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Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation., The histone H3K79 methyltransferase DOT1L plays a central role in B cell development and differentiation. DOT1L maintains B cells naivety by orchestrating critical transcriptional and epigenetic regulators.

DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8.

Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3' end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.

Understanding disease-associated cellular defects at a molecular level is critical for the development of pharmacological intervention strategies. Recent breakthroughs in microarray and sequencing technologies have provided powerful tools to rapidly reveal the cellular defects caused by alterations in the genome or transcriptome. However, the picture of how the cellular proteome is affected in a disease state and how changes in DNA and RNA affect protein function is often incomplete. This is perhaps not surprising because the functions of proteins are not just determined by primary sequence and abundance, but are under the control of many regulatory mechanisms. Here, we highlight several recent advances in proteomics technologies that are being developed to generate comprehensive human proteome maps and discuss them in the context of strategies that have been developed in simple model organisms. Chemical biology will play a critical role in drafting a map of the proteome with functional information. Chemical genetic approaches that use high-throughput small molecule screening have resulted in the public availability of small molecule datasets through web interfaces such as PubChem. With such approaches, the opportunities to investigate disease and to explore the proteome with chemistry are rapidly increasing. In addition, new tools are being developed to probe protein function. Here we highlight recent developments in chemical biology and the exciting opportunities that are arising with them.

Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo-neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 17-beta-estradiol on the activity of the OT gene promoter was examined as well as the effect of 17-beta-estradiol in vivo on OT messenger ribonucleic acid (mRNA) and peptide levels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the vivo studies. The direct transcriptional stimulation of OT gene expression by 17-beta-estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides -363 to +16 of the rat OT gene fused to the firefly luciferase reporter gene was co-transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80-fold by 17-beta-estradiol. In estrogen receptor containing MCF-7 cells transfected with a plasmid having nucleotides -188 to +16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 17-beta-estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 17-beta-estradiol, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non-treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, showing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5' flanking region of the OT gene confers estrogen-sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT-producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.

Vasopressin (VP) cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus and supraoptic and paraventricular nuclei are influenced by gonadal steroids. The present paper examined whether VP cells in the bed nucleus of the stria terminalis, medial amygdaloid nucleus, and supraoptic and paraventricular nuclei contain estrogen receptors. Brains from adult short-term castrated, colchicine-treated male rats were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde. In the immunocytochemical double-staining procedure Vibratome sections were first incubated with an estrogen receptor antibody (#H222) and stained with diaminobenzidine-Ni(+). Following methanol-hydrogen peroxide washes, sections were incubated with anti-neurophysin and stained with diaminobenzidine. Parvocellular cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus were double-stained with a blue-black nucleus (indicating the estrogen receptors) surrounded by brown cytoplasm (resulting from VP-neurophysin-immunoreactivity). Our results provide the first direct anatomical evidence supporting the hypothesis that gonadal steroids' influence of parvocellular VP cells in the bed nucleus of the stria terminalis and medial amygdaloid nucleus is mediated directly via estrogen receptors localized in nuclei of VP neurons. We were unable to co-localize any estrogen receptors in VP and oxytocin cells of magnocellular size in the supraoptic, paraventricular and anterior commissural nuclei, suggesting that estrogen indirectly affects these magnocellular hypothalamic cells.

The distribution of antigenic determinants on human IgA was studied with fragments and mutants of IgA1. F(abc)2 and Fabc, lacking the CH3 domain, F(ab')2 and Fab', lacking the CH2 and CH3 domain, Fab that further lacks most of the hinge region, and Fc fragments were included in our investigations. Antibodies specific for the CH3 domain of IgA1 were found in antisera raised against an alpha 1-HCD protein and in anti-Fc alpha antisera. The antisera detected different antigenic determinants on CH3 as was shown by inhibition with sera from nonhuman primates. An anti-Fc5 mu antiserum detected a determinant on Fc common to IgA and IgM. The serum of an IgA-deficient individual reacted with a determinant on CH2 of four-chain molecules only. A subclass-specific anti-Fabc antiserum detected a determinant which needed interaction of CH2 and CH1 or the hinge region. Anti-Fab antisera reacted with class or subclass specific determinants on CH1. The isoallotype nA2m(2) is probably located on CH1. Its expression requires two alpha-chains stabilized in a conformation attributed to by CH2.

Two groups of Chinese individuals of the city of Wuhan in the People's Republic of China, consisting of 98 blood bank donors and 278 workers in a steel factory, respectively, have been typed for G1m, G2m, G3m, A2m and Km allotypes. The Gm-A2m haplotype frequencies as well as the Km gene frequencies appeared to be quite similar in these two groups of people, the main haplotypes being Gmaf;n;b A2m2 and Gmza;..;g A2m1. After joining the data of the two groups, the frequencies of the main Gm haplotypes and the A2m and Km gene frequencies have been compared with those of six other Asian populations. In Asia, going from the south to the north, an increasing frequency of Gmza;..;g and a decreasing frequency of Gmaf;n;b were observed. The frequencies found in the investigated Chinese population lie between the frequencies found in the Chinese from Taiwan and the Japanese.