Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation
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ABSTRACT:
PROVIDER: PRJNA913946 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22814732.fastq.gz | Fastqsanger.gz | |||
| SRR22814733.fastq.gz | Fastqsanger.gz | |||
| SRR22814734.fastq.gz | Fastqsanger.gz | |||
| SRR22814735.fastq.gz | Fastqsanger.gz | |||
| SRR22814736.fastq.gz | Fastqsanger.gz |
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Project description:The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.
2022-12-23 | GSE221351 | GEO
Project description:The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.
| S-EPMC9869049 | biostudies-literature
Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.
| S-EPMC2196218 | biostudies-literature
Project description:Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.
| S-EPMC3093487 | biostudies-literature
Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation induced deaminase (AID), a single-stranded DNA cytidine deaminase that is thought to engage its substrate in the context of RNA polymerase II (RNAPII) transcription. Through a loss of function genetic screen, we identified numerous potential factors involved in SHM including ELOF1, a component of the RNAPII elongation complex that has been shown to function in DNA repair and transcription elongation. Loss of ELOF1 strongly compromises SHM, CSR, and AID targeting and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.
| S-EPMC11463689 | biostudies-literature
Project description:Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal "blunt end-joining," but impaired end joining with partially complementary (1-3 bp) DNA ends. There was also an increased usage of microhomology at the mu-alpha switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (> or =10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia-mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.
| S-EPMC2118080 | biostudies-literature
Project description:Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6(-/-) clones, whereas mutations were dispersed in Msh3(-/-) and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.
| S-EPMC2213309 | biostudies-literature
Project description:Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARFBP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. MIZ1 and MYC ChIPseq experiments in HUWE1 inhibitor-treated Ls174T cells as well as RNAseq experiments in HUWE1- or MIZ1-depleted Ls174T cells after HUWE1 inhibitor treatment. Sequencing was performed on an Illumina Genome Analyzer IIx.
2014-10-21 | E-GEOD-59223 | biostudies-arrayexpress
Project description:Expression and activity of activation-induced cytidine deaminase (AID) encoded by the aicda gene are essential for immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR). SHM and CSR unfold, in general, in germinal centers and/are central to the maturation of effective antibody responses. AID expression is induced by activated B-cell CD40 signaling, which is critical for the germinal center reaction, and is further enhanced by other stimuli, including interleukin-4 (IL-4) secreted from CD4+ T cells or Toll-like receptor (TLR)-activating bacterial and/or viral molecules. Integration of different intracellular signal transduction pathways, as activated by these stimuli, leads to a dynamic aicda-regulating program, which involves both positively acting trans-factors, such as Pax5, HoxC4, E47, and Irf8, and negative modulators, such as Blimp1 and Id2, to restrict aicda expression primarily to germinal center B cells. The phosphatidylinositol 3-kinase (PI 3-K), which functions downstream of activated B-cell receptor (BCR) signaling, likely plays an important role in triggering the downregulation of aicda expression in postgerminal center B cells and throughout plasmacytoid differentiation. In B cells undergoing SHM and CSR, AID activity, and, possibly, AID targeting to the Ig locus are regulated at a posttranslational level, including AID dimerization/oligomerization, nuclear/cytoplasmic AID translocation, and phosphorylation of the AID Ser38 residue by protein kinase A (PKA). Here, we discuss the role of B-cell activation signals, transcription regulation programs, and posttranslational modifications in controlling aicda expression and AID activity, thereby delineating an integrated model of modulation of SHM and CSR in the germinal center reaction.
| S-EPMC2994649 | biostudies-literature
Project description:Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) gene class switch recombination (CSR), somatic hypermutation (SHM), and somatic hyperconversion. In general, high AID expression is found in mature B cells that are responding to antigens. However, AID expression and SHM have also been detected in developing B cells from transgenic mice that have a limited Ig repertoire. Here we demonstrate that AID expression, ongoing CSR, and active SHM occur in developing B cells from wild-type mice. Further, our results suggest that somatic variants arising from developing B cells in the bone marrow further diversify in the spleen of unimmunized mice. AID expression in developing B cells is T cell independent but involves engagement of B cell receptors and Toll-like receptors. Early AID expression can increase the preimmune repertoire of developing B cells, may provide an innate population of IgG- and IgA-expressing cells, and could be involved in receptor editing of self-reactive immature B cells.
| S-EPMC2082107 | biostudies-literature