The DSIF subunits Spt4 and Spt5 have several unexpected, distinct roles in regulating immunoglobulin class switch recombination
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE33206 | GEO | 2013/10/22
SECONDARY ACCESSION(S): PRJNA149425
REPOSITORIES: GEO
Similar Datasets
Project description:Class-switch recombination (CSR), induced by activation-induced cytidine deaminase, can be divided into two phases: DNA cleavage of the switch (S) regions, and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in CH12F3-2A cells, and Spt4 and Spt5, carry out independent functions in CSR. Depleting Spt5 reduced the H3K4me3 levels and DNA cleavage at the S? region, whereas Spt4 knockdown did not perturb the H3K4me3 status or S region cleavage. Thus, we conclude that Spt5 plays a role similar to the histone chaperone FACT by regulating histone modification and DNA cleavage in CSR. Assay systems using synthetic repair substrates revealed that both Spt4 and Spt5 are required for non-homologous end joining and homologous recombination. Taken together, Spt4 and Spt5 are involved in multiple CSR steps, and can function independently. Spt4 or Spt5 were knocked down in CH12F3-2A cells in the presence of CD40L, IL4, TGFb (CIT) stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi-treated, CIT-stimulated samples.
2013-10-22 | E-GEOD-33206 | biostudies-arrayexpress
Project description:H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation (SHM) in AID target loci. H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci, in both S and variable regions, and in non-IgH loci, such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting approximately 5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Spt6 is a unique histone chaperone, capable of regulating the histone epigenetic state of both AID targets and the AID locus. CH12F3-2A cells were transfected with control and Spt6 siRNAs; 24h later, cells were stimulated with CIT to induce CSR. Total RNA was extracted from control and Spt6 siRNA treated cells for mRNA expression profiling.
2013-02-01 | E-GEOD-35334 | biostudies-arrayexpress
Project description:Aim and Methods: To understand the precise role of Spt4 in budding yeast, various high throughput sequencing techniques were used. Nucleosome positions were studied using MNase-seq in WT and spt4∆ cells. The position of RNAPII was examined using NET-seq in spt4∆ cells or cells in which Spt4 has been anchored away to the cytoplasm (Spt4 AA). RNAPII was also mapped in cells in which Spt5 has been anchored away to the cytoplasm (Spt5 AA). Steady-state transcript levels were assessed using RNA-seq in samples coupled with NET-seq. The position of the Spt4 on RNAPII was mapped at single nucleotide resolution using TEF-seq. With the same technique, the position of Spt5 on RNAPII was investigated in wild type (WT) and spt4 knock-out (spt4∆) cells. The position and levels of the pre-initiation complex were assessed using ChIP-seq on Sua7(TFIIB) in WT and spt4∆ cells. Results: The interaction between the Spt4/5 complex and RNAPII periodically changes as RNAPII transitions through nucleosomes. The dynamic interaction of Spt5 with RNAPII is dependent on Spt4. In spt4∆ and Spt4 AA cells, RNAPII distribution is altered in a similar way compared to their respective controls. After transcribing into nucleosomes, RNAPII accumulates upstream of nucleosome dyads, especially around the +2 nucleosome. In Spt5 AA cells, the distribution and amount of RNAPII on genes are severely affected. Nucleosome positions are altered in spt4∆ cells. Although the position of the +1 nucleosome remains unchanged, nucleosome spacing from this point is increased, and the level of increase in nucleosome spacing correlates with the level of RNAPII accumulation. Conclusion: This work suggests that Spt4 regulates nucleosome positioning by a mechanism related to transcription and promotes RNAPII movement through nucleosome barriers, especially the barrier set by the +2 nucleosome.
2021-09-27 | GSE159291 | GEO
Project description:H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation (SHM) in AID target loci. H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci, in both S and variable regions, and in non-IgH loci, such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting approximately 5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Spt6 is a unique histone chaperone, capable of regulating the histone epigenetic state of both AID targets and the AID locus.
2013-02-01 | GSE35334 | GEO
Project description:The shieldin (SHLD) complex, composed of SHLD1, SHLD2, SHLD3 and MAD2L2/REV7, acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end-resection and promote non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and for repair of RAG-induced DSBs in XLF-deficient cells, the role of SHLD in these activities remains elusive. Here, we report that contrary to 53BP1, SHLD1 is dispensable for lymphocyte development and V(D)J recombination, even in the sensitized XLF-deficient background or for the joining of distant V(D)J segments. By contrast, SHLD1 restricts resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR. Finally, we show that this end-protection function is required for orientation-specific joining of AID-initiated DSBs. We propose that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms; the synapsis of V(D)J segments and switch regions within chromatin independently of SHLD and the protection of AID-DSB ends against resection mediated by SHLD.
2022-05-11 | GSE202567 | GEO
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.
2016-08-19 | E-GEOD-84102 | biostudies-arrayexpress
Project description:The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline (micronucleus - MIC) and somatic nuclei (macronucleus - MAC), massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. As we were mostly interested in transcription of the germline genome in the MIC during meiosis, we decided to probe the composition of the Spt5-Spt4 complex characteristic to this nucleus. Experiments in which Spt4mB-3xFLAG was used as bait demonstrated its interaction with either Spt5m or Spt5v, which is consistent with the Spt4mB-GFP localization pattern. Interestingly, mass spectrometry revealed that Spt5m-3xFLAG (which is present only in the MIC at the analyzed stage) interacts either with the meiotic Spt4mB or with Spt4vA. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.
2022-04-27 | PXD027748 | Pride
Project description:To generate antibodies with different effector functions, B cells undergo Immunoglobulin class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical non-homologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ~25% CSR can be achieved by the alternative end-joining (A-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ mediated repair of endonuclease generated breaks requires DNA end-resection, we show that CtIP-mediated DNA end-resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppress resection without altering the MH-pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ mediated CSR by suppressing inter-chromosomal translocations independent of end-resection. Finally, temperol analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favoriate substrates for MH-mediated end-joining contributing to the robustness and resection-indepndence of A-EJ-mediated CSR.
2020-09-18 | GSE156392 | GEO
Project description:A set of 22 expts. aimed at identifying splicing events dependent upon on the Spt4-5 transcription elongation factors in yeast. Four spt mutants and an mRNA capping mutant were analyzed four times each, including biological and technical (dye-swap) replicates. Two wt vs wt expts. were also performed. Keywords = transcription/spt5/spt4/splicing/DEDS
2010-06-09 | E-GEOD-1610 | biostudies-arrayexpress
Project description:IgH class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical non-homologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining (A-EJ) that is more biased to use longer junctional micro-homologies (MHs). Deficiency for DSB response (DSBR) factors, including ATM and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B lymphoma cells. Compared to wild-type, CSR and c-Myc to S region translocation junctions in the absence of 53BP1, and to a lesser extent other DSBR factors, have increased MH-utilization; indeed, 53BP1-deficient MH-profiles resemble those associated with C-NHEJ deficiency. Yet, translocation junctions between c-Myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-Myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.
2018-01-08 | GSE106922 | GEO