SHER-E-KASHMIR UNIVERSITY OF AGRICULTURAL SCIENCE AND TECHNOLOGY OF

KASHMIRDivision of Biotechnology, Faculty of Veternary Sciences and Animal Husbandry,

Shuhama-Srinagar.

A Lecture on

STRUCTURE AND ORGANIZATION OF

CHROMATIN

Presented by

ZAFAR IQBAL BUHROO(Research Scholar)

CONTENTS

PARTICULARS Page No.

1. INTRODUCTION

2. ULTRASTURCUTRE OF CHROMATIN

2.1. Multistrand Model

2.2. Folded Fibre Model

2.3. Nucleosome Model

3. ORGANIZATION OF CHROMATIN

3.1. The Nucleosome and “Beads on String”

3.2. 30 nm Chromatin Fibre

3.3. Higher level of DNA packing into metaphase-chromosome

4. TYPES OF CHROMATIN

4.1. Euchromatin

4.2. Heterochromatin

4.2.1. Constitutive Heterochromatin

4.2.2. Facultative Heterochromatin

5. COMPOSITION OF CHROMATIN

5.1. DNA

5.2. Histones

5.3. Non-Histones

6. FUNCTIONS OF CHROMATIN

7. CONCLUSION

REFERENCES

STRUCTURE AND ORGANIZATION OF CHROMATIN

1. INTRODUCTION

The nucleus is the heart of the cell, which serves as the main distinguishing

feature of the eukaryotic cells. It is an organelle submerged in its sea of turbulent

cytoplasm which has the genetic information encoding the past history and future

prospects of the cell. Nucleus contains many thread like coiled structures which remain

suspended in the nucleoplasm which are known as chromatin substance.

Chromatin is the complex combination of DNA and proteins that makes up

chromosomes. It can be made visible by staining with specific techniques and stain (thus

the name chromatin which literally means colored material). The major proteins involved

in chromatin are histone proteins; although many other chromosomal proteins have

prominent roles too. The functions of chromatin is to package DNA into smaller volume

to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and to serve as a

mechanism to control gene expression and DNA replication.

Chromatin is thus, the mixture of DNA and proteins present in an organized

manner in the chromosomes (Fig. 1).

2. ULTRASTRUCTURE OF CHROMATIN

The field of ultrastructure of chromatin is still the area where electron microscope

has failed to provide us a clear picture of the organization of DNA in chromatin. For the

study of chromosomes with the help of electron microscope, whole chromosome mounts

as well as sections of chromosome were studied. Such studies demonstrated that

chromosomes have very fine fibrils having a thickness of 2nm. Since, DNA is 2nm wide,

there is possibility that a single fibril corresponds to a single DNA molecule.

Fig.1. Chromatin and condensed structure of chromosome

Various workers have proposed different models to describe the organization of DNA in

the chromosomes. Three such models of chromosome structure are Multi-stranded model

and folded fibre model

2.1. Multi-stranded model

According to this model each chromatin fibre is on an average 100 Ao in diameter.

Each chromatin fibre is composed of 2 strands. Each strand is 35-40Ao in diameter. Each

strand consists of a single double helix structure (The two are separated by 25 Ao). Four

chromatin fibers (each composed of 2 DNA double helix) coil around each other to form

a Quarter chromatid. Quarter chromatid is the smallest sub-unit of the chromosome (400

Ao). Quarter chromatid givesrise to half chromatid (800 Ao). Half chromatid is made up

of 16 DNA double helices. Two half chromatids coil around each other to produce one

chromatid, which is 1600 Ao in diameter and made up of 32 DNA double helices. Thus a

chromosome has 64 DNA double helices, would be 3200 Ao diameter .

2.2 Folded fibre model

A popular model was proposed by E.J. Dupraw in 1965. According to this model,

the chromosome is composed of tightly folded fibre which has a diameter of 200-300 Ao.

Each chromosome fibre contains only one DNA double helix which is in a coiled state.

This DNA coil is coated with histones and non-histone proteins. Folding of the chromatin

fibres drastically reduces their length and at the same time markedly increases their

thickness and stainability. This folded structure normally undergoes super coiling which

further reduces the length and thickness of the chromosome. This is the most popular

model.

2.3 Nucleosome model

This model was proposed by R.D. Kornberg (1974). According to this model

DNA is tightly bound to histone proteins which serve to form a repeating array of DNA-

Protein particles called Nucleosome. This is the most significant and widely accepted

model.

3. ORGANIZATION OF CHROMATIN

Any model of chromatin fibre structure has to account for packing of DNA. The

basic structure shows three levels of organization of chromatin in the chromosome.

i) DNA wrapping around “Nucleosomes” – “the beads on a string” structure.

ii) A 30 nm condensed chromatin fibre consisting of nucleosome arrays in their

most compact form.

iii) Higher level of packing into the metaphase chromosome.

These three levels of organization are illustrated in Fig. 2 and explained below.

Fig.2 Structural organization of chromatin

3.1 The nucleosome and “Beads on string” structure

The first level of packing involves the binding of the chromosomal DNA to

histones. In eukaryotes, DNA is tightly bound to form a repeating array of DNA-protein

particles called nucleosomes. Histones play a crucial role in packing this very long DNA

molecule in an orderly way into nucleus which is only a few micrometers in diameter.

Thus, nucleosomes are the fundamental packing unit particles of the chromatin and give

chromatin a “beads on string” appearance in the electron micrographs. Each nucleosome

bead is separated from the next by a region of linker DNA (Fig.3).

There are five main types of histones called H1, H2a, H2b, H3 and H4. Histones are

very basic proteins, about 25% of their amino acids are lysine and arginine. So histones

have a large number of positively charged amino acid side chains. Their positively

charged groups therefore bind to the negatively charged phosphate groups of DNA.

Fig.3. Nucleosome with histone H1

The nucleosome beads can be removed from the DNA string by digestion with

enzymes that degrade DNA such as bacterial enzyme-micrococcal nuclease. After

digestion for a short period of time with micrococcal nuclease, only the DNA between

the nucleosome beads (linker DNA) is degraded. The rest is protected from digestion and

remains as double stranded DNA. Fragments 146 base pairs long bound to a specific

complex of eight nucleosome histones (the histone Octamer) (Fig 4).

Each nucleosome is a disc shaped particle with a diameter of about 11 nm and a

length of 5.7 nm. It consists of a core histones around which DNA is wound. The core

consists of two discs arranged in parallel, each composed of four histone molecules one

each of H2a, H2b, H3 and H4. The DNA molecule runs along the rim of the discs and a

molecule of H1 sits on the outside of the nucleosome complex acting as a seal; 146 base

pairs of DNA are associated with nucleosome core. Each nucleosome is separated from

the next by a region of linker DNA. The length of the linker between nucleosomes varies

between species, in humans it is about 60 base pairs giving a total length of DNA per

nucleosome of 200 base pairs. Generally DNA makes two complete turns around the

histone Octamer and these two turns (200 base pairs long) are sealed by H1 molecule.

Thus, on an average, nucleosome repeats at intervals of about 200 nucleotides or base

pairs. This is the basic level of packing of DNA in chromatin.

Fig. 4. Histone Octamer (a nucleosome)

3.2 30 nm chromatin fibre

When nuclei are very gently lysed on to the electron microscope grid, most of the

chromatin is seen to be in the form of a fibre, with a diameter of 30 nm. This diameter is

larger than a single nucleosome and suggests that the nucleosomes are organized into a

higher order structure. The 30 nm fibre consists of closely packed nucleosomes. It

probably arises from the folding of the nucleosome chain into a solenoid structure having

about six nucleosomes per turn (Klug and Coworkers 1976-85).

The fibre is formed by a histone H1 molecule binding to the linker DNA of each

nucleosome at the point where it enters and leaves the nucleosome. The histone H1

molecules interact with each other, pulling the nucleosomes together. Thus, H1 molecules

are found responsible for packing nucleosomes into 30 nm fibres. The H1 histone

molecule has an evolutionary conserved globular core or central region linked to

extended amino acid terminal and carbonyl terminal ‘arms’ whose amino acid sequences

has evolved much more rapidly. Each H1 molecule binds through its globular portion to a

unique site on a nucleosome and has arms that are thought to extend to contact with other

sites the histone cores of adjacent nucleosomes, so that the nucleosomes are pulled

together into a regular repeating array, thus giving the chromatin 30 nm fibre structures.

The binding of H1 molecule to chromatin tends to create a local polarity that a chromatin

otherwise lacks (Fig.5).

Fig.5 30 nm chromatin fibre showing solenoid structure

Fig.6. Organization of chromatin in chromosome

3.3 Higher level of DNA packing into the metaphase chromosome

Increasing levels of packing are observed within the nucleus. The highest level of

packing is found in chromosomes at the metaphase stage of cell division.

In an experiment, the histones are removed from the metaphase chromosome by

adding poly anion dextrin sulphate. Histone depleted chromosomes are found to have a

central core of scaffold surrounded by a hallow mode of loops of DNA. The scaffold is

made up of non-histone proteins and retains the general shape of the metaphase

chromosome. Each chromosome has two scaffolds, one for each chromatid and

connected together at the centromere region. When the histones are removed, the DNA

which has packed about 40 folds in the 30 nm chromatin becomes extended and produces

loops with an average length of 25 m with 15,000 base pairs. In each loop the DNA

exists from the scaffold and returns to an adjacent point. On the basis of these

observations, a model of chromosome structure was prepared by Lammli and Coworkers

(1979-1904).

In Lammli’s radial loop model, DNA is arranged in loops anchored to non-histone

scaffold. Because the lateral loops have 25 m DNA, after contracting 40 folds into 30

nm fibre, they would be only about 0.6 m long, a length consistent with the diameter of

metaphase chromosome (1 m) shows how the chromatin is arranged so that the base of

the loop forms a scaffold in the center of the chromatid.

Thus, in the early stage of cell division the chromatin strands become more and

more condensed. They cease to function as accessible genetic material (transcription

stops) and become a compact transportable form. This compact forms makes the

individual chromosomes visible, and they form the classic four arm structure, a pair of

sister chromatids attached to each other at the centromere. During division long

microtubules attach to the centromere at the opposite ends of the cell. The microtubules

then pull the chromatids apart, so that each daughter cell inherits one set of chromatids.

Once the cells have divided the chromatids are uncoiled and can function again as

chromatin. Inspite of their appearance, chromosomes are highly condensed, which

enable these giant DNA structures to be contained with in a cell nucleus.

Fig.4. Higher Levels of DNA packing in Chrosome

4) TYPES OF CHROMATIN

Two distinct types of chromatin have been distinguished depending on their

staining properties as Euchromatin and Heterochromatin

4.1 Euchromatin

It is the lightly packed form of chromatin that is rich in gene concentration. This

chromatin takes up light stain and represent most of the chromatin, that disperse after

mitosis has completed. Euchromatin consists of structural genes which replicate and

transcribe during G1 and S phase of the interphase. Euchromatin is considered

genetically active chromatin, since it has a role in their phenotypic expression of the

genes. In euchromatin, DNA is found packed in 3-8 mm fibre. During metaphase it takes

up dark stain.

4.2 Heterochromatin

It is a tightly packed form of chromatin that takes up deep stain during interphase

and prophase but metaphase takes up light stain. Chromomeres, centromeric regions, and

knobs also take up dark staining, of which centromeric regions and knobs are the true

Heterochromatic. (chromomeres are transcribed so not true H.C.). IN the chromosomes

all the centromeres fuse to form a long Heterochromatic mass called chromocentre.

Heterochromatin consists of highly repetitive DNA sequences. It is late replicating during

the s-phase of the cell and is not transcribed.

Heterochromatin has been further classified into two types: Constitutive

heterochromatin and Facultative heterochromatin.

4.2.1 Constitutive heterochromatin

In such a heterochromatin, the DNA is permanently inactive and remains in the

condensed state throughout the cell cycle. This most common type of heterochromatin

occurs around the centromere, in the telomeres and in the C-bands of the chromosomes. It

takes up deep stain.

Constitutive heterochromatin contains short repeated sequences of DNA called

satellite DNA. This DNA is called satellite DNA because upon ultra centrifugation, it

repeats from the main component of DNA.

4.2.2 Facultative Heterochromatin

This is essential euchromatin that has undergone heterochromatinization. It is not

permanently maintained in the condensed state instead it undergoes periodic dispersal

when ever it becomes transcriptionally active.

Frequently in facultative heterochromatin, one chromosome of the pair becomes

either totally or partially heterochromatic. An example of facultative heterochromatin is

x-chromosome inactivation in female mammals; one x-chromosome is packaged in

facultative heterochromatin and silenced, while the other x-chromosome is packaged in

euchromatin and is expressed. The silenced chromosome is inactive and forms at

interphase-the sex chromatin or Barr body (named after Murray L. Barr). Bar body

contains DNA which is not transcribed and is not found in males.

5. CHEMICAL COMPOSITION OF CHROMATIN

Chromatin is composed of 20-40% of DNA, 50-65% of proteins and 05-10% of

RNA. They vary from species to species and also among the tissue of the same species

5.1 DNA of Chromatin

DNA is the most important chemical constituent of chromatin, since it plays the

central role of controlling heredity. The most convenient measurement of DNA is

picogram.

C-value: The DNA in nuclei was stained using the fulgen reactions and the amount of

stain in single nuclei was measured using a special microscope called cytophotometer.

This technique confirmed that nuclei contain a constant amount of DNA. Thus, all the

cells in an organism contain the same DNA content (2C) provided that they are diploid.

Gametes are haploid and therefore have half the DNA contents (1C). Some tissues such

as liver contains occasional cells that are polyploidy and their nuclei have a

correspondingly higher DNA content (4C or 8C).

Thus, each species has a characteristic content of DNA which is constant in all the

individuals of that species and thus have been called the C-value.

5.2 RNA of Chromatin

Chromatin has 05-10% of RNA, which is associated with chromatin as:

Ribosonal RNA –(rRNA)

Messenger RNA – (mRNA) and

Transfer RNA – (tRNA)

5.3 Proteins of Chromatin

Proteins associated with chromatin are classified into two groups:

i) Histones

ii) Non-histones

5.1.1 Histones

Histones are very basic proteins because they constitute about 60% of total

chromatin protein, almost 1:1 ratio with DNA.

Histones are basic proteins because they are enriched in amino acid arginine and

lysine (they are devoid of tryptophan). There are five types of histones in the eukaryotic

chromosomes, namely H1, H2A, H2B, H3 and H4.

One of the important discoveries that has came from chemical studies is that H2a,

H2b, H3 and H4 are highly conserved during the evolutionary history. They play a primary

role in chromatin organization. While the H1 histone is least rigidity conserved protein. It

is present only once per 200 base pairs of DNA and is rather loosely associated with

DNA. It is absent in yeast (Sacchromyces cervisiae).

5.1.2 Non-histones:

They are 20% of total chromatin protein and the amount is variable. About 50%

non-histone proteins of chromatin have been found to be structural proteins and include

such proteins as actin, L- and B- tubulin and myosin. These contractile proteins function

during chromosome condensation and in the movement of chromosomes during mitosis

and meiosis. Many of the remaining 50% of non-histonse include all the enzymes and

factors that are involved in replication, transcription and regulation of transcription.

These proteins are not as highly conserved among organism.

6. FUNCTION OF CHROMATIN

The function of the chromatin is to carry out the genetic information from one

generation to another, by encoding the past history and future prospects of the cell. DNA,

being the only permanent component of chromatin, is the sole genetic material of

eukaryotes. It never leaves the cell, thus maintaining heredity of the cell.

7. CONCLUSION

Chromatin is the complex combination of DNA and proteins that organizes

chromosomes which appear as many thread like coiled and elongated structures

suspended in the nucleoplasm. So the chromatin contains genetic material instructions

to direct cell function.

The first level of packing in Chromatin involves the binding of DNA to histones into

fundamental packing unit particles called nucleosomes.

The second level of packing involves packing of nucleosomes into 30 nm thick

chromatin fibre.

The highest level of packing of chromatin in the chromosome is found at the

metaphase stage of cell division.

There are two distinct types of chromatin- euchromatin and heterochromatin which

differ on their staining properties.

In the chromatin, DNA and basic proteins called histones are present in about equal

amounts.

DNA is the permanent component of chromosomes and is the sole genetic material of

eukaryotes.

REFERENCES

1. De Robertis and De Robertis (1998) cell and Molecular Biology, Lea & Febiger,

Hongkong.

2. Ringo.J. (2004), Fundamental Genetics, Cambridge University Press.

3. Winter P.C. Hickey G.I and Fletcher. H. L. (1999), Instant notes on Genetics,

Viva Books Pvt. Ltd.

4. Hames B.D. and Hooper N.M. (2001), Instant notes on Biochemistry, Viva Books

Pvt. Ltd.

5. Verma P.S. and Agarwal V.K. (2004), Cell biology, Genetics, Molecular biology

and Evolution, S. Chand and Co. Ltd.