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Chromatin Remodeling. Levels of chromatin organization. 300 nm fiber. nucleosome arrays. Structure of the nucleosome: problems of accessibility for DNA-binding proteins. Histone cores are predominantly alpha helical. Luger et al. (1997) Nature 389, 251.

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slide2

Levels of chromatin organization

300 nm fiber

nucleosome

arrays

slide4

Histone cores are predominantly alpha helical.

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

slide5
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.

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

Transcription

factor

footprint

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

slide7

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.

slide8

Analysis of chromatin changes by micrococcal nuclease

+

-

DNA

transcription

Disappearance of

ordered nucleosomes

upon transcriptional

induction

Li & Reese(2001) JBC 276, 33788.

slide9

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

Chromatin modifying complex

Chromatin remodeling complex

slide10

Examples of histone modification

Berger (2002) Curr. Opin.

Gene. Dev. 12, 142.

slide11

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.
slide13

Histone Acetyl

Transferases

  • Multiple families
  • Gene-specific or global activators of transcription
  • Distinct substrate specificities for different families
  • Could acetylate non-histone proteins (transcription factors)
slide14

How acetylation might contribute to activation

  • 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.
slide15

Non-enzymatic domains in Chromatin Modification

proteins

Marmorstein (2001) Nat. Rev.

Mol. Cell. Biol. 2, 422.

Bromodomain recognition

of acetyl-lysine

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

slide16

Multiplicity of non-enzymatic domains in histone modifying

enzymes

Marmorstein (2001) Nat. Rev.

Mol. Cell. Biol. 2, 422.

chromatin remodeling complexes e g swi snf iswi etc
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.
slide18

Assays for Chromatin Remodeling

Altered positioning

Changes in restriction

enzyme access.

Narlikar et al. (2002) Cell 108, 475

slide19

A chromatin remodeling complex increases the

accessibility of DNA to restriction enzyme cleavage

in an ATP-dependent fashion.

Saha et al. (2002) Genes

& Dev. 16, 2120.

slide20

Structures of representative remodeling complexes

ISWI family

SWI/SNF family

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

Narlikar et al. (2002) Cell 108, 475

mechanisms of remodeling
Mechanisms of Remodeling
  • Sliding Vs. Eviction
  • Translational repositioning
  • Conformational change: induce twisting and/or bending of DNA.
slide22
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.
slide23

Chromatin immunoprecipitation

(ChIP):

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

slide24

ChIP analysis of the ß-interferon gene.

Agalioti et al. (2000)

Cell 103, 667.

slide25

Loading of complexes at the ß-interferon

gene

  • Activator
  • HAT complex
  • SWI/SNF complex
  • GTFs and Pol II

SWI/SNF

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

slide26

Loading of complexes at the HO gene in

yeast

  • SWI/SNF
  • HAT complex
  • SWI/SNF complex
  • GTFs and Pol II

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

slide27

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

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