resetting chromatin structure after
passage of the large RNAPII complex. (B) FACT
enables the displacement of the histone H2A/H2B
dimer from the nucleosome octamer, leaving a
"hexasome" on the DNA. The histone chaperone
activity of FACT might help to redeposit the dimer after
passage of RNAPII, thus resetting chromatin structure.
(C) A possible relationship between histone acetylation
and transcription through the nucleosome. In this
scenario, HATs associated with RNAPII acetylate the
histone that is being traversed, facilitating its disruption
and displacement. Upon redeposition of the displaced
histone dimer or octamer, HDACs immediately
deacetylate the histones, resetting chromatin structure.
For simplicity, only Spt6-mediated displacement of
octamers is shown.
Both activators and
PolII work to
remodel chromatin
Comprehensive interaction map for the RNAPII elongation factors
Chromatin Immunoprecipitation
In vivo formaldeyde crosslinking
RNA
DNA
Pol II
Cell lysis
Pol II
Pol II
sonication
HeLa cells
immunoprecipitation
Reverse crosslinking
and analyses by PCR
“Distribution of acetylated histones resulting from Gal4-VP16
recruitment of SAGA and NuA4 complexes”
Marissa Vignali, David J. Steger, Kristen E. Neely and Jerry L.
Workman.
EMBO J. 2000, 19:2629
How the transcriptional activators work?
What is the role Histone Acetylation on expression?
How the transcriptional activators remodel chromatin?
Non specific
Targeted histone acetylation by SAGA and NuA4 is required for stimulated transcription
In vitro under competitive conditions
array
+/- competitor
+ HAT
+ [3H]-acetil CoA
Electrophoresis
and flurorgraphy
Is specific!
The HAT activity of SAGA and NuA4 but not of NuA3 is recruited by VP16
Gal4-VP16 directs the HAT activity of SAGA complex to promoter proximal
nucleosomes
The domain of acetylation generated by NuA4 upong Gal4-VP16 targeting is
Broader than that observed for SAGA