Combinatorial Transcription:expression/regulation depends on the

combination of elements in the promoter

GC boxMRE- metal response elementBLE- enhancer that responds to activator AP1GRE- Glucocorticoid response element

Human Metallothionine promoter

+ H1

- H1

The role of histone H1

H1 binds to the nucleosome where the DNA enters and exits the core.

5 mM NaCl 1 mM NaCl

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 nucleosome-free 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

Basal factors RNAP II

Enhancer with bound protein

promoter

Models for enhancer function

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.

E- enhancerPsi40- rRNA promoter

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

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.

Confining Gene Expression

- 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

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.

Yellow gene

The gypsy Retrotransposon of Drosophila

One example of a specific insulator trought enhancer-promoter interaction!

Where is the insulator activity?

HpaII, Sma and Hae meth sensitiveMspI (isoesch of Hpa II) meth insensitive

The core is not methylated (Fig5), but doesn’t work as a promoter (Fig6)

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

CTD of large subunit of Pol II

Pol IIa

CTD of large subunit of Pol IIP

PPP

PP

Pol IIokinase + ATP

phosphatase

Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation.

CTD has repeat of (YSPTSPT)26-50

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 thefrequency 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 nucleosomes2. 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 ImmunoprecipitationChromatin ImmunoprecipitationIn vivo formaldeyde crosslinking

Cell lysis sonication

immunoprecipitation

Reverse crosslinking and analyses by PCR

HeLa cells

Pol II Pol II Pol IIDNA

RNA

“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?

Targeted histone acetylation by SAGA and NuA4 is required for stimulated transcriptionIn vitro under competitive conditions

Non specific

The HAT activity of SAGA and NuA4 but not of NuA3 is recruited by VP16

array +/- competitor + HAT+ [3H]-acetil CoA

Electrophoresisand flurorgraphy

Is specific!

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