Bio221

April 04, 2016

Chromatin and Epigenetics
Shelley Berger
University of Pennsylvania
CDB, Biology, Genetics
Epigenetics Program
 Outline
Chromatin structure
How are nucleosomes positioned?
How is chromatin regulated?
Histone variants
DNA methylation and demethylation
Histone chemical modifications
Introduce epigenetics
Epigenetic mechanisms:
mitotic memory
transgenerational memory
Human disease and epigenetic therapy
Chromatin Mechanisms

ATP-dependent
remodeling

Histone variants

Histone
modifications
DNA methylation
Chromatin
Closed
chromatin

Open
chromatin
 Outline
Chromatin structure
How are nucleosomes positioned?
How is chromatin regulated?
Histone variants
DNA methylation and demethylation
Histone chemical modifications
Introduce epigenetics
Epigenetic mechanisms:
mitotic memory
transgenerational memory
Human disease and epigenetic therapy
ATP-dpdnt remodeling enzymes
ATP-dpdnt remodeling enzymes
What are histone variants?
- Proteins highly structurally
related to the core histones
- Specific locations in
the genome
- Specialized functions in
genomic regulation
H3 variants
Consideration of following issues:
- sequence variation
- nucleosome structure
- genomic function
- location within the genome
H2A

H2B

H3

H4
 Gene
activation
H2A

H2B
Gene
activatio
n
H3

H4
H2A

Gene repression
X chrom inact
H2B

H3

H4
 DNA repair

H2A

H2B

H3

H4
H2A

H2B

H3

H4 Centromere
identity
 Histone H3.1 and H3.3

H3/H3.1
H3.3

H4
H3.3 is very similar to H3.1 in sequence
 What genomic process does
H3.3 regulate?

during outside of
Replication: Replication:
either only H3.3
H3.1 or H3.3
(mostly H3.1)
 What genomic process does
H3.3 regulate?

during outside of
Replication: Replication:
either only H3.3
H3.1 or H3.3 made
(mostly H3.1)
 H3.3 localizes with RNAPII at genes

H3.3

H3.1

RNAP

genes
 H3.3 localizes with RNAPII at genes

H3.3

H3.1

RNAP

genes
 H3.3 localizes with RNAPII at genes

H3.3

H3.1

RNAP

genes
 H3.3 localizes with RNAPII at genes

H3.3

H3.1

RNAP

genes
Histone H3.3 and H2AZ Variants
Located at specific genomic sites
- H3.1 all over genome
- H3.3 at genes
- CENPA at centromeres
Have specific genomic functions:
- H3.1 standard nucleosomes
- H3.3 gene activation
(both H2A.Z and H3.3 destabilize)
- CENPA at centromere
(spindle attachment)
 Histone CENPA

H3
CENPA
H4 Centromere
identity
What are centromeres?
What are centromeres?
Centromeric
DNA from
lower to

Sequence complexity
higher
eukaryotes
CENPA, an
H3 variant,
is in a

Sequence complexity
specialized
NCP at
centromeres
in all
eukaryotes
What is it about CENPA
compared to H3 that
localizes CENPA
specifically to
centromeric DNA?
Targeting of CENPA to centromeres

Mapping in vitro and

in vivo defined a
region in the histone
fold as CATD
Targeting of CENPA to centromeres

Mapping in vitro and

in vivo defined a
region in the histone
fold as CATD
Targeting of CENPA to centromeres

Mapping in vitro and

in vivo defined a
region in the histone
fold as CATD
Targeting of CENPA to centromeres

Mapping in vitro and

in vivo defined a
region in the histone
fold as CATD
Summary H3 Histone Variants
Located at specific genomic sites
- H3.1 all over genome
- H3.3 at genes
- CENPA at centromeres
Have specific genomic functions:
- H3.1 standard nucleosomes
- H3.3 gene activation
(both H2A.Z and H3.3 destabilize)
- CENPA at centromere
(spindle attachment)
 Outline
Chromatin structure
How are nucleosomes positioned?
How is chromatin regulated?
Histone variants
DNA methylation and demethylation
Histone chemical modifications
Introduce epigenetics
Epigenetic mechanisms:
mitotic memory
transgenerational memory
Human disease and epigenetic therapy
DNA Methylation
 Eukaryotic DNA methylation
Cytosines are methylated at
CG or CNG dinucleotides

Function:
Generally is repressive to transcription
Reduces DNA binding of many proteins
Condenses chromatin structure
Binding site for methyl binding proteins
De Novo vs. Maintenance
DNA methylation

De Novo methylation

Maintenance methylation
DNA Methyltransferases
 DNMT enzymes establish
and maintain DNA methylation
Hemimethylated
CH3
DNMT3a/b
CG CG
GC GC
De novo

CH3

CG
GC Methylated
CH3
 DNMT enzymes establish
and maintain DNA methylation
Hemimethylated Methylated
CH3 CH3
DNMT3a/b DNMT1
CG CG CG
GC GC GC
De novo Maintenance CH3
 DNMT enzymes establish
and maintain DNA methylation
Hemimethylated Methylated
CH3 CH3
DNMT3a/b DNMT1
CG CG CG
GC GC GC
De novo Maintenance CH3

Epigenetic Memory:
Pathway to reinstate a chromatin state
in newly synthesized DNA
 Where is DNAme in the genome?
Bisulfite Mutagenesis and Sequencing
 Where is DNAme in the genome?
Bisulfite Mutagenesis and Sequencing
 Where is DNAme in the genome?
Bisulfite Mutagenesis and Sequencing
Genome-wide mapping of
DNAme in human cells
Genome-wide mapping of
DNAme in human cells
General roles
- regulation of gene expression

- cell differentiation: high in ESCs, neurons

- parental genome regulation/imprinting

- X inactivation in mammals

- defense against transposons, viruses,

counteracts recombination of repetitive DNA

- aging and cancer

How does CpG methylation
interfere with transcription?

1. Direct interference with transcription:

DNA binding transcription factors
cannot bind to their sites

2. Indirect interference with transcription:

Methyl binding proteins bring in
repressive activities
DNA Methylation

Bird, GenesDev, 2002

How does CpG methylation
interfere with transcription?

1. Direct interference with transcription:

DNA binding transcription factors
cannot bind to their sites

2. Indirect interference with transcription:

Methyl binding proteins bring in
repressive activities
Methyl-CpG-Binding proteins

Bogdanovic, Chromosoma, 2009

 Methyl-CpG-binding proteins
MeCP2: first identified has MBD (methyl binding domain),
binds mSin3a (HDAC)

MBD1: interacts with SETDB1 (H3K9 HMT)

MBD2 : in MeCP1 complex:

1: heterochromatin, binds HP1, Suv39, p150 (CAF1)
2: also in NuRD (HDAC)

MBD3: NuRD complex component

MBD4: DNA glycosylase; Cme-T repair

KAISO: no MBD, binds methylated DNA via Zn-finger

Important for amphibian development
DNA Methylation

Bird, GenesDev, 2002

General roles
- regulation of gene expression

- cell differentiation: high in ESCs, neurons

- parental genome regulation/imprinting

- X inactivation in mammals

- defense against transposons, viruses,

counteracts recombination of repetitive DNA

- aging and cancer

General roles
- regulation of gene expression

- cell differentiation: high in ESCs, neurons

- parental genome regulation/imprinting

- X inactivation in mammals

- defense against transposons, viruses,

counteracts recombination of repetitive DNA

- aging and cancer