Chromatin Structure Chromatin Structure & Dynamics& Dynamics

Victor JinDepartment of Biomedical InformaticsThe Ohio State University

Chromatin Walther Flemming first used the term Chromatin in 1882. At that

time, Flemming assumed that within the nucleus there was some kind of a nuclear-scaffold.

Chromatin is the complex of DNA and protein that makes up chromosomes.

Chromatin structure: DNA wrapping around nucleosomes – a

“beads on a string” structure.

In non-dividing cells there are two types of chromatin: euchromatin and heterochromatin.

Chromatin Fibers

30 nmchromatin fiber

11 nm(beads)

Chromatin as seen in the electron microscope. (source: Alberts et al., Molecular Biology of The Cell, 3rd Edition)

The basic repeating unit of chromatin.

It is made up by five histone proteins: H2A, H2B, H3, H4 as core histones and H1 as a linker.

It provides the lowest level of compaction of double-strand DNA into the cell nucleus.

It often associates with transcription.

Nucleosome

H2A H2BH3

H4

1974: Roger Kornberg discovers nucleosome who won Nobel Prize in 2006.

Core Histones are highly conserved proteins - share a structural motif called a histone fold including three α helices connected by two loops and an N-terminal tail

Histone Octamer

Each core histone forms pairs as a dimer contains 3 regions of interaction with dsDNA; H3 and H4 further assemble tetramers. The histone octamer organizes 146 bp of DNA in 1.65 helical turn of DNA: 48 nm of DNA packaged in a disc of 6 x 11nm

< 6 nm >

<

11

nm

>

Nucleosome Assembly In Vitro

4 core histones + 1 naked DNA template at 4C at 2M salt concentration, from Dyer et al, Methods in Enzymology (2004), 375:23-44.

DNA compaction compaction in a human cell nucleus

1bp (0.3nm)

10,000 nm

30nm

11 nm

The N-terminal tails protrude from the core

Histone Modifications

Me

P

Ub

Su

Ac Me

Acetylation

Methylation

Ubiquitination

Sumoylation

Phosphorylation

‘Histone Code’

Acetylation of LysinesAcetylation of the lysines at the N terminus of histones removes positive charges, thereby reducing the affinity between histones and DNA.

This makes RNA polymerase and transcription factors easier to access the promoter region. 

Histone acetylation enhances transcription while histone deacetylation represses transcription.

Methylation of Arginines and Lysines

Arginine can be methylated to form mono-methyl, symmetrical di-methyl and asymmetrical di-methylarginine.

Lysine can be methylated to form mono-methyl,

di-methyl and tri-methylarginine.

Methylation of Histone H3-K27

K27

PCDNMT

SUZ12HDACEED

EZH2

Functional Consequences of Histone Modification

Establishing global chromatin environment, such as Euchromatin, Heterochromatin and Bivalent domains in embryonic stem cells (ESCs).

Orchestration of DNA-based process transcription.

Euchromatin

A lightly packed form of chromatin; Gene-rich; At chromosome arms; Associated with active transcription.

Heterochromatin

A tightly packed form of chromatin; At centromeres and telomeres; Contains repetitious sequences; Gene-poor; Associated with repressed transcription.

Bivalent Domains

Poised state. The chromatin of embryonic stem cells has “bivalent” domains with marks of both gene activation and repression. In these domains, the tail of histone protein H3 has a methyl group attached to lysine 4 (K4) that is activating and a methyl group at lysine 27 (K27) that is repressive (above). This contradictory state may keep the genes silenced but poised to activate if needed. When the cell differentiates (right), only one tag or the other remains, depending on whether the gene is expressed or not.

DNA Methylation

5-methylcytosine

S-adenosylmethionine

DNA methyltransferase

deoxycytosine

N

N

O

OH H

-OO

N

N

N

O

OH H

-OO

NCH3

CpG Islands

CpG island: a cluster of CpG residues often found near gene promoters (at least 200 bp and with a GC percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 0.6).

~29,000 CpG islands in human genome (~60% of all genes are associated with CpG islands)

Most CpG islands are unmethylated in normal cells.

Mark Transcriptionally relevant sites Biological RoleMethylated

cytosine(meC)

CpG islands Transcriptional Repression

Acetylated lysine (Kac)

H3 (9,14,18,56), H4 (5,8,13,16), H2A, H2B

Transcriptional Activation

Phosphorylated serine/threonine

(S/Tph)

H3 (3,10,28), H2A, H2B Transcriptional Activation

Methylated argine (Rme)

H3 (17,23), H4 (3) Transcriptional Activation

Methylated lysine (Kme)

H3 (4,36,79)H3 (9,27), H4 (20)

Transcriptional Activation

Transcriptional Repression

Ubiquitylated lysine(Kub)

H2B (123/120)H2A (119)

Transcriptional Activation

Transcriptional Repression

Sumoylated lysine (Ksu)

H2B (6/7), H2A (126) Transcriptional Repression

Chromatin modifications

Genome-wide Distribution Pattern of Histone Modification Associated with Transcription

Li et al. Cell (review) 128, 707-719Source: Li et al. Cell (Review, 2007), 128:707-719

EpigeneticsModifications of DNA (cytosine methylation) and proteins (histones) define the epigenetic profile.

In 1942, Conrad Waddington first used “epigenetics” to describe the interactions between genome and environment that give rise to differences between cells during embryonic development.

Currently, Epigenetics is the study of heritable changes in gene function that occur without a change in DNA sequence.

Summarizes mechanisms and phenomena that affect the phenotype of a cell or an organism without affecting the genotype.

Epigenomics is the study of these epigenetic changes on a genome-wide scale.

Normal Cellular Functions Regulated by Epigenetic Mechanisms

Correct organization of chromatin Genomic imprintingSilencing of repetitive elementsX chromosome inactivation

X-chromosome Inactivation

Source: Jones et al. Nat.Genet. 19, 187 (1998)

X-inactivation (also called lyonization) is a process by which one of the two copies of the X chromosome present in female mammals is inactivated.

The inactive X chromosome is silenced by packaging in repressive heterochromatin.

The choice of which X chromosome will be inactivated is random in higher mammals such as mice and humans. Once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell.

Silencing initiated at Xic/XIC and spreads along chromososme.

5meC CpG DNA modification is observed in inactivated X chromosomes.

5meC binds transcriptional repressor MeCP2 (MethylC-binding Protein-2).

MeCP2 binds Sin3 with RPD3 histone deacetylase.transcriptional repressor

Histone DeacetylaseSin3

RPD3

MeCP25’..pCpGp..3’me

5

3’..pGpCp..5’5me

co-repressor

Epigenetic Diseases

Some human disorders such as Angelman syndrome and Prader-Willi syndrom are associated with genomic imprinting.

Involvement in cancer and development abnormalities.

The emerging hypothesis of cancer stem cells (CSC).

DNA Methylation and Gene Silencing in Cancer Cells

1 32 4

1 2 3 4

X

CGCG CG CG CG MCG

MCGNorma

l

Cancer

CG CG CGMCG

MCG

MCG

MCG

C: cytosinemC: methylcytosine

CpG island

Normal Cancer

Region-Specific Hypermethylatio

n

Accumulation of

Epigenetic Abnormalities

Global Hypomethylation

+

Progressive Alterations in DNA Methylation in Cancer

DNMT

Histone-modifying Proteins

Methyl-Binding Domain Proteins

Polycomb Repressors

Epigenetic Mediation of Gene Silencing

CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

Normal Epithelia Dysplasia Carcinoma

in situ

Carcinoma

Metastasis

1

2

3

4

5

Normal Epithelia Dysplasia Carcinoma

in situ

Carcinoma

Metastasis

1

2

3

4

5

CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

Normal Epithelia Dysplasia Carcinoma

in situ

Carcinoma

Metastasis

1

2

3

4

5

CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

Normal Epithelia Dysplasia Carcinoma

in situ

Carcinoma

Metastasis

1

2

3

4

5

CpG Island Methylation: A Stable, Heritable and Positively Detectable Signal

Epigenetic Alterations in Cancer Stem Cells

Cancer Stem Cells: Stem cells arising through the malignant transformation of adult stem cells.

Cancer Stem Cells Hypothesis: Cancer stem cells are the main driving force behind tumor proliferation and progression.

Hallmarks of Cancer Stem Cells

A cell residing in a tumor that – 1. has a capacity to remain in an undifferentiated state 2. has properties of asymmetric divisions and self-renewal 3. has metastatic and repopulation capacities at specific niches

(microenvironment) in the body4. gives rise to a tumor that is histologically identical to the one

from which the CSC is derived

The Evidence of Cancer Stem Cells

First isolated from the patients of acute myeloid leukemia in 1997 by John Dick and colleagues at the University of Toronto.

Isolated from two solid tumors, breast and brain cancers.

~1% cancer cells may be really cancer stem cells.

More ChIP-chipStep 1: Rapid fixation of cells chemically cross-links DNA binding proteins to their genomic targets in vivo.

Step 2: Cell lysis releases the DNA-protein complexes, and sonication fragments the DNA.

Step 3: Immunoprecipitation (IP) purifies the protein-DNA fragments, with specificity dictated by antibody choice.

Step 4: Hydrolysis reverses the cross-links within the released DNA fragments.

Step 5: PCR amplification of ChIP DNA

Step 6: PCR amplification on a known binding-site region for that protein will need to be performed using either conventional PCR methods followed by agarose gel electrophoresis or by quantitative PCR.

Step 7: Labeling pool of protein-DNA fragments.

Step 8: Hybridization of DNA onto microarrays featuring 60-mer oligonucleotide probes.

Major types of array platforms

NimbleGen Arrays: tiling arrays, promoter arrays, whole

genome arrays.

(http://www.nimblegen.com/products/chip/index.html)

Agilent Arrays: promoter arrays, whole genome arrays.

(http://www.chem.agilent.com/Scripts/Phome.asp)

Affymetrix Arrays: tiling arrays, Chr21,22 arrays, whole

genome arrays.

(http://www.affymetrix.com/index.affx)

Measurement of intensity of probes on the array

The hybridized arrays were scanned on an Axon GenePix 4000B scanner (Axon Instruments Inc.) at wavelengths of 532 nm for control (Cy3), and 635 nm (Cy5) for each experimental sample. Data points were extracted from the scanned images using the NimbleScan 2.0 program (NimbleGen Systems, Inc.). Each pair of N probe signals was normalized by converting into a scaled log ratio using the following formula:

•Si = Log2 (Cy5l(i) /Cy3(i))

Confirming on a known target

Different antibodies to same factor

Antibodies to different family members

siRNA-ChIP

Antibodies to two components of a complex

Antibodies to an enzyme/modification pair

Antibody Validation

Confirming on a known target

Comparison of biological replicates and antibodies to different E2Fs

Loss of E2F6 ChIP signal after knockdown of E2F6 siRNA

•Promoter 1 •Promoter 2

Reproducibility of promoter arrays using biological replicates

•Top 1000 overlap

•Top 1000 overlap

•H3me3K27; Ntera2 cells

•500 kb region of chromosome 6

•500 kb region of chromosome 1

Amount of Sample Per ChIP

Number of cells Chromatin input

ChIP output

1x107 200 µg 150 ng

1x106 20 µg 10 ng

5x105 10 µg 1.3 ng

1x105 2 µg 300 pg

1x104 200 ng 30 pg

Amount of Sample Per ChIP

Number of cells Chromatin input

ChIP output

1x107 200 µg 150 ng

1x106 20 µg 10 ng

5x105 10 µg 1.3 ng

1x105 2 µg 300 pg

1x104 200 ng 30 pg

•Standard ChIP Protocol (1x107 cells; WGA2)

• Promoter Arrays

• Genome Tiling Arrays

•MicroChIP Protocol (10,000-100,000 cells; WGA4)

• Promoter Arrays

• Genome Tiling Arrays

Miniaturization

Reproducibility of MicroChIP Protocol