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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.
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. ( Histone-histone interactions within chromatin-preliminary characterization of presumptive H2B-H2A and H2B-H4 binding-sites, Martinson, H; Maccarthy, B. Biochemistry. Vol 15.Issue 18, Pgs:4126-4131.(1976) Nucleosomal Positioning:
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, E. A., Moqtaderi, Z. & Struhl, K. Intrinsic histone-DNA interactions and low nucleosome density are important for preferential accessibility of promoter regions in yeast. Mol. Cell 18, 735-748 (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. Also, since, AT-rich sequences are easier to compress within the minor groove,
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:
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. (SWI/SNF chromatin remodelling complex and retroviral gene silencing)
Hideo Iba*, Taketoshi Mizutani and Taiji Ito.
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) (Purification and properties of an ATP-dependent nucleosome remodeling factor (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 prompter region, resulting in transcription in vitro ( Tsukiyama, 1995).
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