Biol/Chem 473

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Biol/Chem 473
Schulze lecture 8 Chromatin remodelling
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Spreading of heterochromatin in flies (and
humans!)
K9 of H3 (H3K9me)
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Spreading of silenced chromatin on a homeotic gene
E(z) (SET)
K27 of H3 H3K27me
Polycomb
Polycomb
Polycomb
Polycomb-complexed chromatin
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Heterochromatin vs. euchromatin
adapted from Jenuwein Allis (2001) Science 293,
1074
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Heterochromatin vs. euchromatin
H3K4me
adapted from Jenuwein Allis (2001) Science 293,
1074
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Heterochromatin vs. euchromatin
H3K9me
adapted from Jenuwein Allis (2001) Science 293,
1074
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Regulation of homeotic genes
H3K27me
Repressed homeotic gene
active homeotic gene
adapted from Jenuwein Allis (2001) Science 293,
1074
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Three big questions

• How are the PcG proteins targeted to their
  cognate sequences?
• What is the maintenance mark that functions to
  keep specific genes active/inactive?
• How is the maintenance mark maintained through
  cell division?

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Deposition of histones during replication
During replication, parental histones are
distributed randomly onto the replicated DNA
strands, and newly synthesized histones fill the
gaps.
http//www.umassmed.edu/faculty/show.cfm?start0f
aculty912
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Enzymes that regulate chromatin

• Two classes of enzymes that regulate chromatin
  structure histone modifiers (last two lectures)
  and chromatin remodelers (today).
• Histone modifiers dont alter nucleosome
  position they make passive marks that recruit
  more active functions (histone code).
• Chromatin remodelers hydrolize ATP to actively
  remodel chromatin shift nucleosome position with
  respect to DNA, exposing or occluding regulatory
  sequences.
• These enzymes function within larger complexes of
  subunits that collectively act to enhance and/or
  target the remodeling activity.

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How to plough through chromatin?
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Ground state is restrictiveSilent state is MORE
restrictive
GROUND STATE
Struhl, 1999
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Chromatin remodeling is an active process

• Chromatin remodeling describes the
  energy-dependent (ATP) displacement or
  reorganization of nucleosomes that occurs in
  conjunction with activation (or repression) of
  genes for transcription
• Chromatin remodelers also play a role in
  recombination and repair

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Chromatin remodelers move nucleosomes around

• Remodeling improves access to DNA or histone
  binding sites recognized by transcriptional
  regulators or histone modifiers
• This remodeling process can lead to activation or
  repression

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Why do you need ATP to move nucleosomes around?

• Bending DNA around a bunch of histones depends on
  DNA sequence to some extent
• Some sequences bend more easily than others
• Nucleosomes do have strong affinity for DNA and
  even have sequence preferences
• This preference may be exploited in nature to
  organize a chromatin landscape energetically
  conducive (or not) to gene expression
• (Widom et al. (2006) Nature 442(17)772
  computational paper)
• But that landscape must change as gene expression
  requirements change
• and this requires energy

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Stamp collecting classifying chromatin remodelers

• Chromatin remodeling complexes are classified
  based on protein motifs found in addition to the
  ATPase domain, or on how the ATPase domain itself
  is structured
• This classification is purely structural,
  designed to make it easier for us humans to sort
  them all out it may not accord with functional
  criteria
• There is a great deal of mixing and matching of
  subunits between various groups of remodelers
  (combinatorials again.)

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SWI2/SNF2 ATPase SUPERFAMILY
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SWI2/SNF2 ATPase SUPERFAMILY
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Chromatin remodeling complexes
SWI2/SNF2 subfamily
ISWI subfamily
hBrm
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Conservation of SWI/SNF subfamily complexes
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Cloned Members of the Pc-G and trx-G
trx-G
Genetically SWI/SNF subfamily members isolated as
activators of gene expression
(PRE/TRE)
Homeotic gene expression
trx Mll, Mll2 trl brm Brm,
Brg1 mo Baf155, Baf170 osa Baf250 snr1 Baf47/Snf5
z ash-1 Ash1l ash-2 Ash2l lid kis lawc
Pc M33/MPc1, MPc2, MPc3 ph Rae28/Mph1 Psc Mel18,
Bmi-1 Scm Scmh1, Scmh2 Pcl M96 Pho YY1 Su(z)2
Mel18, Bmi-1 dRING Ring1a, Ring1b E(z) Enx-1,
-2/Ezh-1, -2 esc eed Asx Asxl1, Asxl2 crm
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Method nucleosomal mobility assay using EMSA
(electrophoretic mobility shift assay)
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Experiment how do SWI/SNF subfamily chromatin
remodelers shift nucleosomes?

• Amplify template (215bp bit longer than normal
  so you can assay movement)
• Purify histones
• Reconstitute end-labeled mononucleosomes in a
  test tube (bunch of chemistry)
• Nucleosomes at the end of a DNA fragment will
  migrate on a gel more rapidly than those in the
  middle of a DNA fragment
• Extract those stable nucleosomal fragments
• Run again in the presence of SWI/SNF, with () or
  without (-) ATP
• What happens?

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SWI/SNF sub family remodelers can slide
nucleosomes along DNA
Ramachandran et al. (2003) J. Biol. Chem.
278(49)48590
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Protein domains in a SWI/SNF complex
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Drosophila polytene chromosomes allow
visualization of chromosomal activity during
interphase
Chromosomes replicate without nuclear division
endoreplication
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Drosophila polytene chromosomes allow
visualization of chromosomal activity during
interphase
Chromosomes replicate without nuclear division
endoreplication
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Where does Brahma localize in the interphase
nucleus?
Armstrong et al. (2002) Embo J. 21(19)5245

• The Drosophila SWI/SNF ATPase Brahma localizes to
  active regions of the genome
• There is little or no colocalization with the
  homeotic repressor Polycomb

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SWI/SNF and cancer

• The tumor suppressor BRCA1 co-purifies with the
  human SWI/SNF Brg1 complex
• Is the SWI/SNF remodeling complex functioning as
  a transcriptional co-activator?

Bochar et al. (2000) Cell 102257
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Chromatin remodeling complexes
SWI2/SNF2 subfamily
ISWI subfamily
hBrm
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A comparison of SWI/SNF and ISWI subfamily ATPases
BRAHMA
ATPase somewhere in here
ISWI
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ISWI complexes are VERY diverse
Nucleosome remodeling factor
Chromatin accessibility complex
ATP-dependent chromatin assembly and remodeling
factor
Nucleolar remodeling complex
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ISWI complexes are VERY diverse
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Where does ISWI localize in the interphase
nucleus?
Deuring et al. (2000) Mol. Cell 5355

• Distribution of ISWI and RNA PolII do NOT overlap
  (much)
• Do ISWI remodelers play a role in chromatin
  repression?

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Method microccocal digestion measures nucleosome
repeat lengths
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Silent vs. active chromatin show different
micrococcal nuclease digestion patterns

• Transgene in heterochromatin or euchromatin
• Cut genomic DNA with micrococcal nuclease (cuts
  between nucleosomes)
• Blot DNA and incubate with transgene sequence
  probe (Southern!)

MNase
Cryderman et al. (1999) NAR 27(16) 3364
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ISWI ATPases can induce regular OR irregular
nucleosomal spacing

• The implication is that ISWI chromatin remodelers
  can organize chromatin into a more repressive
  configuration, OR a more active one
• Since yeast has two ISWI ATPases with different
  effects on chromatin in vitro, they may have
  evolved contrasting activities

Gelbart et al. (2001) Mol. Cell Biol. 21(6) 2098
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Summary SWI/SNF and ISWI

• Two types of ATPases that form large
  multi-subunit chromatin remodeling complexes.
• These complexes use the energy of ATP to remodel
  nucleosomal DNA.
• SWI/SNF subfamily members are more often
  associated with activation of chromatin but they
  can silence as well (I didnt show you evidence
  but its out there)
• ISWI subfamily members correlate with repressed
  chromatin but they can activate as well.
• Chromatin remodelers contrast with chromatin
  modifiers make sure you know how!