Transcription and chromatin

General Transcription Factors +Promoter-specific factors +

Co-activators

Cofactor or Coactivator1. work with DNA specific transcription factors to

make them more effective - examples A. TFIID B. yeast mediator - contains 20 polypeptides

binds to the CTD of RNA polymerase IIC. CBP/p300 works with CREB proteinD. TRAPS - steroid receptor protein cofactorE. OCA-B with Oct 1 proteinF. SAGA complex - contains Ada proteins

(adaptor protein) one of which is also known as Gcn5; works with acidic activators such as Gal4 protein or Gal4-VP16

Cofactor or Coactivator

2. Several of these proteins modify chromatin; acetylation of histone tails

A. TAFII 250B. Gcn5 is one of the first

characterized histone acetyl transferases(HAT)

C. CBP/p300

Gaining Access to the DNA

Large multi-subunit protein complexes alter the state of

chromatin

Organization of Chromatin

Gary Felsenfeld & Mark Groudine, Nature, 2003; 421:448-53.

Schalch, T et. al., Nature. 2005;436:138-41.

Proposed 30nm fiber structure

What is chromatin remodeling?

The process of making DNA more or less accessible in the eukaryotic genome using

a series of specialized proteins

Chromatin Remodeling • ATP-dependent chromatin remodeling

– SWI/SNF and RSC– ISWI: CHRAC, NURF, ACF– CHD/Mi-2– INO80 and SWR1

• Covalent modification– ACETYLATION– METHYLATION– PHOSPHORYLATION– UBIQUITINATION– Poly ADP RIBOSYLATION– SUMOLYATION

What is the functional role of chromatin remodeling

• Global regulation of chromatin structure• Transcription activation and repression• Transcription elongation and termination• DNA repair • DNA recombination

– including immunoglobulin gene rearrangement

• DNA replication

ATP-dependent chromatin remodeling machines

A. they alter the structure of the nucleosome i. the nucleosomal DNA is made more

accessible for binding to transcription factors ii. changes can also be observed by measuring

access to cutting by restriction endonucleasesiii. changes have also been observed in DNA

supercoiling suggesting a change in the path of DNA around the nucleosome

iv. electron microscopy studies have suggested a reduction on the amount of DNA wrapped in the remodeled nucleosome

ATP-dependent chromatin remodeling machines

b. Slides nucleosomes along DNA, changes their translational position

i. changes in electrophoretic mobility is observedii. micrococcal nuclease mapping also shows

changes in translational positioningc. Spaces and assembles nucleosomes

Sliding Nucleosomes

Spacing nucleosomes

Nucleosomes are randomly distributed along DNA

Located equal distances from each other (uniformly spaced, but no uniquely positioned)

These complexes can be recruited to specific chromatin sites by:

A. transcriptional activators such as SWI/SNF by Gal4, Gcn5 and the glucocorticoid receptor protein

B. repressors such as the ISWI complex from yeast called ISW2 by the Ume6 repressor protein

Accessibility is Key Regulatory Step

ATP-dependent chromatin remodeling machinesDifferent classes of chromatin

remodeling complexes

Different classes of ATP-dependent chromatin remodeling complexes

A. SWI/SNF or SWI-SNF like complexes– required for activation of a small subset of

genes– found in humans, flies, and yeast– at least two different forms of the complex in

yeast or humans– in yeast it has 11 or 17 subunits– total complex size is 1.5 -2 Megadaltons

Wiley-Liss, Inc.

Mammalian SWI/SNF

Different classes of ATP-dependent chromatin remodeling complexes

B. ISWI or imitation SWI complexes-its DNA dependent ATPase subunit is

similar to the ATPase subunit of SWI/SNF-has the distinguishing protein domain called

SANT-smaller complexes: only 1-4 subunits-more abundant and play a more global role-are involved in transcriptional repression- there are three distinct ISWI complexes in

flies and yeast

ISWI Subfamily of Chromatin Remodeling Complexes

Corona, D.F. and J.W. Tamkun, Multiple roles for ISWI in transcription, chromosome organization and DNA replication.Biochim Biophys Acta, 2004. 1677(1-3): p. 113-9.

Remodeling Activities of ISWI Subfamily

Langst, G. and P.B. Becker, Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors.J Cell Sci, 2001. 114: p. 2561-8.

Different classes of ATP-dependent chromatin remodeling complexes

C. Mi-2 or CHD complexes-these chromatin remodeling complexes are

associated with histone deacetylases (HDAC) - ATPase subunit has sequence homology to

the ATPase subunit of SWI/SNF belongs to the Swi2 superfamily, but makes up its own subfamily

Different classes of ATP-dependent chromatin remodeling complexes

D. INO80 complex-involved in DNA repair-has DNA helicase activity associated

with the complexE. SWR1 complex

-promotes the exchange of H2A from the nucleosome

-brings in a H2A variant called H2AZ

SNF2

ISWI

Mi-2

INO80

Helicase Bromo AT Hook SANT

Chromo PHD

Chromatin Remodeling Complexes Are Classified by the ATPase Subunits

HAND

SLIDE DBINO

Mechanisms of ATP-dependent chromatin remodeling

Several models involving DNA bulges or DNA twist

Flaus and Owen-Hughes Biopolymers 2003

Twist diffusion model

Would move nucleosomes in 1 bp increments

Least disruptive of histone-DNA contacts

Bulge Propagation Model

Flaus and Owen-Hughes Biopolymers 2003

Propagation of bulge in a wave-like manner around the nucleosome

Planar bulge

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