CHROMATIN

REMODELLING
 DNA Molecules are highly condensed in chromosomes
Nucleosomes of interphase under electron microscope
Nucleosome: basic level of chromosome/chromatin organization
Chromatin: protein-DNA complex
Histone: DNA binding protein
A: diameter 30 nm; B: further unfolding, beads on a string conformation
Nucleosome :
-146 bp of DNA
-an octamere of histone proteins
SARs are very AT-rich fragments several hundred base
pairs in length that were first identified as DNA fragments
that are retained by nuclear scaffold/matrix
preparations. They define the bases of the DNA
loops that become visible as a halo around extracted
nuclei and that can be traced in suitable electron micro-graphs
of histone-depleted metaphase chromosomes.
They are possibly best described as being composed of
numerous clustered, irregularly spaced runs of As and Ts
R-bands are known to replicate early, to contain most
housekeeping genes and are enriched in hyperacetylated
histone H4 and DNase I-sensitive chromatin.
This suggests they have a more open chromatin conformation,
consistent with a central AT-queue with longer loops that reach
the nuclear periphery.
In contrast, Q-bands contain fewer genes and
are proposed to have loops that are shorter and more tightly
folded, resulting in an AT-queue path resembling a
coiled spring.
Zigzag model of the 30-nm chromatin fiber
 The bending of DNA in a nucleosome
1. Flexibility of DNAs: A-T riched minor groove inside and G-C
riched groove outside
2. DNA bound protein can also help
Nucleosome Structures
Histone octamer
2 H2A
2 H2B
2 H3
2 H4
Structural Organization of the Core Histones
The Assembly of the Core Histones
Notice the long tails of the octamer
 Irregularities in the 30-nm fiber
Flexible linker, DNA binding proteins
Structural modulators: H1 histone, ATP-driven Chromatin
remodeling machine, covalent modification of histone tails
 Chromatin Remodeling
1. In eukaryotes, binding of histones to form chromatin generally represses
gene expression, making specific repressor proteins unnecessary.
2. Evidence for the role of chromatin structure includes:
a. Increased sensitivity to DNaseI of transcriptionally active genes.
b. Hypersensitive DNaseI digestion sites upstream of transcription start sites,
corresponding to promoter regions.
c. In vitro experiments showing directly that histones can repress gene
expression.
i. If DNA is simultaneously mixed with both histones and promoter-binding
proteins, it binds more readily to the histones, forming nucleosomes at the
TATA box and preventing transcription.
ii. If DNA is first mixed with promoter-binding proteins, adding histones does
not produce nucleosomes, and transcription occurs.
iii. If DNA is simultaneously mixed with histones, binding proteins:
(1) Enhancer-binding proteins bind the enhancer sequences.
(2) Promoter-binding proteins bind the promoter sequences.
(3) Histones are unable to bind, and so transcription occurs.
d. Histones, therefore, are effective repressors, but other proteins
can overcome that repression.
 Activating Genes by Remodeling Chromatin
1. Activation of eukaryotic genes requires alteration of the chromatin structure near the core
promoter, a process called chromatin remodeling. Two classes of protein complexes cause
chromatin remodeling (Figure 20.3):
a. Acetylating and deacetylating enzymes act on core histones. Histone acetyl transferases
(HATs) are part of multiprotein complexes recruited to chromatin when activators bind
DNA.
i. HATs acetylate lysines in the amino-terminus of core histones.
ii. The negative charges of acetyl groups decrease the positive charges of the histones,
reducing their affinity for DNA.
iii. Acetylation of histones changes 30-nm chromatin to 10-nm fiber, making promoter
more accessible for transcription.
iv. The effect is reversible. When histone deacetylases (HDACs) remove acetyl groups,
30-nm chromatin reforms.
b. Nucloesome remodeling complexes are ATP-dependent multiprotein complexes that alter
nucleosome positions on the chromatin in response to binding of activators to DNA,
increasing transcription. Different types of nucleosome remodeling complexes are known,
and some have more than one function:
i. Some slide a nucleosome along the DNA, exposing DNA-binding sites for proteins.
ii. Some restructure the nucleosome in place.
iii. Some transfer the nucleosome from one DNA molecule to another.
iv. An example is SWI/SNF, which can remodel using all three methods. Originally
discovered in yeasts, where it affects mating type switch and sucrose fermentation
pathways, this complex is now known in many eukaryotes, including mammals.
Fig. 20.3 Chropmatin modeling by (a) histone acetylases and (b)
nucleosome remodeling complexes
 Histone acetyl transferases bound
to activation domains

Decondensed chromatin structure

Chromatin decondensation appears to require two
types of protein complexes each made of several
polypeptide subunits:

1. Histone acetylase complexes.

These are often referred to as HATS for histone
acetylases.

2. Chromatin remodeling factors.

These are often refered to as Swi/Snf factors
because they were first identified as yeast mutants
defective in mating type switching and in the ability
to metabolize sucrose (sucrose non-fermenting).
Chromatin remodeling factors use energy from
ATP hydrolysis to rearrange the packing of
nucleosomes in higher order chromatin structures

There are several different chromatin remodeling

complexes in cells.

Some of these bind to activation domains and

de-condense the associated chromatin.

Some bind to repression domains and condense

the associated chromatin.
 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?
SWI/SNF sub family remodelers
can slide nucleosomes along
DNA

Ramachandran et al. (2003) J. Biol. Chem. 278(49):48590

 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….)
 SWI2/SNF2 ATPase BROMO
subfamily

ATPase SANT
ISWI
subfamily
SWI2/SNF2
ATPase
SUPERFAMILY ATPase
CHD/Mi2
subfamily CHROMO DNA binding

ATPase ATPase
Ino80
subfamily
 SWI2/SNF2 ATPase BROMO

Originally isolated subfamily

genetically in budding
yeast as mutants in mating BROMO
type switching and sucrose
non-fermenting functions
from Drosophila: Brahma
ATPase SANT
ISWI
subfamily SANT
SWI2/SNF2 SWI3 ADA2 N-CoR TFIIIB
ATPase
SUPERFAMILY ATPase
CHD/Mi2
subfamily CHROMO DNA binding

ATPase ATPase
Ino80
subfamily
Chromatin remodeling
complexes
SWI2/SNF2 subfamily ISWI subfamily

hBrm
 Variations in Human SWI/SNF Complexes

BRG1/BAF BRM/BAF PBAF

BAF170 BAF170
BAF57
BAF170
BAF60 BAF57 BAF60 BAF57 BAF60
a Actin Actin Actin
a a
BRG1 BRM BRG1 BAF180
BAF53a a Actin BAF53a BAF53a
BAF47 BAF250a BAF47 BAF250a BAF47 a&b
BAF155 BAF155 BAF155

EBAFa EBAFb

ENL ENL
EBAF70 BAF170 EBAF70
BAF170
BAF57 BAF60 BAF57 BAF60
a & b Actin a & b Actin
BAF53a BRG1 BAF53a BRG1
BAF47 BAF250a BAF47 BAF250b
BAF155 BAF155
EBAF140 EBAF140
EBAF100 EBAF100
Conservation of SWI/SNF
subfamily complexes
Protein domains in a SWI/SNF
complex
Binds DNA
(sequence Recognizes acetylated
dependant OR lysines in histone tails
independent)

Binds DNA
AND protein

Binds DNA
(sequence
dependant OR
independent)
Binds DNA and
maybe also
histone tails
what’s this
doing in the
nucleus???

dunno
Chromatin remodeling
complexes
SWI2/SNF2 subfamily ISWI subfamily
“Imitation
switch” –
isolated first
in
Drosophila
by homology
with
SWI/SNF
from yeast

hBrm
 A comparison of SWI/SNF and
ISWI subfamily ATPases

BRAHMA

ATPase somewhere in here Associates with

acetylated histones

ISWI

Once upon a time, a DNA

binding domain. Now?
 ISWI complexes are VERY
diverse
Nucleosome remodeling factor
Chromatin accessibility complex

ATP-dependent
chromatin assembly
and remodeling factor

Nucleolar remodeling complex

ISWI complexes are VERY
diverse Transcriptional activation of some
homeotic genes

involved in replication
of heterchromatin?
Bozhenok et al.
(2002)Embo J 21(9) 2231

keeping sister
chromatids
together during
mitosis
rDNA repression
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 didn’t show you
evidence but it’s 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!
Evidence for BRG1’s Role in
Human Tumor Development
- Mutations found in 10% of human tumor cell lines

- LOH in region surrounding BRG1 locus (19p13.2)

- BRG1+/- mice develop adenocarcinomas

- Expression lost in ~50% of NSCLC tumor cell lines, ~20% of

adenocarcinoma cell lines & 10% of primary tumors.

- Required for RB-mediated growth arrest

 Histone Deacetylases Histone Acetylases
Chromatin Remodeling Chromatin Remodeling
Complexes Complexes
Fig. 11-36
Fig. 11-37
Fig. 11-37
Fig. 11-37
Fig. 11-37
Fig. 11-37
Fig. 11-37
SWI/SNF-Mediated Chromatin Remodeling
BAF170
BAF57
BAF60
BAF53
BRG1
hSNF5
BAF155 BAF250

c-Myc
p53
SWI/SNF-Mediated Chromatin Remodeling

BAF170
BAF57
BAF60
BAF53
BRG1
hSNF5
c-Myc BAF155 BAF250
p53
SWI/SNF-Mediated Chromatin Remodeling

ATP
BAF170
BAF57 ADP+Pi
BAF60
BAF53
BRG1
hSNF5
c-Myc BAF155 BAF250
p53
SWI/SNF-Mediated Chromatin Remodeling

BAF170
BAF57
BAF60
BAF53
BRG1
hSNF5
c-Myc BAF155 BAF250
p53
 Exchanging histone types
• Octamer interrupted.
– In yeast, the transcriptionally silenced
chromatin of telomeres is formed by
the association of the Sir2, Sir3, and
Sir4 proteins (dark blue) with
nucleosomes.
– Replacement of H2A/H2B histone
dimers (yellow) with H2A.Z/H2B
dimers (red) containing the variant
histone protein H2A.Z prevents the
spread of Sir proteins into adjacent
regions of unsilenced chromatin.
– This histone replacement acts as a
buffer halting the spread of chromatin
silencing.
– The Swr1 complex mediates the
removal of H2A/H2B dimers from
nucleosomes and their replacement
with H2A.Z/H2B dimers.
 Cyclic Diagram for
nucleosome formation and
disruption
 Euchromatin versus
heterochromatin
• Euchromatin
– Open, genes inducible
– Modifications: histones hyperacetylated, H3-mK4
present, cytosine hypomethylation, irregular
nucleosome packing dispersed with HS (DNase
hypersensitive) sites
• Silenced chromatin = facultative chromatin
• Heterochromatin = constitutive heterochromatin
– Condensed, rich in repetitive DNA with low gene
density, regular nucleosome arrays, genes silenced,
histone hypoacetylated, H3 mK9 present, cytosine
hypermethylatoin
Heterochromatin vs.
euchromatin

adapted from Jenuwein & Allis (2001) Science 293, 1074

Characteristics of Hetero- and Eu-chromatin

Euchromatin Heterochromatin
(constitutive)
Staining Dispersed Condensed (speckles)

DNA sequence Predominantly unique Repetitive & gene poor

& gene rich

Replication Early to Late S-Phase Late S-Phase

Recombination Abundant Limited

Chromatin Irregular nucleosomes Regular nucleosomes

Accessible to nucleases Less accessible to nucleases
Hypersensitive Sites Few Hypersensitive Sites

Modifications H3-K4 methylation H3-K9 methylation

Low CpG methylation More CpG methylation
Anders H. Lund et al. Genes Dev. 2004; 18: 2315-2335
Cloned Members of the Pc-G and trx-G
Pc-G trx-G
Genetically SWI/SNF
subfamily members
isolated as activators of
(PRE/TRE) gene expression

Homeotic gene expression

Pc M33/MPc1, MPc2, MPc3 trx Mll, Mll2
ph Rae28/Mph1 trl
PRC1 Psc Mel18, Bmi-1 brm Brm, Brg1
Scm Scmh1, Scmh2 mo Baf155,
SWI/SNF
Pcl M96 Baf170
Pho YY1 osa Baf250
Su(z)2 Mel18, Bmi-1 snr1 Baf47/Snf5
dRING Ring1a, Ring1b z
E(z) Enx-1, -2/Ezh-1, -2 ash-1 Ash1l
PRC2 esc eed ash-2 Ash2l
Asx Asxl1, Asxl2 lid
crm kis
lawc
 Formation of heterochromatin
- interplay between three systems
• Histone deacetylation
Form together a self-
• Histone H3 K9 methylation
reinforcing network
• DNA methylation
 Histone hypoacetylation as a mark for
Heterochromatin and silent domains
• Hypoacetylation (H3 + H4) associated with
hetero-chromatin domains
• Facilitate repressive structures
• Model: the yeast SIR complex
– A repressive complex that interacts (Sir3
& Sir4) specifically with histone tails in
hypoacetylated form
– Sir2 is a NAD-dependent protein
deacetylase
– Recruitment, deacetylation and spreading
• TSA (HDAC inhibitor) causes relaxation of
silencing
 Methylation of H3 on K9 and assoc.
with HP1 mark heterochromatin

• Modification of H3 Lys9
- a molecular mark for
heterochromatin?
– Repression also
associated with
methylation of H3-K27
and H4-K20
T. Kouzarides
Dinshaw Patel
F.Ochsenein
 The « Histone Code » hypothesis

The residue and the type of modification define chromatine state:

Silencing
Me

NH2 ARTKQTARK9STGGKAPRQKQL----- H3

Transcriptional activation

me3 Ac

ARTK4QTARKSTGGK14APRQKQL----- H3
 Histone code translators

HP1 Chromodomain Silencing

Me

NH2 ARTKQTARK9STGGKAPRQKQL-----
H3

Swi/Snf Transcriptional
Bromodomain
activation
Ac

NH2ARTKQTARKSTGGK14APRQKQL----- H3
 Cytosine methylation patterns
as a mark for Heterochromatin
• Cytosine hypermethylation in
heterochromatin
– Patterns established &
maintained in a dynamic
process
• Enzymes: Dnmt1, 3a and 3b
• How patterns are generated??
- elusive
• Recent evidence suggest that
H3 mK9 heterochromatin code
may determine mC-DNA
pattern
– Cytosine methylation may
lay downstream of histone
methylation
Heterochromatin vs.
euchromatin
H3K4me

adapted from Jenuwein & Allis (2001) Science 293, 1074

Heterochromatin vs.
euchromatin
H3K9me

adapted from Jenuwein & Allis (2001) Science 293, 1074

 Regulation of homeotic genes

active homeotic gene H3K27me Repressed homeotic gene

adapted from Jenuwein & Allis (2001) Science 293, 1074

Spreading of heterochromatin
Spreading of heterochromatin
in flies (and humans!)

K9 of H3
(H3K9me)
 Methylated H3 K9 recognized
by a specific protein: HP1
• HP1 is bifunctional
reagent
– Heterochromatin
protein 1 (HP1) is a
methyl-lysine
binding protein
specific for H3-
mK9.
– HP1 recruits
SUV39H1 leading to
propargation of
methylation
Also used for local repression by
Rb, first deacetylation, then
methylation
Step 1: deacetylation Step 2: methylation
Spreading of silenced chromatin on
a homeotic gene

E(z)
(SET)
Polycomb Polycomb Polycomb
K27 of H3
H3K27me

Polycomb-complexed chromatin
Action of PcG and trxG complexes on chromatin
Nucleosome trxG
remodeling
Ac (BRM complex) ON Maintenance of
Histone active states
acetylation and (open
methylation
(TAC1 and Me K4 H3 chromatin)
ASH1
complexes)

Target
PRE gene

OFF PcG
Deacetylation and Me K27 H3
methylation Maintenance of
(ESC-E(Z) repressed states
complex) (compact
- Chromatin chromatin)
compaction Ub H2A
- H2A
Ubiquitination
(PRC1 complex)
 Histones post-translational modifications

B. Turner
S. Dent
Covalent Modification
of core histone tails
Acetylation of lysines
Methylation of lysines
Phosphorylation of
serines

Histone acetyl
transferase (HAT)
Histone deacetylase
(HDAC)
Fig. 20.15 Effect of 5-methylcytosine on cleavage of DNA with HpaII
and MspI
 Summary
• Histone code
– Heterochromatin - H3K9me3
– Repressed promoter - H3K27me3
– Active promoters - H3K4me3, H3Ac, H4Ac
The regulatory sequences in the
genome

Co-activator complex Initiation machinery Co-repressor complex

CTCF

Promoter
Enhancers Repressor Insulator
 Activation/Repression
ACTIVATION
CBP pCAF

SRC SRC chromosome remodeling

histone acetylation
SR SR TFII-B Transcription
TBP
 RNA
Pol.

REPRESSION
Sin3 mRPD3
Sin3 chromosome condensation
NCoR/SMRT histone deacetylation
SR SR
NO transcription
Structural Domains of Corepressors
1 305 783 ****** 2473
RD1 DAD RD2 RD3 RID1 RID2
•SMRT and NCOR are coregulators that activate histone
deacyetalases (HDACs)

•RD1-3: Transcription repression domains. Sites of

interaction and recruitment of HDACs. Some HDACs are
recruited directly( HDAC 3,4,5,7), others (HDAC1,2) indirectly
through Sin3.
•RID1,2: Steroid Receptor interaction domains (CoRNR
boxes)*** “IXXI/VI” similar to LXXLL but extended sequence
•DAD: deacetylase activation domain (HDAC3 is inactive until
it binds to SMART/NCOR and activation is dependent on DAD
domain)
 Gene Silencing and Telomeres
1. In gene silencing, a gene is not transcribed because of its location, rather than by the action of a
specific repressor. Heterochromatin is commonly involved in gene silencing, and affects large
sections of DNA.
2. An example is found in gene silencing at the yeast telomere (TEL) (Figure 20.13).
a. Yeast telomeres are telomere repeat sequences with complex hairpin structure, and no
protein-coding sequences.
b. If active genes are moved to the telomeres, they are silenced. This telomere position effect is
associated with binding of telomeres in groups to the nuclear envelope.
3. Active genes moved to the telomeres and thus silenced are used to find mutations that relieve
silencing, which define the SIR (silent information regulation) genes.
a. Rap1p (produced by the repressor-activator protein gene, RAP1) binds telomere repeat
sequences.
b. The bound Rap1p recruits the SIR silencing complex (SIR2p, SIR3p, SIR4p).
c. The SIR silencing complex contacts the histones, and Sir2p (a histone deacetylase) removes
acetyl groups from histone tails.
d. Deacetylated histones are recognized by the silencing complex, and a wave of binding and
deacetylation moves along the chromosome. Heterochromatin is generated.
e. The spread of silencing is stopped by methylation of histone H3 tails by histone methyl
transferases (HMTs).
Fig. 20.13 Gene silencing at a yeast telomere
 Yku70/Sir4

Sir3,4/Rap1 Rif1
Sir2 Rif2
Sir3 Sir4
Single strand DNA 3’ (G rich)
Rap1Rap1
Rap1Rap1
Rap1
Histones Yku70
Cdc13
H3/H4 C1-3A/TG1-3 Yku80
Rap1 binding sites

Tel1p
MRXcomplex