Chromatin modeling gene expression regulation

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					Chromatin modeling-gene
 expression regulation
          Yi Jia
       Mar. 28, 2006

• Introduction
• Activities and mechanisms of ATP-
  dependent remodeling complexes
• Complex interactions in chromatin
• Summary
• Post-translational modifications of histone tails
  acetylation, phosphorylation, methylation etc.

• ATP-dependent remodeling
  ATP-dependent chromatin remodeling complex
Jennifer A. Armstrong lab website
ATP-dependent chromatin remodeling complex

   acetyl-lysine–binding domain   Binding histone tail   Chromatin organisation modifier

                                          Narlikar GJ, et al. Cell, 2002, 108:475-487
Relationship between SWI/SNF-class chromatin remodeling complexes
                 from yeast, Drosophila and mammals

                                   Mohrmann L and Verrijzer CP. B.B.A., 2005, 1681:59
Diverse functions of different complexes in one family

Complex     Functional specificity
            Non-essential for viability
            Low abundance
            Facilitates expression of several inducible genes
            Involved in exit from mitosis through stimulation of expression of some
            mitotic genes
            Implicated in transcriptional activation and repression
            Essential for viability
            Highly abundant
            Target genes distinct from those of ySWI/SNF
   RSC      Involved in transcriptional activation and repression
            Sth1, Sfh1, Rsc3 and Rsc9 are essential for cell cycle progression
            through G2/M
            Involved in sister chromatid cohesion and segregation

                               Modified from Mohrmann L and Verrijzer CP. B.B.A., 2005, 1681:59
Nucleosome Disruption by Human SWI/SNF Is Maintained in the
          Absence of Continued ATP Hydrolysis

                               Imbalzano AN, et al. J.B.C., 1996, 271:20726
      Various activities of ATP-dependent chromatin remodeling complexes

                                                          •All families can change position of nucleosomes on
                                                          DNA (“sliding” nucleosomes)
                                                          •All can also form regularly spaced nucleosomes in a
SNF2 subfamily   SNF2, not ISWI       SNF2, not ISWI      nucleosomal array
                                                          •ISWI-containing complexes appear to act primarily by
                                                          nucleosome sliding
                                                          •SWI/SNF can change conformation of nucleosomes
                                                          to expose nucleosomal DNA on surface of histone
                                                          octamer without sliding nucleosome.

                  ISWI family

                            Disassembly of nucleosomes?

                                  SNF2 subfamily
                                                              Narlikar GJ, et al. Cell, 2002, 108:475-487
Mechanisms for nucleosome

 Flaus A and Owen-Hughes T. Current Opinion in Genetics & Development, 2004, 14:165-173
Mohrmann L and Verrijzer CP. B.B.A., 2005, 1681:59
           Chromatin Remodeling -Nucleosome Sliding

1) Used Isw2- mutant yeast strain grown on raffinose, where 3 nuclesomes in promoter region appear to be
shifted, leading to partial deregulation of gene.
2) Constructed gal-inducible allele of ISW2 w/ FLAG tag integrated into normal ISWI locus.
3) Analyzed POT1 locus, a previously identified target of Isw2.
4) Performed ChIP assays w/ anti-FLAG Ab for association of POT1 locus with Iswi-FLAG (i.e, Iswi complex).
5) Showed Iswi2 complex increased assoc. with POT1 locus during course of gal induction of Iswi.

                                                            Fazzio TC, et al. Molecular Cell, 2003, 12:1333-1340
Fazzio TC, et al. Molecular Cell, 2003, 12:1333-1340
Fazzio TC, et al. Molecular Cell, 2003, 12:1333-1340
    Chromatin remodeling by nucleosome disassembly

Nucleosome transaction catalyzed     Reversal of histone depletion by RSC
by RSC and ATP in the presence       and Asf1 and of nucleosome disassembly
of histone chaperones (Asf1, Nap1)   by RSC and Nap1.

                                           Lorch Y, et al. PNAS, 2006, 103:3090-3093
   Chromatin remodeling by nucleosome disassembly

Time course of nucleosome     RSC transfers histone octamers
disassembly by RSC and Nap1   to Nap1 but not to DNA or RNA

                                  Lorch Y, et al. PNAS, 2006, 103:3090-3093
      How are remodeling complexes targeted to
              particular nucleosomes?

1) Non-targeting model: SWI/SNF introduces transient changes in
   chromatin structure by a catalytic and random fashion throughout the
   genome, and that persistent, targeted changes in chromatin only
   occur in the presence of a DNA-binding transcription factor

2) Pol II association: SWI/SNF may be targeted to specific genes by
    association with RNA pol II holoenzyme. But only a fraction of all pol
    II holoenzyme contains SWI/SNF.

3) Targeting by transcription factors: SWI/SNF may be targeted to the
    correct genes by direct interaction and recruitment by gene-specific
    activators (and repressors).

                             Yudkovsky N, et al. Genes & Development, 1999, 13:2369-2374
         Other Mechanisms for Targeting Remodeling Complexes

Chromatin Remodeling

                        a) Targeting by DNA-binding factors

                        b) Targeting by methylated DNA (for silencing)

                        c) Targeting by interaction w/ nuclear matrix and
                        actin-like proteins

                        d) Targeting and stabilization by modification
                           pattern of histones

                       Becker PB and Horz W, Annu Rev Biochem, 2002, 71:247-273
   Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators

                                                                                           Srb2 – prevents pol II
                                                                                           recruitment to promoter
                                                                                           rSrb – recombinant Srb2
                                                                                           TBP1143N – TBP mutation
                                                                                           rTBP – recombinant TBP

                                                                                              SWI/SNF components

        Immobilized templates used in assay

                                                                                                 pol II components
1) Used template of His4 promoter w/ TATA box (& promoterless control)
   attached to magnetic beads
2) Formed PIC’s w/ various yeast nuclear extracts & GAl4; washed away
   uncomplexed proteins (using templates immobilized by magnetic beads)
3) Detached promoter/PIC from beads by PstI digestion
4) Western blotted PIC w/ Ab’s to Swi3, Snf5, Rpb3, Gal11, TBP, Gal4

                                                  Yudkovsky N, et al, Genes & Development, 1999, 13:2369-2374
SWI/SNF is recruited to DNA by activators independent of promoter sequences

                                                        G = Gal4 DNA-binding domain only
                                                        A = Gal4-AH (weak activation domain)
                                                        V = Gal4-VP16 (strong activation domain)
                                                        W = His4 promoter template
                                                        Δ = promoterless template

                                                                        STH1 = component of RSC
                                                                               remodeling complex

                             Yudkovsky N, et al, Genes & Development, 1999, 13:2369-2374
   Repressor target ATP-dependent remodeling complex

                                                      Decreased expression of the SUC2 gene in isw2
Increased DNase I accessibility and
                                                      mutants despite increased DNase I accessibility
improperly positioned nucleosomes
at the POT1 locus in ISW2 mutant cells                of the upstream regulatory region

                                         Fazzio TG, et al, Molecular and cellular biology, 2001, 21:6450-6460
           Interactions between Complexes

IFN-ß promoter                                    HO promoter

                           Narlikar GJ, et al, Cell, 2002, 108:475-487
Proteins interacting with the SWI/SNF chromatin
              remodelling complex

                                                 Regulation of cell cycle

                                                 Differentiation

                                                 Transcription regulation

                                                 Cancer

                                                 Immunity

                     Iba H, Mizutani T and Ito T. Rev Med Virol, 2003, 13:99-110
• ATP-dependent chromatin remodeling complexes is
  conserved and diverse

• Remodeling actions can be classified into sliding and
  disassemble mechanisms

• Remodeling complexes can be targeted into specific
  chromatin by binding specific regulator and histone
  modification complex

• Many biologically important proteins can interact with
  chromatin complexes