Eukaryotic cells contain a definite membrane-bound organelle, the nucleus which carries genetic material, organized in the form of multiple DNA molecules with a variety of proteins to form chromosomes. Chromosomes contain genes, their regulatory elements and other DNA binding proteins, which serve to package the DNA and control its functioning. Since the total length of cellular DNA is upto hundred thousand times a cell's length, the entire chromosomal DNA is held in the nucleus in the form of a nucleoprotein complex called chromatin during a significant part of the cell cycle. Nuclear proteins called histones are responsible for compaction and organization of chromosomal DNA into structural units called nucleosomes. Each human cell contains about 1.8 metres of DNA, but when wound on histones, it has about 90 nanometres of chromatin, ultimately resulting in approximately 120 micrometres of chromosomes after duplication and condensation. (Redon C et al, 2002) Thus the major components of chromatin are DNA and histone proteins, alongwith a variety of other chromosomal proteins such as transcription factors, hormone receptors and other nuclear enzymes which play a major role in regulation of gene expression. Thus, though chromatin is a compacted structure, it is organized in a way such that specific DNA sequences within the chromatin are readily available for cellular processes like transcription, DNA replication, repair and recombination.
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Changes in chromatin structure such as methylation, acetylation, phosphorylation and ubiquitination are brought about by histone proteins during post-transcriptional modifications. (Horn P.J et al, 2002). Also, other biological processes such as X chromosome inactivation and genomic imprinting also result in alterations in chromatin structure.
Basics of Chromatin Structure:
Histones are principle structural proteins of Eukaryotic chromatin:
Histones are relatively small, alkaline nuclear proteins with a very high proportion of positively charged amino acids (lysine and arginine). Compact binding of histones to the negatively charged DNA is possible due to this positive charge. Histones can be grouped into five major classes, H1/H5, H2A, H2B, H3 and H4. They are further organised into super classes; core histones, H2A, H2B, H3 and H4 and linker histones, H1 and H5. The core histones also known as nucleosomal proteins are small proteins (120-135 amino acids) and are responsible for coiling the DNA into nucleosomes. The four types of histone chains have very low sequence homology, but they share a common motif called the 'histone fold'. It is a dimerizing, structurally conserved domain consisting of about 70 amino acids, which is flanked by -NH2 and -COOH unstructured terminal domains, called the 'histone tail'. The linker or H1 proteins are large proteins (containing about 220 amino acids) and consist of a winged helix motif flanked by -NH2 and -COOH terminal tails. They bind to the linker DNA connecting adjacent nucleosomes and are responsible for the formation of higher order structures. Although only the linker histone globular domain is essential for binding to nucleosomes, the tail domains play a significant role in the formation of higher order structures. (Ausio Juan, 2006).
The Nucleosome Core Particle:
The fundamental packaging unit of chromatin, nucleosomes, consists of a protein core with DNA wound around its surface and gives a 'beads-on-a-string' appearance in electron micrographs. (Lodish H et al, 2008) On digestion of the long DNA string with nuclease, only DNA between the nucleosomes beads is degraded and the rest remains as double-stranded DNA. Each nucleosomal bead is a disc-shaped particle of about 11nm diameter, containing two copies of each of the four nucleosomal histones, H2A, H2B, H3 and H4 bound to 146 nucleotide base pairs of double stranded DNA. Thus, the core of each nucleosomal particle is a wedge-shaped protein octamer around which two turns of DNA are wound in a left-handed super helix with about 0-80 nucleotide base pairs per turn. The two turns are sealed by a molecule of the linker histone, H1.
On salt treatment, the histones form H2A-H2B heterodimers and H3-H4 heterotetramers. The H2A/H2B dimer binds onto the H3/H4 tetramer due to interactions between H4 and H2B, resulting in the formation of a hydrophobic cluster. Cyanogen bromide peptide analyses of a histone cluster indicate that the core histone H2B contains separate binding sites for H2A and H4 with H2A being connected to the -COOH terminal and H4 being linked to the -NH2 terminal of H2B. The histone octamer thus formed contains a H3/H4 tetramer in the centre sandwiched between two H2A/H2B dimers.
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Due to the compaction of DNA within the nucleosome, certain regions within the nucleosome are not readily accessible to transcription factors. Thus, sequences located in the linker DNA are easily transcribed as compared to those lying within the nucleosome. The ability of histone octamer binding to different DNA sequences depends upon the DNA bending ability, which in turn is highly sequence-specific. (Sekinger et al, 2005) The nucleosome code predicts that regions that are highly expressed should be low in nucleosomes e.g. genes coding for rRNA and tRNA. Also, transcription initiation sites are low on nucleosomes, making them available to transcription factors. On the contrary, centromeric and telomeric regions should be densely occupied with nucleosomes.
Higher order Chromatin structures:
Eukaryotic chromatin, being a highly compact structure is responsible for the structural organization and packaging of genetic material into the genome. Depending upon the degree of compaction, different chromatin structures are involved in this process. These structures are referred to as 'orders'.
Nucleosomes are the basic structural units of chromatin, which undergo folding events through a series of successively higher order structures and result in the formation of a chromosome. Small segments of linker DNA join adjacent nucleosomes together and form a polynucleosome fibre. The 10 nm diameter polynucleosome fibre is formed by binding of core histone H1 to each nucleosome and to the linker DNA. Most of the chromatin exists in the form of a compacted fibre with a diameter of about 30 nm. The nucleosomes are stacked upon each other by coiling the nucleosomal array into a solenoid structure.
Other higher order structures include the coils, loops and folds which condense the 30nm fibre and result in organized structures (chromosomes) within the genome.
Nucleosomes package DNA into tightly compacted structures called nucleosomes. Although nucleosomes present the DNA sequences to DNA binding proteins such as transcription factors and other regulatory proteins, they are also responsible for preventing the interaction of certain regions of DNA with other proteins such as RNA polymerase. This is done by unfolding of higher order chromatin structures. Chromatin remodeling occurs in a cell prior to transcriptional activation. This is a prerequisite as DNA becomes accessible to nuclease attack. Chromatin remodelling involves shifting of the entire nucleosome assembly without disruption of the histone octamer, but by a phenomenon known as nucleosomes sliding. The core of histone octamers slides along the length of the DNA molecule. ATP-dependent remodeling enzymes use chemical energy released by ATP hydrolysis to disrupt the histone-DNA interactions. This allows access to the nucleosomal DNA and facilitates the displacement of histone octamers to neighbouring DNA segment or to a different DNA segment. Thus, ATP-dependent remodeling opens up the chromatin structure and also assists in chromatin assembly and maintenance of higher order chromatin structures.
Chromatin remodeling enzymes: All enzymes belonging to the ATP-dependent family of remodelling enzymes contain an ATPase subunit, which is related to the SWI/SNF family of nucleic acid dependent ATPases alongwith a variety of polypeptides that are responsible for the regulation and efficient functioning of each complex. The SWI/SNF i.e. SWitch/Sucrose Non-Fermentable complex consists of protein products of the SWI and SNF genes. This complex can alter the histone-DNA interactions in a nucleosome assembly in an ATP-dependent manner. The DNA on the edges of the nucleosomes is dissociated with simultaneous reassociation of DNA within the nucleosome. This results in the formation of a bulge on the surface of the histone octamer. Repositioning of the DNA would then take place by translocation of the loop along the surface of the histone octamer.
Three different groups of chromatin remodeling complexes have been identified in the basis of associated ATPase; SNF1/SNF2, ISWI and the Mi-2/CHD group. Each of these complexes have a unique motif which interacts with the chromatin substrates.
SW1/SNF2 group includes the Drosophila brahma gene, the yeast SWI/SNF, yeast RSC and the mammalian homologues of the brahma gene, Brm amd BRG1. These subunits have DNA-dependent ATPase activity and each complex contains either the Brm or BRG1. The remodelling complex interacts with protein products of viral genes, oncogenes and proteins associated with apoptosis and cell cycle control.
Role of SWI/SNF complex in viral gene silencing:
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It has been previously studied that vesicular stomatitis virus G protein based- retrovirus vector is capable of successful transduction into human tumour cell lines. A vesicular stomatitis virus-G protein pseudotyped vector encoding the Î²-galactosidase (LacZ) gene into tumour cell lines was examined for its transduction frequency. It was found that the transduction frequency was significantly low in some tumour lines, like C33A and SW13. (Ito et al, 1997)
To reassess LacZ expression, these cell cultures as well as MDA-MB435 (positive control) were allowed to transducer with the LacZ virus. It was seen that the MDA-MB435 colonies obtained were successfully transduced with LacZ, indicating that all progenies expressed LacZ. Surprisingly, most of the colonies formed by virus-transduced C33A and SW13 cell lines contained mixed populations of positive and negative cells. Thus, it was indicated that retroviruses are capable of integrating into the host chromosome after reverse transcription and initiate transcription in C33A and SW13 cell lines. Also, the retrovirus gene expression is affected as random, discontinuous gene silencing takes place.
ISWI group: Chromatin remodelling complexes belonging to this group contain the ISWI protein as the ATPase subunit. Based on their ability to disrupt or generate nucleosomal arrangements, three complexes were isolated from Drosophila extracts; namely, ACF (ATP-utilizing chromatin assembly and remodelling factor) (Ito et.al,1997), NURF (Nucleosome Remodeling factor) (Varga-Wiesz et al, 1997) and the CHRAC (chromatin accessibility complex), (Tsukiyama et al, 1995) ACF and CHRAC assist in nucleosome assembly and sliding, while NURF-induced sliding disrupts the nucleosome chain. It binds to transcription factors, resulting in the translocation of nucleosomes to the promoter region, resulting in transcription in vitro ( Tsukiyama, 1995). NURF consists of four subunits, NURF 301, which is the largest subunit, NURF140, the ISWI ATPase, NURF55, a protein involved with histone metabolism, and NURF38, a pyrophosphate.
A chromatin remodelling complex hNURD has been recently purified from human cells. It contains the Swi2/Snf2 ATPase homologue known as CHD4 or commonly named as Mi-2Î². The complex comprises of MTA1 and MTA2, which are found in metastatic tissue. The MTA2 subunit is involved with deacetylase activity of the hNURD complex. (Zhang et al., 1998) A distinct feature of the Mi-2/CHD complexes is that they are involved in deacetylation alongwith chromatin remodelling. (Xue et al, 1998). Also, unlike other remodelling complexes which are involved with enhancement of gene expression, hNURD, being a deacetylase is associated with repression of gene expression. The human and Xenopus Mi-2 complexes contain methyl-CpG binding proteins, indicating that the deacetylase activity of the hNURD complex must be directed towards the methyl-rich regions within the genome, thus leading to repression of gene expression due to specific-binding to repressor proteins. (Kehle et al,1998)
Modifications in chromatin structure:
Chromatin is functionally divided into euchromatin and heterochromatin. Generally, euchromatin exists in the decondensed form and contains those those regions that possess actively transcribed genes or potentially active ones. The promoter regions in euchromatin are easily accessible to DNA binding proteins such as polymerases and nucleases. Heterochromatin refers to the highly compact and transcriptionally inactive regions within the genome.
Gene regulation involves binding of transcription factors to the promoter region of genes, followed by a series of modifications, resulting in either activation or silencing of genes. Histone proteins within the nucleosome contain a -NH2 terminus and a -COOH terminus extending from the octameric core. These terminal regions are commonly termed as histone tails. They are the sites for many post translational modifications such as methylation, acetylation, phosphorylation and ubiquitination.
Types of histone modifications:
Transcription, Repair, Replication, Condensation
Transcription, Repair, Condensation
Chromatin structure affects gene expression as well as replication, recombination and DNA repair. Several human diseases are linked to or are even based on defects in the machinery maintaining and/or modifying chromatin structure. For instance, DNA methylation patterns are severely altered in tumors with a bias for overall hypo-methylation of the genome and hypermethylation of specific CpG rich regions. Locally restricted hypermethylation can lead to gene repression, while genomic hypo-methylation particularly of repetitive sequences was suggested to relate to increased genomic instability. (Gaudet et al, 2003).
Research performed in recent years, however, has shed some light on the mechanism leading to locally restricted hypermethylation of some promoters and the consequential silencing of their genes. Such a hypermethylation can result from the abnormal recruitment of DNA methyltransferases by transcription factors which confer locus specificity. Deregulated transcription factors are considered to play a dominant role in the development of leukemias. This idea is supported by analysis of gene-knockout mice, which uncovered crucial roles of several transcription factors in normal haematopoiesis, and of individuals with leukemia, in whom transcription factors are frequently miss-expressed or mutated. Several chromosomal translocations, which are associated with specific forms of leukemia, generate fusion genes that encode altered transcription factors. (Look et al, 2004)
Sekinger, E. A., Moqtaderi, Z. & Struhl, K.Â Intrinsic histone-DNA interactions and low nucleosome density are important for preferential accessibility of promoter regions in yeast.Â Mol. CellÂ 18, 735-748 (2005).