This document provides information on the structure and organization of chromatin. It discusses three main levels of chromatin organization: 1) Nucleosomes, which involve DNA wrapping around histone proteins in "beads on a string" structures. 2) The 30nm chromatin fiber, where nucleosomes are further compacted into a solenoid structure through histone H1 binding. 3) Higher-order compaction into metaphase chromosomes, where DNA is organized into loops anchored to a protein scaffold. The document also describes different chromatin types (euchromatin and heterochromatin), chromatin composition, and models of chromatin structure including the nucleosome model.

1. SHER-E-KASHMIR
UNIVERSITY OF AGRICULTURAL SCIENCE AND TECHNOLOGY OF
KASHMIR
Division of Biotechnology, Faculty of Veternary Sciences and Animal Husbandry,
Shuhama-Srinagar.
A Lecture on

2. STRUCTURE AND ORGANIZATION OF
CHROMATIN
Presented by
ZAFAR IQBAL BUHROO
(Research Scholar)

3. 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

4. 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.

5. 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

6. 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.

7. 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.

8. 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.

9. 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)

10. from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)
Nucleosomes contain 2 copies
of H2A, H2B, H3 and H4
147 bp of DNA is wrapped
around nucleosome
Histone tails emanate from core
Some nucleosomes
contain histone variants
H1 is a linker histone
Nucleosome Structure
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.

11. 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

12. Fig.6. Organization of chromatin in chromosome

13. 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 µm 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 µm DNA, after contracting 40 folds into 30 nm
fibre, they would be only about 0.6 µm long, a length consistent with the diameter of
metaphase chromosome (1 µm) 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.

14. Fig.4. Higher Levels of DNA packing in Chrosome
4) TYPES OF CHROMATIN

15. 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.

16. 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

17. 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).

18. 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.

19. • 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.

20. 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.