Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences [GRO-seq]
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PROVIDER: PRJNA746967 | ENA |
REPOSITORIES: ENA
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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 | GSE180176 | GEO
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
Project description:Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences [ChIP-Seq]
| PRJNA746968 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below.
2021-10-22 | GSE180178 | GEO
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:Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes
| PRJNA705367 | ENA
Project description:Immunoglobulin gene rearrangement and somatic hypermutation have the potential to create neoantigens in non-Hodgkin B cell lymphoma. However, the presentation of these putative immunoglobulin neoantigens by B cell lymphomas has not been proven. We used MHC immunoprecipitation followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) to define antigens presented by follicular lymphomas (FL), chronic lymphocytic leukemias (CLL), diffuse large B cell lymphoma (DLBCL) and mantle cell lymphomas (MCL). We found presentation of the clonal immunoglobulin molecule, including neoantigens by both class I and class II MHC, though more commonly in class II MHC. To determine whether B cell activation could promote presentation of immunoglobulin neoantigens, we used a toll-like receptor 9 (TLR9) agonists to upregulate expression of MHC-II. This resulted in enhanced class II MHC presentation of the immunoglobulin variable region including neoantigens. These findings demonstrate that immunoglobulin neoantigens are presented across most subtypes of B cell lymphomas. Activation of lymphoma cells to upregulate antigen presentation boosts presentation of immunoglobulin neoantigens and represents a strategy for augmenting lymphoma immunotherapies.
2019-01-07 | PXD010808 | Pride
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:The H3.3 histone variant and its chaperone HIRA are involved in active transcription but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells is not yet fully understood. In this study we show that, knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of immunoglobulin heavy chain (IGH) in the human Ramos B cell line without changing the levels of AID and other major proteins known to be involved in SHM. Except for H3K79me2/3, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells and this was accompanied by decreased nascent transcription in IGH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the Ig locus was also reduced in the HIRA KO. Somewhat surprisingly HIRA loss increased the chromatin accessibility of Ig V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IGH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription coupled chromatin structure and accessibility contribute to SHM in human B cells.
2021-07-29 | GSE180899 | GEO