The histones of some human tissues.
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ABSTRACT:
SUBMITTER: MacGillivray AJ
PROVIDER: S-EPMC1187195 | biostudies-other | 1968 Nov
REPOSITORIES: biostudies-other
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Project description:1. The relative amounts of incorporation in vivo of l-lysine, and in one experiment l-arginine, into different histone fractions from Krebs ascites and a lymphoma ascites cells of mice and a ;solid' tumour and liver of rats have been determined. 2. No marked differences in the incorporations of the amino acids into the fractions F1, F2a, F2b and F3 from the tumours were generally observed, although in some experiments there was a greater incorporation into fraction F2b, which could be decreased by further purification. 3. In the tumours the incorporations into all cell protein fractions obtained were approximately the same, indicating that the amount of incorporation was that required for the increase of cell mass. 4. In rat liver, the incorporations into fractions F1, F2a and F3 were not greatly different. That into fraction F2b was variable. The incorporation into the histone fractions was much less than that into the acid-insoluble nuclear residue, indicating that considerable turnover of amino acids in the latter occurs. 5. The decrease in radioactivity of labelled histone and acid-insoluble nuclear protein in vivo during several days confirmed the relatively small turnover of the histone fraction. The time taken for liver whole histone to lose half its radioactivity was about 1 week. A histone fraction of slower metabolism was also detected. 6. It is concluded that no appreciable turnover of protein occurs in any one histone fraction, the somewhat higher values obtained in certain cases being associated with acidic impurities. The apparently high rate of incorporation into histone of resting liver is discussed in relation to recent evidence on DNA metabolism of resting liver.
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Project description:Bidirectional promoters have received considerable attention because of their ability to regulate two downstream genes (divergent genes). They are also highly abundant, directing the transcription of approximately 11% of genes in the human genome. We categorized the presence of DNA sequence motifs, binding of transcription factors, and modified histones as overrepresented, shared, or underrepresented in bidirectional promoters with respect to unidirectional promoters. We found that a small set of motifs, including GABPA, MYC, E2F1, E2F4, NRF-1, CCAAT, YY1, and ACTACAnnTCC are overrepresented in bidirectional promoters, while the majority (73%) of known vertebrate motifs are underrepresented. We performed chromatin-immunoprecipitation (ChIP), followed by quantitative PCR for GABPA, on 118 regions in the human genome and showed that it binds to bidirectional promoters more frequently than unidirectional promoters, and its position-specific scoring matrix is highly predictive of binding. Signatures of active transcription, such as occupancy of RNA polymerase II and the modified histones H3K4me2, H3K4me3, and H3ac, are overrepresented in regions around bidirectional promoters, suggesting that a higher fraction of divergent genes are transcribed in a given cell than the fraction of other genes. Accordingly, analysis of whole-genome microarray data indicates that 68% of divergent genes are transcribed compared with 44% of all human genes. By combining the analysis of publicly available ENCODE data and a detailed study of GABPA, we survey bidirectional promoters with breadth and depth, leading to biological insights concerning their motif composition and bidirectional regulatory mode.
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Project description:Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites.
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Project description:How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2.
| S-EPMC4946945 | biostudies-literature
Project description:BACKGROUND:Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. METHODS:To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. RESULTS:We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. CONCLUSIONS:This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.
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Project description:Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A-H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3-H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg(2+)-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin.
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Project description:Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.
| S-EPMC1196040 | biostudies-literature
Project description:CONTEXT:?-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of ?-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy. OBJECTIVE:This is the first study to characterize systemwide tissue expression of transmembrane ?-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human ?-Klotho expression in health. RESULTS:The distribution of ?-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong ?-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, ?-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane ?-Klotho isoform. CONCLUSIONS:The data presented confirm ?-Klotho expression in the kidney tubule and in the artery and provide evidence of ?-Klotho expression across organ systems and cell types that has not previously been described in humans.
| S-EPMC4596032 | biostudies-literature
Project description:BackgroundCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.Methods and findingsTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.ConclusionsCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.
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Project description:Immunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies.By immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3.At metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.
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