Transcription and chromatin General Transcription Factors + Promoter-specific factors + Co-activators
Cofactor or Coactivator 1. 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 II C. CBP/p300 works with CREB protein D. TRAPS - steroid receptor protein cofactor E. OCA-B with Oct 1 protein F. 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 250 B. 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 Proposed 30nm fiber structure Gary Felsenfeld & Mark Groudine, Nature, 2003; 421:448-53. Schalch, T et. al., Nature. 2005;436:138-41.
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 endonucleases iii. 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 observed ii. micrococcal nuclease mapping also shows changes in translational positioning c. 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 machines Different 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 complex E. SWR1 complex -promotes the exchange of H2A from the nucleosome -brings in a H2A variant called H2AZ
Chromatin Remodeling Complexes Are Classified by the ATPase Subunits SNF2 ISWI Mi-2 INO80 Helicase Bromo AT Hook HAND SANT SLIDE Chromo PHD DBINO
Mechanisms of ATP-dependent chromatin remodeling Several models involving DNA bulges or DNA twist
Twist diffusion model Would move nucleosomes in 1 bp increments Flaus and Owen-Hughes Biopolymers 2003 Least disruptive of histone-dna contacts
Bulge Propagation Model Planar bulge Propagation of bulge in a wave-like manner around the nucleosome Flaus and Owen-Hughes Biopolymers 2003
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