Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of Gsk-3-Deficient Embryonic Stem Cells Reveals Large-Scale Alterations in Gene Expression

ABSTRACT: The two vertebrate Gsk-3 isoforms, Gsk-3a and Gsk-3b, are encoded by distinct genetic loci and exhibit mostly redundant function in murine embryonic stem cells (ESCs). Here we report that deletion of both Gsk-3a and Gsk-3b in mouse ESCs results in significant changes in gene expression. In contrast, deletion of either Gsk-3a or Gsk-3b individually had little effect on gene expression. These data support the notion that Gsk-3 isoforms are functionally redundant in embryonic stem cells. In addition, we did not find the expected upregulation of known Wnt target genes. Our data suggests that Gsk-3-meidated regulation of gene expression in embryonic stem cells is complex, and likely involves affects on numerous signaling pathways. The study was designed to examine the changes in gene expression between wild-type, Gsk-3a-/-, Gsk-3b-/-, and Gsk-3a-/-;Gsk-3b-/- mouse embryonic stem cells.

ORGANISM(S): Mus musculus

SUBMITTER: Christopher Phiel

PROVIDER: E-GEOD-27395 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:GSK-3b plays a critical role in regulating the Wnt/b-catenin signaling pathway, and manipulating GSK-3b in dendritic cells (DCs) has been shown to improve anti-tumor efficacy of DC vaccines. Since inhibition of GSK-3b leads to activation of b-catenin, we hypothesize that blocking GSK-3b in DCs negatively regulates DC-mediated CD8 T cell immunity and anti-tumor immunity. Using CD11c-GSK-3b-/- conditional knockout mice in which GSK-3b is genetically deleted in CD11c-expressing DCs, we surprisingly found that deletion of GSK-3b in DCs resulted in increased anti-tumor immunity, which contradicted our initial expectation of reduced anti-tumor immunity due to presumed upregulation of b-catenin in DCs. Indeed, we found by both western blot and flow cytometry that deletion of GSK-3b in DCs did not lead to augmented expression of b-catenin protein, suggesting that GSK-3b exerts its function independent of b-catenin. Supporting this notion, our single cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3b-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated b-catenin. This suggests that deletion of GSK-3b in DCs is unlikely to lead to upregulation of b-catenin at the transcriptional level. Consistent with enhanced anti-tumor immunity, we also found that CD11c-GSK-3b-/- mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that deletion of GSK-3b in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3b in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although deletion of GSK-3b in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3b-/- mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3b in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3b exerts its functions independent of its regulation of b-catenin.

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Analysis of Gsk-3-Deficient Embryonic Stem Cells Reveals Large-Scale Alterations in Gene Expression

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