Chromatin Remodeling

Levels of chromatin organization

nucleosome arrays

300 nm fiber

Structure of the nucleosome: problems of accessibility for DNA-binding proteins.

Histone cores are predominantly alpha helical.

Luger et al. (1997) Nature 389, 251.

Transcriptionfactorfootprint

Analysis of binding of transcription factor to naked DNA and nucleosomal DNA

• Assembling the DNA into a nucleosome strongly inhibits the binding of a sequence-specific transcription factor.

• Assembling the DNA into a nucleosome leads to cleavage by DNase I at 10 nucleotide intervals.

Taylor et al (1991) Gene & Dev 5, 1285.

DNase Footprinting

DNase I binds the minor groove and cuts the phosphodiester backbone. When DNA rests against a surface, the minor groove is maximally accessible at ~10 base intervals.

transcription - + DNA

Disappearance ofordered nucleosomesupon transcriptionalinduction

Analysis of chromatin changes by micrococcal nuclease

Li & Reese(2001) JBC 276, 33788.

“Chromatin remodeling complexes” and “Chromatin modifying complexes” are important for transcriptional activation

Chromatin modifying complex

Chromatin remodeling complex

Examples of histone modification

Berger (2002) Curr. Opin.Gene. Dev. 12, 142.

The “histone code” hypothesis : the pattern of post-translational modifications occurring on the histone tails

serves as binding sites for specific proteins.

• Note that other chromatin modifying complexes include kinases, methylases and ubiquitin conjugating proteins.

• Acetylation typically correlates with transcriptional activation while deacetylation correlates with repression.

Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422.

Histone AcetylTransferases

• Multiple families

• Gene-specific or global activators of transcription • Distinct substrate specificities for different families

• Could acetylate non-histone proteins (transcription factors)

• Weakens interaction of basic tails with negatively charged phosphate backbone of DNA.

• Weakens interactions that occur between nucleosomes, thus promoting decondensation of the chromatin fiber.

• Provide a marker for recognition by other proteins. For example, a conserved “bromo” domain found in SWI/SNF and other transcription factors recognizes this marker.

How acetylation might contribute to activation

Non-enzymatic domains in Chromatin Modificationproteins

Bromodomain recognitionof acetyl-lysine

Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422.

Dhalluin et al. (1999) Nature 399, 491.

Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422.

Multiplicity of non-enzymatic domains in histone modifyingenzymes

Chromatin remodeling complexes. (e.g. SWI/SNF, ISWI, etc.)

• Couples ATP hydrolysis with altering the nucleosome structure so that DNA binding proteins can access the DNA.

• DNase I footprinting analysis shows that the 10 base periodicity of cutting disappears.

• Gel shift and DNase I footprinting assays like those shown previously show that the chromatin remodeling complexes decrease the binding constant of proteins for nucleosomal DNA.

Assays for Chromatin Remodeling

Altered positioning

Changes in restrictionenzyme access.

Narlikar et al. (2002) Cell 108, 475

A chromatin remodeling complex increases theaccessibility of DNA to restriction enzyme cleavage

in an ATP-dependent fashion.

Saha et al. (2002) Genes& Dev. 16, 2120.

SWI/SNF family ISWI family

Structures of representative remodeling complexes

Narlikar et al. (2002) Cell 108, 475

• Generally multi-component.

• The large catalytic subunits contains both ATPase and non-enzymatic domains.

Mechanisms of Remodeling

• Sliding Vs. Eviction

• Translational repositioning

• Conformational change: induce twisting and/or bending of DNA.

How is the activation process initiated by a DNA binding protein if the protein can’t bind the DNA in the first place?

• Some DNA binding protein recognize their sites of the surface of a nucleosome - e.g. Glucocorticoid receptor.

• Position the binding site in linker DNA.

• In some cases, nucleosome associations are quite dynamic in the absence of activities that constrain their locations on the DNA so that DNA binding proteins are provide “windows of opportunity” to associate.

an assay for interaction of proteins with regulatory sequences in vivo.

Chromatin immunoprecipitation (ChIP):

ChIP analysis of the ß-interferon gene.

Agalioti et al. (2000)Cell 103, 667.

Loading of complexes at the ß-interferongene

i. Activator

ii. HAT complex

iii. SWI/SNF complex

iv. GTFs and Pol II

SWI/SNF

Narlikar et al. (2002) Cell 108, 475.

Loading of complexes at the HO gene inyeast

i. SWI/SNF

ii. HAT complex

iii. SWI/SNF complex

iv. GTFs and Pol II

Narlikar et al. (2002) Cell 108, 475.

The chromatin remodeling complex binds to the promoter prior to the HAT, followed by Pol II and GTF’s.

The time course of association of factors with the HO endonuclease gene in yeast.

The remodeling process may depend on the initial status of the chromatin