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DYRK1A regulates B cell class switch recombination through phosphorylation of MSH6

ABSTRACT: Effective protection from viral infections depends on the antibody-specific isotype. Here, we found that the protein kinase, DYRK1A, is essential for B cell-mediated protection from viral infection and vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells were impaired in CSR activity in vivo and in vitro. Phospho-proteomic screens and kinase-activity assays identified MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site attenuated CSR. After CSR and germinal center seeding, DYRK1A was required for proper clonal expansion of antigen-specific B cells through attenuation of proliferation. These findings reveal DYRK1A-mediated biological mechanisms of antibody-mediated immune responses that may be used for manipulation in antibody-mediated autoimmunity

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, B Cell

SUBMITTER: Meital Kupervaser

LAB HEAD: Ziv Shulman

PROVIDER: PXD034156 | Pride | 2023-03-16

REPOSITORIES: Pride

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			Action	DRS
	HF2_ZS_12620_KO_10_IgM_03062021.raw	Raw
	HF2_ZS_12620_KO_10_LPS_03062021.raw	Raw
	HF2_ZS_12620_KO_11_IgM_03062021.raw	Raw
	HF2_ZS_12620_KO_11_LPS_03062021.raw	Raw
	HF2_ZS_12620_KO_2_IgM_03062021.raw	Raw

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DYRK1A regulates B cell class switch recombination through phosphorylation of MSH6

Project description:Effective protection from viral infections depends on the antibody-specific isotype. Here, we found that the protein kinase, DYRK1A, is essential for B cell-mediated protection from viral infection and vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells were impaired in CSR activity in vivo and in vitro. Phospho-proteomic screens and kinase-activity assays identified MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site attenuated CSR. After CSR and germinal center seeding, DYRK1A was required for proper clonal expansion of antigen-specific B cells through attenuation of proliferation. These findings reveal DYRK1A-mediated biological mechanisms of antibody-mediated immune responses that may be used for manipulation in antibody-mediated autoimmunity

2022-11-27 | GSE206146 | GEO

Transcription profiling of primary human astrocytes from brain treated by stathmin, poly IC and ultrapure LPS to examine the effects of different Toll-like receptor agonists

Project description:Goal of the experiment was to examine the effects of different Toll-like receptor agonists on the human astrocyte response, in termsof levels of transcripts encoding cytokines, chemokines and theirreceptors. Stathmin and poly IC are considered as agonist for TLR3, LPSas an agonist for TLR4. The manuscript we are about to submit to Journal of Immunology contains these data, and identifies stathmin, aprotein, as a novel TLR3 agonist. We all know that poly IC is, and bycomparing the transcript responses in astrocytes to either poly IC andstathmin, and observing the similarity in these responses, the experiment adds to the evidence that stathmin indeed activates a verysimilar cellular response as poly IC. The LPS-induced response,mediated by TLR4, is included as a reference, to illustrate thatanother TLR-mediated reaction produces quite something different.

2010-02-11 | E-TABM-851 | biostudies-arrayexpress

A damage independent role for 53BP1 that impacts break order and Igh architecture during CSR

Project description:During Class Switch Recombination (CSR) B cells replace the Igh CÎ¼/Î´ exons with another downstream constant region exon (CH), altering the antibody isotype. CSR occurs through the introduction of AID-mediated double strand break (DSBs) in switch regions and subsequent ligation of broken ends. Here we developed an assay to investigate the dynamics of DSB introduction in individual cells. We demonstrate that the upstream switch region SÎ¼ is always targeted first during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR. High Resolution 4C was performed in resting and activated B cells using a bait on the EÎ¼ enhancer

2016-06-16 | E-GEOD-81180 | biostudies-arrayexpress

Identification of genes involved in class switch recombination by genome-wide CRISPR/Cas9 knock-out screen

Project description:This screen aims to identify genes involved in class switch recombination (CSR), a process that allows B cells to change the isotype they express from IgM/IgD to IgG, IgA or IgE. For that purpose, the murine B cell line CH12 stably expressing Cas9 was used. This cell line can be induced to undergo CSR from IgM to IgA very efficiently in vitro when cultured with TGF-b, IL-4 and an anti-CD40 antibody. CH12Cas9 cells were transduced with the second-generation mouse Brie (mBrie) retroviral gRNA library (pMX-mBrie) and selected with puromycin for two weeks to allow for the Cas9-mediated generation of knockouts. Cells were then stimulated to undergo CSR for 72h. Those which failed to undergo CSR (which remained IgM+) or that succeed (which became IgA+) were sorted using magnetic beads. Genes, whose loss-of-function affect the efficiency of CSR, were identified by looking at gRNA enrichment in the IgM+ versus IgA+ populations through deep sequencing.

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MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation

Project description:Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

2023-12-14 | PXD047325 | Pride

RNA-sequencing of DYRK1A-deficient (CKO) pre-B and pre-T cells

Project description:Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells’ proliferation, but little is known about its post-translational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a non-phosphorylatable Cyclin D3 T283A mutant recapitulated these defects, while inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development. 5 cell populations were analyzed (small pre-B cells, large pre-B cells, quiescent CD4+CD8+ thymocytes, cycling CD4+CD+ thymocytes, and mature granulocytes) from 2 Control mice (pooled) and 2 DYRK1A-deficient mice (pooled) for a total of 10 samples.

2015-05-01 | E-GEOD-67052 | biostudies-arrayexpress

DNA Double-strand Break Response Factors Influence End-joining Features of IgH Class Switch and General Translocation junctions

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

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2021-04-23 | PXD020293 | Pride

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

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

Comparison of phosphorylation status of Wnt proteins in control and Fto deficient cells upon Wnt 3a stimulation

Project description:Wnt Phospho protein profiling comparing control and Fto deficient cell after Wnt3a stimulation Control vs Fto deficient MEFs treated with Wnt 3a condioned medium for 40 minutes. 6 replicates per array

2013-11-21 | E-GEOD-52572 | biostudies-arrayexpress

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