Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

Developmental Patterns of Catechol-O-Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies

Abstract: Catechol- O -methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50–60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.     

“绿色革命”新进展:赤霉素与氮营养双重调控的表观修饰助力水稻高产高效育种

以半矮秆育种为代表的“绿色革命”极大地提高了作物产量,但也带来氮营养利用效率降低的严重问题。“绿色革命”主要基于调控赤霉素的代谢和信号转导而实现。前期的研究发现,赤霉素信号转导关键因子DELLA蛋白通过调控GRF4而负调控氮素的吸收利用,为半矮秆品系氮利用效率低的问题提供了解决方案。最近的一项研究进一步揭示了GA信号途径与氮响应交叉互作的新机制。该研究发现水稻(Oryza sativa)NGR5是氮素调控分蘖数目的一个关键基因,其表达受氮诱导。通过招募PRC2,NGR5对D14和OsSPL14等分蘖抑制基因所在位点进行H3K27me3甲基化修饰,从而抑制其表达。而在半矮秆背景下超表达NGR5可以提高低氮水平下的水稻产量。NGR5同时也被发现为赤霉素受体GID1的一个新靶标,受到其负调控。该研究发现了调控赤霉素信号通路的新机制,并对高产高效的新一代“绿色革命”育种实践具有重要启示。     

A Dimeric Structural Scaffold for PRC2-PCL Targeting to CpG Island Chromatin

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Introduction: Metals in Biology: α-Ketoglutarate/Iron-Dependent Dioxygenases

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.     

H1° histones of normal and cancer human cells

Summary H1° histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1° histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1° histones showed that: (a) all the H1° preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; (b) H1° is distinguishable from other H1 histones by the presence of methionine and histidine; (c) H1° histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1° histone is not inversely related with cell growth rate nor with the expression of the -fetoprotein gene.     

Effects of 5'deoxy-5'-methylthioadenosine on the metabolism of S-adenosyl methionine

The treatment of transformed rat cells with micromolar amounts of 5'deoxy 5'methyl thioadenosine induces rapid effects on the rate of methylation of DNA concomitantly with alterations of intracellular pools of S-adenosyl methionine and S-adenosyl homocysteine. Pulse chase labelling experiments indicate that 5'deoxy 5'methylthioadenosine does not inhibit the degradation of S-adenosyl homocysteine but inhibits the consumption of S-adenosyl methionine. In vitro transmethylation assays performed with heterologous DNA show that low doses of the thioethernucleoside do not significantly affect the DNA methyltransferase activity of cellular extracts. The biological role of 5'deoxy 5'methylthioadenosine, a natural molecule formed during the synthesis of polyamines is discussed.