Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast
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Histone H2B
Histone Methylation
Histone code
Histone octamer
Epigenomics
The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.
Histone octamer
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Histone octamer
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Epigenomics
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Histone H4
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Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.
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Histone H2B
Histone Methylation
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Histone octamer
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The topography of the interaction between histone H1 and the histone octamer has been investigated. Bovine thymus nuclei or enzymatically fragmented chromatin were treated 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, which catalyzes the formation of covalent bonds between residues of proteins in electrostatic contact. Histone H1-core histone dimers were identified and the segments of molecules participating in crosslinking were elucidated. The results demonstrate that the major histone H1-core histone dimer generated upon carbodiimide crosslinking of intact nuclei, chromatin, or mononucleosomes consists of the segment of histone H1 containing amino acids 74-106 crosslinked to the segment of histone H2A containing amino acids 58-129. Thus, the central globular region of histone H1 intimately contacts the histone octamer. Besides histone H1-H2 dimers, two other histone H1-containing crosslinked products were detected. In these instances, the segments of histone H1 molecules containing amino acids 1-72 were shown to participate in crosslinking. The histone H1 contact points defined here all occur within mononucleosomes and not between nucleosomes. These results permit the formulation of a testable model for the arrangement of histone H1 along polynucleosome chains.
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In vitro studies on nucleosome core particles (NCPs) and nucleosomes have generally been limited to the use of histone proteins isolated from chromatin. Numerous reliable and well-established methods have been described of obtaining single histone proteins in significant quantity (e.g., refs. 1 and 2, and references therein). Briefly, the histone complexes (histone octamer, or histone tetramer and histone dimer) are isolated from "long chromatin," which is extracted from nuclei. The histone complexes can be further fractionated into individual histone proteins. This approach suffers from several disadvantages. First, the procedure is time-consuming and depends on the availability of fresh tissue or blood from the organism of choice. Second, histone proteins isolated from natural sources are often degraded by contaminating proteases (3). Third, histone isotypes and posttranslational modifications of histone proteins give rise to heterogeneity. The extent of heterogeneity and modification strongly depend on the type and developmental state of the tissue from which chromatin is isolated and can vary significantly between different batches. Fourth, and most important, only naturally occurring histone proteins can be obtained by this method.
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The genetic information of eukaryotes is organised in a nucleoprotein complex
called chromatin. The fundamental repeating unit of chromatin is nucleosome in
which 146 bp of DNA is wrapped around octamer of core histone proteins H2A,
H2B, H3 and H4. Histone H1 (linker histone) associates with DNA between two
nucleosomes. Core histones are highly conserved proteins among all eukaryotes.
The N-terminus of the core histones extends outwards from the nucleosome and
is flexible in nature whereas the globular C-terminus forms the scaffold of the
nucleosome (Fischle et al., 2003). Nature has evolved mechanisms to dynamically
alter chromatin structure like chromatin remodelling by ATP dependent chromatin
remodellers, covalent histone modifications, and replacement of histone proteins by
their respective variants.
Histone octamer
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Histone octamer
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