Chromatin Structure & Genome Organization

What is so special about chromosomes ?

1.They are huge:One bp = 600 dalton, an average chromosome is 107 bp long = 109- 1010 dalton ! (for comparison a protein of 3x105 is considered very big.

What is so special about chromosomes ?

1.They are huge:One bp = 600 dalton, an average chromosome is 107 bp long = 109- 1010 dalton ! (for comparison a protein of 3x105 is considered very big.

2. They contain a huge amount of non- redundant information (it is not just a big repetitive polymer but it has a unique sequence) .

• Philosophically - the cell is there to serve, protect and propagate the chromosomes.

• Practically - the chromosome must be protected at the ends - telomers

and it must have “something” that will enable it to be moved to daughter cells - centromers

Genome Complexity

Chromosome structure

• The DNA compaction problem• The nucleosome histones (H2A, H2B,

H3, H4)• The histone octamere • Histone H1 the linker histone• Higher order compactions• Chromatin loops and scaffolds (SAR)• Non histone chromatin proteins• Heterochromatin and euchromatin• Chromosome G and R bands• Centromeres

Lesson 2 - Chromosome structure

• The DNA compaction problem• The nucleosome histones (H2A,

H2B, H3, H4)• The histone octamere• Histone H1 the linker histone• Higher order compactions• Chromatin loops and scaffolds

(SAR)• Non histone chromatin proteins• Heterochromatin and euchromatin• Chromosome G and R bands• Centromeres

Chromosome is a compact form of the DNA that readily fits inside the cell

To protect DNA from damageDNA in a chromosome can be

transmitted efficiently to both daughter cells during cell division

Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression as well as recombination

Importance

• Take 4 meters of DNA (string) and compact them into

a ball of 10M. Now 10M are 1/100 of a mm and a

bit small to imagine – so now walk from here to the

main entrance let say 400 meters and try to compact

it all into 1 mm.

• This compaction is very complex and the DNA isn’t

just crammed into the nucleus but is organized in a

very orderly fashion from the smallest unit - the

nucleosome, via loops, chromosomal domains and

bands to the entire chromosome which has a fixed

space in the nucleus.

Chromatin The complexes between eukaryotic DNA and proteins are called 

chromatin, which typically contains about twice as much protein as DNA.

The major proteins of chromatin are the histones—small proteins containing a high proportion of basic amino acids (arginine and lysine) that facilitate binding to the negatively charged DNA molecule.

There are five major types of histones—called H1, H2A, H2B, H3, and H4—which are very similar among different species of eukaryotes .

The histones are extremely abundant proteins in eukaryotic cells; together, their mass is approximately equal to that of the cell's DNA.

chromatin• In addition, chromatin contains an approximately equal

mass of a wide variety of nonhistone chromosomal proteins. There are more than a thousand different types of these proteins, which are involved in a range of activities, including DNA replication and gene expression.

• Histones are not found in eubacteria (e.g., E. coli), although the DNA of these bacteria is associated with other proteins that presumably function like histones to package the DNA within the bacterial cell. Archaebacteria, however, do contain histones that package their DNAs in structures similar to eukaryotic chromatin

The nucleosome

Nucleosome & histone structures

The basic structural unit of chromatin, the nucleosome, was described by Roger Kornberg in 1974 . Two types of experiments led to Kornberg's proposal of the nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with proteins) yielded a continuous smear of randomly sized fragments. These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion, so that the enzyme can attack DNA only at sites separated by approximately 200 base pairs. Consistent with this notion, electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs. Thus, both the nuclease digestion and the electron microscopic studies suggested that chromatin is composed of repeating 200-base-pair units, which were called nucleosomes.

Discovery of nucleosomes

Nucleosomes

• DNA associated with individual nucleosomes determined by DNAse I digestion

(1) Nucleosomes are the building blocks of chromosomesThe nucleosom

e

The nucleosome is composed of a core of eight histone proteins and the DNA (core DNA, 147 bp) wrapped around them. The DNA between each nucleosome is called a linker DNA. Each eukaryote has a characteristic average linker DNA length (20-60 bp)

Figure 7-18 DNA packaged into nucleosome

Six-fold DNA compaction

Five abundant histones are H1 (linker histone, 20 kd), H2A, H2B, H3 and H4 (core histones, 11-15 kd).

The core histones share a common structural fold, called histone-fold domain

The core histones each have an N-terminal “tail”, the sites of extensive modifications

(2) Histones are small, positively charged (basic) proteinsThe nucleosom

e

Histonea Molecular weight

Number of amino acids

Percentage Lysine + Arginine

H1 22,500 244 30.8

H2A 13,960 129 20.2

H2B 13,774 125 22.4

H3 15,273 135 22.9

H4 11,236 102 24.5

The Major Histone Proteins

The core histones share a common structural fold

(1)(2)

(3) Many DNA sequence-independent contacts (?) mediate interaction between between the core histones and DNA

The nucleosome

(4) The histone N-terminal tails stabilize DNA wrapping around the octamer

The histone tails emerge from the core of the nucleosome at specific positions, serving as the grooves of a screw to direct the DNA wrapping around the histone core in a left-handed manner.

Lesson 2 - Chromosome structure

• The DNA compaction problem• The nucleosome histones (H2A, H2B,

H3, H4)• The histone octamere• Histone H1 the linker histone • Higher order compactions• Chromatin loops and scaffolds (SAR)• Non histone chromatin proteins• Heterochromatin and euchromatin• Chromosome G and R bands• Telomeres• Centromeres

Chromosome structure• The DNA compaction problem• The nucleosome histones (H2A, H2B, H3,

H4)• The histone octamere• Histone H1 the linker histone • Higher order compactions• Chromatin loops and scaffolds (SAR)• Non histone chromatin proteins• Heterochromatin and euchromatin• Chromosome G and R bands• Centromeres

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

Scaffold attachment regions

Compaction level of interphase chromosomes is not uniform

Euchromatin Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains

Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further

Heterochromatin vs Euchromatin

Types of Heterochromatin

Figure 10.20

• Constitutive heterochromatin– Always heterochromatic– Permanently inactive with regard to transcription

• Facultative heterochromatin– Regions that can interconvert between euchromatin

and heterochromatin– Example: Barr body

Most of the euchromatin in interphase nuclei appears to be in the form of 30-nm fibers, organized into large loops containing approximately 50 to 100 kb of DNA.

About 10% of the euchromatin, containing the genes that are actively transcribed, is in a more decondensed state (the 10-nm conformation) that allows transcription.

Chromatin structure is thus intimately linked to the control of gene expression in eukaryotes.

Dynamics of chromatin

The extent of chromatin condensation varies during the life cycle of the cell.

In interphase (nondividing) cells, most of the chromatin (called euchromatin) is relatively decondensed and distributed throughout the nucleus .

During this period of the cell cycle, genes are transcribed and the DNA is replicated in preparation for cell division.

Dynamics of chromatin

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.

The Molecular Structure of Centromeres and TelomeresThe Molecular Structure of Centromeres and Telomeres

Bhaumik, Smith, and Shilatifard, 2007.

Types of Histone Modifications

• Covalently attached groups (usually to histone tails)

• Reversible and Dynamic– Enzymes that add/remove modification– Signals

• Have diverse biological functions

Cell, 111:285-91, Nov. 1, 2002

Methyl Acetyl Phospho Ubiquitin SUMO

Features of Histone Modifications

• Small vs. Large groups

• One or up to three groups per residue

Features of Histone Modifications

Jason L J M et al. J. Biol. Chem. 2005

Ub = ~8.5 kDaH4 = 14 kDa

• Others: Sumoylation (Lysine), ADP Ribosylation (Glutamate)

Histone Modifications and Modifers

Residue Modification Modiying Enzyme

Lysine AcetylationDeacetylation

HAT HDAC

Lysine MethylationDemethylation

HMT HDM

Lysine UbiquitylationDeubiquitylation

Ub ligaseUb protease

Serine/Threonine PhosphorylationDephosphorylation

KinasePhosphatase

Arginine MethylationDemethylation

PRMTDeiminase/

Demethylase

General Roles of Histone Modifications• Intrinsic

– Single nucleosome changes– Example: histone variant specific modifications (H2AX)

• Extrinsic – Chromatin organization: nucleosome/nucleosome interactions– Alter chromatin packaging, electrostatic charge– Example: H4 acetylation

• Effector-mediated – Recruitment of other proteins to the chromatin via

• Bromodomains bind acetylation• Chromo-like royal domains (chromo, tudor, MBT) and PHD bind methylation• 14-3-3 proteins bind phosphorylation

Kouzarides, Cell 2007

- Prevent binding: H3S10P prevents Heterochromatin Protein 1 binding

Acetylation

• Many lysine residues can be acetylated• mainly on histone tails (sometimes in core)

• Can be part of large acetylation domains

• Modifying enzymes: • often multi-enzyme complexes • can modify multiple residues

• Well correlated with transcriptional activation

• Other roles (chromatin assembly, DNA repair, etc.)

• Two general types:• Type B: cytoplasmic (newly synthesized histones)

• HAT1

Kouzarides, Cell, 2007.

Histone Acetyl Transferases (HATs/KATs)

Type B

1. Opens up chromatin: – Reduces charge interactions of histones with DNA

(K has a positive charge)– Prevents chromatin compaction (H4K16ac prevents

30nm fiber formation)

2. Recruits chromatin proteins with bromodomains (SWI/SNF, HATs: GCN5, p300)

3. May occur at same residues as methylation with repressive effect (competitive antagonism)

Roles of Acetylation

Robinson et al., J. Mol. Biol., 2008.

Mujtaba et al., Oncogene, 2007.

PCAF

Yang and Chen, Cell Research, 2011.

H3K27ac

H3K27me3

4. Highly correlated with active transcription i.e. enriched at TSS of actively transcribed genes

Roles of Acetylation -- Continued

Expression: P1<P2<P3<P4

Heintzman N et al., Nature Genetics, 2007.

Roles of Acetylation

H4Ac H3Ac H3 RNAPII

208 TSS investigated

5. Correlated with binding of activating transcription factors i.e. enriched at promoters and enhancers

Roles of Acetylation

Heintzman N et al., Nature Genetics, 2007.

H4Ac H3Ac p300

Expression: E1<E2<E3 74 enhancers

(distal p300 binding sites)