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CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors.

ABSTRACT: The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4(+) splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.

SUBMITTER: Blonska M

PROVIDER: S-EPMC3311756 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors.

Blonska Marzenna M Joo Donghyun D Zweidler-McKay Patrick A PA Zhao Qingyu Q Lin Xin X

Journal of immunology (Baltimore, Md. : 1950) 20120227 7

The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARM ...[more]

PMID: 22371397

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Project description:The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional activator but also as a repressor.

Project description:BackgroundThe transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.MethodsImmunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).ResultsGATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.ConclusionsWe propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.

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CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors.

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CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors.

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