Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes
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PROVIDER: PRJNA705367 | ENA |
REPOSITORIES: ENA
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Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than the constant (C) region which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout (KO) and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of Pol II and its S2 phosphorylated form at the IgH locus. KO of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of P-TEFb complex) on the V region and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreased SHM and the abundance of Pol II S2P at the IgH locus. With all these factors playing a role in transcription elongation, our studies extend our insights into the chromatin context and dynamics of transcription required for SHM.
2021-02-28 | GSE167873 | GEO
Project description:Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.
| S-EPMC8307847 | biostudies-literature
Project description:Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L’s functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.
2020-10-21 | GSE134083 | GEO
Project description:The recognition of modified histones by “reader” proteins constitutes a key mechanism regulating gene expression in the chromatin context. Compared with the great variety of readers for histone methylation, few protein modules that recognize histone acetylation are known. Here we show that the evolutionarily conserved YEATS domains constitute a novel family of acetyllysine readers. The human AF9 YEATS domain binds strongly to histone H3K9 acetylation and, to a lesser extent, H3K27 and H3K18 acetylation. Crystal structural studies revealed that AF9 YEATS adopts an eight-stranded immunoglobin fold and utilizes a serine-lined aromatic “sandwiching” cage for acetyllysine readout, representing a novel recognition mechanism that is distinct from that of known acetyllysine readers. Histone acetylation recognition by AF9 is important for the chromatin recruitment of the H3K79 methyltransferase DOT1L. Together, our studies identify the YEATS domain as a novel acetyllysine-binding module, thereby establishing the first direct link between histone acetylation and DOTL1-mediated H3K79 methylation in transcription control. ChIP-seq analysis of AF9, H3K79me3, H3K9ac in Hela cells and H3K79me3 in Hela AF9 knockdown and Hela Dot1L knockdown cells.
2014-12-11 | E-GEOD-60366 | biostudies-arrayexpress
Project description:The transcription factor Bach2 is required for germinal center formation and somatic hypermutation (SHM) of immunoglobulins, both central to an efficient antibody-mediated immune response. Activation-induced cytidine deaminase (AID) initiates SHM and CSR in germinal centers and has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID-mediated immunoglobulin gene diversification processes, we established a Bach2-deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), an important AID-dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp-1 and that Bach2-deficient cells have altered expression of several genes regulating AID expression, stability and function. These results demonstrate that Bach2 has a previously unappreciated role in the production of high-affinity antibodies.
2017-03-27 | GSE92430 | GEO
Project description:The recognition of modified histones by “reader” proteins constitutes a key mechanism regulating gene expression in the chromatin context. Compared with the great variety of readers for histone methylation, few protein modules that recognize histone acetylation are known. Here we show that the evolutionarily conserved YEATS domains constitute a novel family of acetyllysine readers. The human AF9 YEATS domain binds strongly to histone H3K9 acetylation and, to a lesser extent, H3K27 and H3K18 acetylation. Crystal structural studies revealed that AF9 YEATS adopts an eight-stranded immunoglobin fold and utilizes a serine-lined aromatic “sandwiching” cage for acetyllysine readout, representing a novel recognition mechanism that is distinct from that of known acetyllysine readers. Histone acetylation recognition by AF9 is important for the chromatin recruitment of the H3K79 methyltransferase DOT1L. Together, our studies identify the YEATS domain as a novel acetyllysine-binding module, thereby establishing the first direct link between histone acetylation and DOTL1-mediated H3K79 methylation in transcription control.
2014-12-11 | GSE60366 | GEO
Project description:We wanted to investigate the effects of Dot1l deletion on gene expression in LSKs and GMPs of C57/BL6 mice Aberrant Hox gene activation is a recurrent feature in several different types of human leukemia, including leukemias with rearrangements of the mixed lineage leukemia (MLL) gene. In this study, we demonstrate that Hox gene expression is controlled by higher degree H3K79 methylation in acute myeloid leukemia (AML). We show that the deposition of progressive H3K79 methylation states at the genomic loci of critical Hox genes is dependent on the interaction of the H3K79 methyltransferase Dot1l with Af10, a protein that is found in the Dot1l complex isolated from diverse cell types. Furthermore, abrogation of the Dot1l-Af10 interaction reverses aberrant epigenetic profiles found in the leukemia epigenome and impairs the transforming ability of mechanistically distinct AML oncogenes. Lineage negative Sca-1 positive Kit positive (LSK) cells and granulocyte macrophage progenitors (GMPs) were sorted from Dot1 wt/wt x Mx-Cre mice or Dot1l fl/fl x Mx-Cre mice were injected with PIPC. PIPC injection induced biallelic deletion of the Dot1l allele in the Dot1l fl/fl mice but not the Dot1l wt/wt mice. The Dot1l wt/wt LSKs and GMPs were compared to the Dot1l -/- counterparts by RNA extraction and Microarrays.
2014-09-08 | E-GEOD-54498 | biostudies-arrayexpress
Project description:Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5+ mouse intestinal stem cells and mature intestinal villus epithelium correlated with mRNA expression levels but not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5+ stem cells or in all intestinal epithelial cells eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. Absence of methylated H3K79 did not impair health, intestinal homeostasis or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo. Examination of differential gene expression between Dot1l control (Dot1 f/f) and Dot1l mutant (Villin-Cre, Dot1l f/f) villus cells.
2012-10-19 | E-GEOD-41710 | biostudies-arrayexpress
Project description:Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences
| PRJNA746964 | ENA
Project description:Somatic hypermutation (SHM) drives the genetic diversity of immunoglobulin genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators) but how the enhancers mediate this activity is unknown. We show that DIVACs that strongly stimulate SHM increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full length transcripts, indicating the accumulation of stalled Pol2. DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for anti-sense transcription or an association of H3K27ac with DIVAC activity in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVACs make the target gene a suitable platform for AID-mediated mutation without the need for increasing transcriptional output.
2021-10-22 | GSE180177 | GEO