Higher order organization of eukaryotic chromatin :
In 1974, Roger Kornberg on the basis of micro coccal nuclease digestion pattern of genomic DNA proposed that the lowest level of chromatin organization consists of wrapping of about 200 bp of DNA double helix around a histone octamer. In strict sense, about 147 bp or 1 and ¾ turn of DNA is wound around a block of two copies of histone proteins. H2A, H2B, H3 and H4 each and constitutes the nucleosome core particle. These nucleosomes are connected to each other like beads of pearls on a string through variable length of linker DNA. Recent observations on x-ray crystallographic structure of nucleosome reveal that the architectural motif of nucleosome consists of I,islone folds having (H3),-(l-14), tetramer at the centre and H2A and H2B dimers at the end of the length. The core fold contains 121 bp DNA. Rest 26 bp DNA is contained by N-terminal end of histone units, having about 13 bp at each end. Histone proteins interact with phosphodiestcr backbone and deoxy ribose moiety at minor grooves of DNA
through electrostatic interactions and hydrogen bonding, but do not have any base specific contacts. Each of the histone unit has 10-40 amino acid long N terminal tail sections which can be extended, remains outside the core section and plays important role in chromatin modeling and transcriptional regulation. Another histone protein HI and H5 (in birds) serves as the linker histone by binding to the nucleosome near one end of core DNA and influence the entry and exit of DNA into nucleosome. HI was considered to stabilize additional 10 bp DNA at each end, thereby putting 167 (147 +10+10) bp DNA around nucleosome. The
assembly of 167 bp DNA, Hi and core histone octamer is sometimes referred as chronzatosome. However, recent studies reveal that the repeating unit of higher order chromatin structure contains 156-157 bp DNA in the nucleosome and is known as the 10 nm fiber of DNA due to the nucleosome unit diameter of 10 nm. Nucleosomes are often positioned in specific genomic regions (promoters, regulatory elements, specific sequences etc.) which is gene sequence dependant, a phenomenon known as nucleosome positioning that is suggested to be involved in regulation of gene expression. It has been observed that this positioning is not static; rather nucleosomes remain in a dynamic state in the chromatin.This preferential positioning helps nucleosome to repress transcription. The flexible tails are basic in nature and serve as substrates for many enzymes involved in methylation, acetylation, phosphorylation and other post-translational modifications. This
has led to the birth of a his tone code hypothesis which postulates that the specific arrangements and sequences of histone tail are read as a code to unfold and remodel chrornatin, and in gene expression. It has also been observed that the core histone proteins are also extensively modified and thereby can also be viewed as apart of histone code. Two types of posttranslational modifications, class I and class II modifications are suggested. Class I is involved in histonc-non histone chromatin associated protein interaction, while class II involves histone-DNA and histone-histone interactions.
Second level of chromatin organization constitutes of 30 nm fiber, as observed as a filament of diameter 30 nm under electron microscope. It is a package of t of nucleosomes and is maintained by inter histone interactions, which can be classified into eight distinct types. The interaction between N terminal of histone H4 and part of H2A-H2B dimer is essential for maintenance of 30 nm structure. Previous idea that the 30 nm fiber comprises of an one start solenoid structure of 6-8 nucleosome units is being challenged by recent microscopic and crystallographic observations. Recent findings suggested that 30 nm fiber may be comprised of stacks of nucleosomes arranged in a hc’o start helical model or one start helical imiodel with a very high nucleosome packing ratio (15-22 nucleosomes per unit instead of 6-8 in solenoid model).
Still, the packing ratio, 6 for each nucleosome and about 120 for each 30 nm fiber unit having 20 nucleosomes each (20 x 6) is insufficient for packing DNA into small nucleus. The 30 nm fiber again folds and refolds to form loops of 40-90 kb and the torsion created therein is relieved by action of topoisomerases. The higher order nuclear scaffold structures are considered to be held by structural maintenance proteins (SMCs). Theses proteins also play major role in holding of two chromatids during divisional phases. Recent observations suggest that the organization of chromatin material in higher as well as lower eukaryotes is dynamic in nature in the sense that the individual chromosomes can reposition themselves in the nuclear interior. The chromosomes occupy specific regions in the nucleus known as chromosome territories with euchromatic regions oriented to the interior and heterochrornatic regions at the peripheral space. These chromatins can move within the nucleus to a certain extent, observed to be more at interphase. This spatial distribution and movement of chromatin material is suggested to provide a mechanism for controlling gene expression by allowing binding of genes with localized transcriptional factors, intcrchromosomal interactions between loci present on different chromosomes (gene kissing) and by inducing epigenetic regulations. A number of protein and RNA molecules are involved in prevention of movement of chrorna tin material (chroina tin immobilization), thereby imposing restriction on random movement of the chromosomes in the nucleus.Genome organization in eukaryotic chromosomes at metaphase as well as at interphase is non uniform. A chromosome based on metaphase morphology shows vanous landmarks with distinctive features. Tlie cent romere or primary constriction, where microtubules are attached during anaphasic movement constitutes mostly of hetcrochromatin and are attached to protein complexes known as kinetochore that act as a bridge between microtubules and centromere. For correct chromosome segregation it is e.ential that only one centromere he present in a chromosome. Size of a centromere in eukaryotes may vary greatly, from as little as 125 bp in yeast to several Mb in human. The two ends of the linear chromosomes are known as telo,neres, having complex looped structures involving non-Watson Crick base pairing, hcterochromatinized regions and unusual replication mechanism. A number of proteins arc associated with telomere to prevent DNA damage at chromosome ends. Between centromere and telomere, further constricted regions or secondary constrictions max’ exist. The area between a telomere and nearest secondary constrictions is sometimes called as satellite. Nucleolus is preferentially attached to a region of chromosome known as nuclolare organiser region (NOR).
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