GANP regulates recruitment of AID to immunoglobulin variable regions by modulating transcription and nucleosome occupancy

Singh, Shailendra Kumar; Maeda, Kengo; Eid, Mohammed Mansour Abbas; Almofty, Sarah Ameen; Ono, M.; Pham, Phuong; Goodman, Myron F.; Sakaguchi, Nobuo

doi:10.1038/ncomms2823

Cited by 47 publications

(55 citation statements)

References 52 publications

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“…Further, strong nucleosome positioning sequences have been shown to impair diversification (Kodgire et al , 2012). We therefore asked whether the reduced R‐loop formation in h3.3 cells reflected a change in the distribution of nucleosomes across the locus by mapping the average nucleosome positions in IGVL in wild‐type and h3.3 cells using micrococcal nuclease treatment coupled to quantitative PCR (Singh et al , 2013). This revealed two centres of nucleosome occupancy in IGVL of wild‐type cells (Fig 4B).…”

Section: Resultsmentioning

confidence: 99%

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

et al. 2016

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Immunoglobulin diversification is driven by activation‐induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single‐stranded DNA (ssDNA), the enzymatic substrate of AID. Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R‐loop formation. However, reducing IgV R‐loops by RNase HI overexpression in wild‐type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID‐accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single‐stranded, thereby creating an effective AID substrate.

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Section: Resultsmentioning

confidence: 99%

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

et al. 2016

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“…DNA regions with DSBs initiated by intensive targeting of AID must be repaired during the interphase before the mitotic phase of the cell cycle to rescue and generate IgV region diversity in daughter Ag-reactive B cells. Our proteomics analysis revealed that GANP is associated with DNA-PKcs prominently among the DNA repair molecules (34). Given that GANP interacts with DNA-PKcs, it might be the initial member of the DNA repair pathway interacting with the AID/GANP complex in situ at the IgV region loci in germinal center B cells.…”

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confidence: 88%

“…Samples were prepared with anti-H4 and anti-acetyl H4 Abs (Cell Signaling Technology), as previously described (33,34), and examined using Mesa Blue qPCR MasterMix Plus (Eurogentec) on the Applied Biosystems ViiA 7 with reported primers (37).…”

Section: Ganpmentioning

confidence: 99%

“…Recently, we demonstrated that GANP interacts with cytoplasmic AID and assists its nuclear localization to augment its transport to the transcribed IgV region segment in B cells (33). In this process, the histone acetyltransferase region of GANP is involved in the selective recruitment of GANP toward the terminal region of the rearranged IgV region exons and the site-selective acetylation of the linker histone H1 (34). Thus, AID is targeted to the selective DNA region of the rearranged IgV loci with the assistance of GANP.…”

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confidence: 99%

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GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

Eid

Maeda

Almofty

et al. 2014

The Journal of Immunology

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RNA export factor germinal center–associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining–proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment.

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“…B2 cells are selected as naive B cells of the non-self-reactive primary repertoire in the bone marrow, which are distributed to peripheral lymphoid organs. Ag stimulation with the help of Th cells induces rapid expansion of naive B cells in the germinal centers (GCs) of lymphoid follicles, where Ag-reactive B cells undergo somatic hypermutation (SHM) of IgV region genes and class switch recombination (CSR) at the S regions by AID (24)(25)(26)(27)(28)(29). Thus, B2 cells gain IgV region SHM and CSR in the GCs of lymphoid follicles to become high-affinity Ag-reactive and IgG Ab-producing plasma cells (30).…”

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confidence: 99%

JNK Regulatory Molecule G5PR Induces IgG Autoantibody–Producing Plasmablasts from Peritoneal B1a Cells

Kitabatake

Soma

Zhang

et al. 2015

The Journal of Immunology

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Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody–secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.

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GANP regulates recruitment of AID to immunoglobulin variable regions by modulating transcription and nucleosome occupancy

Cited by 47 publications

References 52 publications

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

JNK Regulatory Molecule G5PR Induces IgG Autoantibody–Producing Plasmablasts from Peritoneal B1a Cells

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