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Gene expression in mature CD4 and CD8 from WT mice and mature CD4 from Lz-Thpok or Mta2-Lz-Thpok retrogenic mice

ABSTRACT: Gene expression in mature CD4 and CD8 from WT mice and mature CD4 from Lz-Thpok or Mta2-Lz-Thpok retrogenic mice

PROVIDER: PRJNA720298 | ENA |

REPOSITORIES: ENA

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Gene expression in mature CD4 and CD8 from WT mice and mature CD4 from DBTBLZ or Mta2-DBTBLZ retrogenic mice

Project description:T cell precursors develop into cytotoxic CD8 and helper CD4+ T cells in thymus. Thpok, a BTB/POZ family transcription factor, enforces commitment to the CD4 lineage and suppresses cytotoxic gene expression. However, it is still not fully understood how Thpok directs CD4 T cell development. Here, using mass-spectrometry, we identify nucleosome remodeling and deacetylase (NuRD) complex as a novel Thpok cofactor. We demonstrate that the Thpok BTB domain is essential for NuRD recruitment. Reconstituting NuRD binding to a BTB-less version of Thpok (which cannot bind NuRD) restored CD4 T cell in vivo. RNA sequencing showed that CD4 T cells expressing the reconstituted protein express a transcriptome similar to that of CD4 T cells expressing Thpok. Thus, our results demonstrate that NuRD recruitment is both necessary and sufficient for the function of the Thpok BTB domain in CD4 T cell development.

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Project description:The transcription factor Thpok is essential for CD4 T cell development in the thymus and remains expressed in post-thymic CD4 T cells. We post-thymically inactivated Thpok and compared microarray gene expression in Thpok-deficient CD4 T cells to that in their wildtype CD4 or CD8 counterparts We show that Thpok constrains the transcriptional circuitry to maintain CD4+-lineage integrity in naM-CM-/ve cells and to couple effector differentiation to environmental cues after antigenic stimulation. Redundantly with the related factor LRF, Thpok is continuously needed to prevent the trans-differentiation of mature CD4+ into -CD8+ T cells. We activated naM-CM-/ve CD4 T cells (either wild-type or Thpok-deficient) and CD8 T cells (wild-type) in vitro under Th1 conditions. Differentiated effectors were sorted 4 days after activation into CD4+CD8- and CD4-CD8+ (wild-type) and CD4+CD8- and CD4+CD8+ (Thpok-deficient) subsets. Total RNA was extracted from sorted subsets and processed for microarray analyses (Affymetrix Mouse Exon 1.0 ST array) at the NCI microarray facility, following the manufacturerM-bM-^@M-^Ys recommendation. Data is from 3 replicates (except wild-type CD4-CD8+ cells, for which two samples only were processed), generated from two distinct cell preparations.

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Transcriptional Reprogramming of Mature CD4 T helper Cells generates distinct MHC class II restricted Cytotoxic T Lymphocytes

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Gene Expression in Effector T cells, wildtype of Thpok-deficient

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NuRD complex recruitment to Thpok mediates CD4+ T cell lineage differentiation

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The NuRD complex is required for Thpok-mediated CD4+ T cell development

Project description:This data represent characterization of the interactome of murine Thpok. A version of Thpok (Thpok-Bio) subject to biotinylation in vivo by the BirA biotin ligase was used; to assess Thpok interactions in primary cells, Thpok-Bio was retrovirally expressed in in vitro activated CD4+ T cells from Thpokfl/fl Ox40-Cre+ mice carrying a Rosa26BirA allele. Expression of Ox40-Cre, initiated upon T cell activation, causes the deletion of endogenous Thpokfl alleles, so that transduced cells only express Thpok-Bio. Two Thpok deletion constructs (BKK and RFK) were also analyzed. Following streptavidin pulldown, proteins were separated by SDS-PAGE, the full lane cut into 9 slices, and in-gel digested. FIles are: 1384 - empty vector; 1385 - Thpok; 1386 - Thpok + Ethidium Bromide; 1387 - Thpok BKK; 1388 - Thpok RFK.

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2022-06-10 | GSE185217 | GEO

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