Transcript Access Slides

Chromatin Remodeling
Levels of chromatin organization
300 nm fiber
nucleosome
arrays
Structure of the nucleosome: problems of accessibility for DNAbinding proteins.
Histone cores are predominantly alpha helical.
Luger et al. (1997) Nature 389, 251.
Analysis of binding of transcription factor to naked DNA and
nucleosomal DNA
•
Assembling the DNA
into a nucleosome
strongly inhibits the
binding of a sequencespecific transcription
factor.
•
Assembling the DNA
into a nucleosome
leads to cleavage by
DNase I at 10
nucleotide intervals.
Transcription
factor
footprint
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.
Analysis of chromatin changes by micrococcal nuclease
transcription
-
+
DNA
Disappearance of
ordered nucleosomes
upon transcriptional
induction
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 Acetyl
Transferases
• Multiple families
• Gene-specific or global activators of transcription
• Distinct substrate specificities for different families
• Could acetylate non-histone proteins (transcription
factors)
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.
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.
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.)
• 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 restriction
enzyme access.
Narlikar et al. (2002) Cell 108, 475
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.
Structures of representative remodeling complexes
ISWI family
SWI/SNF family
•
Generally multi-component.
•
The large catalytic subunits
contains both ATPase and nonenzymatic domains.
Narlikar et al. (2002) Cell 108, 475
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.
Chromatin immunoprecipitation
(ChIP):
an assay for interaction of
proteins with regulatory
sequences in vivo.
ChIP analysis
of the ßinterferon
gene.
Agalioti et al. (2000)
Cell 103, 667.
Loading of complexes at the ß-interferon
gene
i.
Activator
ii. HAT complex
SWI/SNF
iii. SWI/SNF complex
iv. GTFs and Pol II
Narlikar et al. (2002) Cell 108, 475.
Loading of complexes at the HO gene in
yeast
i.
SWI/SNF
ii.
HAT complex
iii. SWI/SNF
complex
iv. GTFs and Pol II
Narlikar et al. (2002) Cell 108, 475.
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.
The remodeling process may depend on the initial
status of the chromatin