Structure of Chromatin in the Light of Nucleosome | Genetics

In this article we will discuss about the structure of chromatin in the light of nucleosome.

Preamble:

A supercoil model for chro­matin structure was originally described by Pardon and Wilkins in 1972. Later studies of Boldwin (1975), Kornberg (1974) and others have shown that such a sub-unit struc­ture of chromatin is not only present in mam­malian cells but also found in wide variety of non-mammals including plants and yeasts.

The emergence of a repeating sub-unit model (nucleosome) of chromatin has provided an existing new perspective for examining the relationship between transcription and chro­matin anatomy on a finer scale than was pos­sible before.

The Bead and Bridge Concept:

Two main proposals received much attention which tried to explain the nature of a structure of repeating unit:

1) Kornberg (1974) suggested a structure in which 200 bp (base pair) of DNA associat­ed in some manner with a group of nine his- tone molecules, comprising two each of his- tones H₄, H₃, H₂A and H₂B one H₁ molecule to generate the bead.

2) Van Holde (1974) favoured a model whereby 140 bp of DNA combine with two molecules each of H₄, H₃, H₂A and H₂B to form bead which are then separated by a linker region where the existence of H₁ is noted .

Such a unique nucleoprotein particles which constitute a distinct sub-unit structure of the chromatin were referred to as ‘NU bodies’ by Olins and Olins in 1973 and Nucleosome by Oudet (1975).

The Nucleosome Paradigm:

Detailed nuclease digestion study, EM-study and bio­chemical studies suggested a compromised model according to which each chromatin sub- unit is a histone octamer consisting of a (H₃ – H₄)2 tetramers and 2 molecules each of H₂A and H₂B with the arginine-rich tetramer apparently constituting an inner core.

Each nucleosome is associated with approximately 190 bp of DNA, 140 of which are apparently associated with the H₃-H₄ tetramer core in a particularly nuclease resistant form.

The total length of nucleosome associat­ed DNA varies phylogenetically, but the length of the core associated DNA appears to be constant with 140 bp unit in all the species examined from protozoa to mammals .

Physical Nature of the Core:

The ear­liest physical studies of nucleosome core struc­ture suggested a particle roughly spherical in shape, about 100Å in diameter. But recent study of Nutron and X-ray scattering behaviour of nucleosome in solution has led Pardon (1977) to suggest that nucleo­some is a flattened cylindrical structure about 100Å in diameter and 50Å in height, with the DNA wrapped around it to form a pair of rings at the top and bottom.

More direct and detailed information about nucleosome (N) core shape has now been provided by the works of Finch, Klug and others (1977). The crystallographic data of these workers show that the N core is actually a flat disc about 110Å in diameter and 57Å of height. These are all close agreement with the studies of van Holde (1978).

It is now assumed from the accumulated data that the 140 bp of DNA form an uniform per helix wrapped around a cylinder and the superhelix have a diameter of about 90Å and a pitch of about 28Å, corresponding to between 75 and 82 bp per superhelical turn of B-form DNA .

Using scanning and transmission electron microscopy, Langmore and Wooley (1973) also confirmed the disc-shaped structure prob­ably about 100 to 120Å wide and 50 to 6OÅ thick for the beads.

Are the Beads Contiguous—”The Spacer Concept”

Although the low angle X-ray scattering study of sugges­ted a contiguous beaded model for nucleo- somes yet more recent biochemical and EM studies showed that under appropriate condi­tions of spreading the chromatin fibres contain regularly spaced nu bodies interconnected by the filaments.

Later studies of Oudet (1977) and Tsanev (1978) demonstrated that these connective DNA threads or spacers cor­respond to the nuclease sensitive nucleosomal DNA. More recent EM data show that in the native chromatin the nucleosomes are so closely packed that no connecting spacers are visible.

Unlike cores the length of the spacer varies according to species, in different tissues and sometimes even within the same tissue. The variable spacer length could be correlated with the variability and heterogeneity of the very lysine rich histones (H₁ and/or H₅) while the evolutionary conservative core nucleo­some may reflect the well known evolutionary stability of the four smaller histone species.

Involvement of H₁:

Several lines of evi­dence suggest that H₁ is not a part of the core (140 bp) nucleosome structure .It may be associated with the 50 bp moncore or spacer DNA, since its selective removal renders this DNA particular­ly nuclease sensitive.

How Spacers are Arranged:

Controversy still exists regarding the exact array of the spacers in between the beads.

3 different possibilities have been suggested:

a) The spacer regions are all of equal length;

b) The spacer regions are ‘quantized’ into a limited number of allowed length;

c) The spacer regions are random in length and the core particles are thus irregularly spaced along the DNA chain. The best evidence available at present suggest that there is a considerable variability in spacer length even in the chromatin of one nucleus, but that there are at least some por­tions of the chromatin in which a 10 bp quantization of spacer length occurs.

Possible Arrangement of Core and H₁Relative to Each Other:

On the basis of various enzymatic studies van Holde et al (1978) favoured a model which allows H₁ and/or H₅ to take a random position on the spacer DNA with 3 possible arrangements:

1. Core and H₁ are separated by naked region;

2. Core and H₁ are in close proximity; or, in contact, such that the probability of strand breakage between them is reduced;

3. The core is in contact with 2H₁ molecules, one each on both ends of DNA.

A Binary Structure of the Core:

Although at present the available data are still not sufficient to propose a detailed model of the internal molecular organization of the core but recent EM, bio-chemical and physical stu­dies have suggested a binary substructure of the Nucleosome (N).

The first evidence for the existence of two sub-units in the N come from the occurrence of two N particles of two different size classes in different preparations/methods of extrac­tion: small size N particles of 80Å diameter and a relatively larger size particle of about 120Å.

Since two types of particles can be obtained from the same chromatin material it has been proposed that N is built up of two small sub-units. Under the influence of low ionic strength and some specific stretching fac­tors the large N can be opened and trans­formed into two smaller particles still inter­connected by a thin DNA filament represent­ing a short intra-nucleosomal fibre. Recent experiment of Oudet et al (1977), with sv 40 minichromosome also support this view.

More detailed EM study of Tsanev and Petrov (1976), revealed that each sub-unit of N may contain a coil of DNA cor­responding to 60 bp and these two subunits are connected with an intranucleosomal spacer of about 30 bp long. Thus a total DNA length in this model corresponds to about 150 bp, which is in close agreement with the length of core DNA (140 bp established by nuclease probe).

Models of Molecular Organisation of Chromosomal Fibre in Relation to Nucleo­some:

Three molecular models for the struc­ture of the native chromosomal fibre have been proposed, each considering nucleosome as the basic structural unit:

1. A simple chain model of nucleosome:

According to this model, each nucleo­some is in close contact with only two other nucleosomes, the one immediately adjacent to it on the DNA backbone. The simple chain of nucleosome is almost always seen by EM at very low ionic strength and after removal of H₁. The most interesting example of simple chain nucleosomes found in sv-40 minichro­mosome.

However, there are only a few cases in which this simple chain model is visible under conditions of ionic strength that would give native structure.

2. A helical model of nucleosome:

A simple helical model of nucleosome organization to form a high order structure has been proposed by several workers.

(a) Finch and Klug (1976) have presen­ted a model which suggests a close packing of nucleosomes in the 400Å fibre which they call a “nucleofilament”. In presence of MgCl₂ and H₁ the nucleofilament coils up to form a thicker fibre (about 300-500Å) which they called a ‘solenoid’.

They also proposed that 110Å ring in X-ray diffraction studies arises from the spacing between the nucleofilament in the regular helical higher order structure. They estimate about 6N per turn of the helix to produce an overall packing ratio of 40: 1 along the fibre axis.

(b) A similar model is proposed by Alberts (1977) where instead of H₁ crys­tal packing forces generated by direct interac­tions among the nucleosomes along the chain would stabilize the helix. They suggested that modification of histones would lead to slightly different helical arrays of nucleosomes which might correspond different units.

3. The superbead model:

By EM and biochemical studies Hoizen (1977 and ’79) favoured a discontinuous super structure of native chromatin fibre, in which the DNA and chromosomal protein are not distributed uniformly along the fibre axis. Instead, a discreet assemblies of N are present.

The term “superbead” has, therefore, been proposed to describe the individual 200Å unit (super denotes the super-structural character and the bead refers to both to the shape .of the super-structural unit and to its nucleosomal makeup).

In this model, tandem arrangement of superbeads in the native chromatin fibre or relatively resistant to micrococcal nuclease attack, while the DNA connecting them is sen­sitive. Micrococcal nuclease digestion followed by differential centrifugation it has been observed that each super-bead 40s contains of about 8N with a repeat length of 180 bp of DNA.

This superbead model has recently been supported by Becak (1979) from their studies on amphibian nuclei. But, in their experiment, treatment with calcium ion reveals the existence of globules of 300Å formed by 8-10N.

Nucleosome and Transcription:

The question whether the nucleosome modify characteristic of bulk chromatin applies also to the organisation of transcribed chromatin regions is a matter of controversy. All histones with the possible exception of H₁ are observed in biochemical fractions of active chromatin.

The possibility that these histones are confined to small amounts of contaminating inactive chromatin is ruled out by the observed DNA histone ratio of these fractions and of template active fractions behaves like nucleohistone, not lined naked DNA. Since active chromatin contains the 4 histone building blocks of the nucleosome, it follows that these regions also contain nucleosome per se.

Although it has been suggested by Foe (1976) that active ribosomal genes do not contain beaded structure analogous to N. The biochemical evidences provided by Reeves and Jones (1976) indicated that the ribosomal genes are organised into the familiar pattern of alternating nuclease sensitive and nuclease resistant DNA i.e. the hall-mark of nucleosome (N) structure.

From the studies of Oncopeltus and Drosophila embryo chromatin it seems per­fectly possible that histones may be present on transcribed DNA but in a sufficiently modified form that their mutual protein-protein associa­tion are no longer recognizable ultrastructurally as nucleosome.

This interpretation receives support from the nucleohistone appearance of DNA under­lined nascent ribosomal RNP matrixes in a negatively stained spreads of New chromatin as well as from the bio­chemical observation that histones are present in purified nucleolar chromatin fraction. If these histones were appropriately disposed along the active ribosomal DNA, they could still considerably generate the characteristic nuclease digestion pattern.

These observations suggest that the histone-histone interactions which constitute the cornerstone of normal N structure are in fact rather dynamic. In some cases (egg’s active ribosomal gene) it seems that nucleosome structure can be so significantly perturbed dur­ing transcription as to eliminate the typical beaded morphology detected by EM.

In contrast to reports of non-beaded ultra- structural appearance of active ribosomal chromatin, studied by Larid and others (1976) revealed that, non-ribosomal transcription units in both Oncopeltus and Drosophila embryo chromatin have a definite beaded morphology .

Both the size and staining texture of these beads are the same as those are seen throughout the nascent RNP free i.e. inactive regions of chro­matin, reinforcing their interpretation as nucleosome.

The DNA chromatin packing ratio of beaded non-ribosomal transcription unit in Oncopeltus is estimated to be 1.6-1.9 m µ of 3-conformation DNA as compared to a com­parison value of 2.3 for beaded inactive regions .It, therefore, seems probable that some longitudinal expansion of the usual (i.e. inactive) DNA folding pattern occurs during the transcription of non-riboso- mal genes.

According to McKnight and Miller (1976) the nearly complete unfolding of active ribosomal DNA may simply represent an extreme case, possible due to the intense rate of RNA polymerase initiation at this particular loci.