GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T

GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling at the Th2 cytokine gene loci. genes and inhibition of locus in an Ruvbl2-dependent manner. The defect in the proliferation of (p16 ink4a) (p15 ink4b) (p18 ink4c) and (p19 ink4d) negatively regulate the activity of the cyclin D-CDK4/6 complex and block the G1-S phase transition halting cellular proliferation in nonimmune cells (17). has been implicated in the regulation of T-cell proliferation supported by the observation that T cells from is restrained by GATA3 in mammary luminal progenitor cells the transcriptional regulation of this gene in Th2 cells is yet to be fully elucidated (19). We herein identified a Gata3/RuvB-like protein 2 (Ruvbl2) FASN complex as a key regulatory mechanism of Th2 cell proliferation via repression of locus and together they repress the expression of the mRNA expression was detected (Fig. S2 Asaraldehyde (Asaronaldehyde) and knockout (Is Repressed in a Gata3- and Ruvbl2-Dependent Manner. Earlier reports demonstrated that Gata3 regulates cell cycle in luminal progenitor cells and neuroblastoma cell via control of and expression respectively (19 21 Thus we next assessed the expression of and in primary Th1 and Th2 cells from wild-type or expression was not detected in primary Th1 and Th2 cells the expression of was lower in Th2 cells compared with Th1 cells and the depletion of in Th2 cells resulted in increased expression of (Fig. 3expression was up-regulated in primary Th2 cells when Ruvbl2 was silenced by siRNA (Fig. 3is repressed in primary Th2 cells in a Gata3- and Ruvbl2-dependent manner. Fig. 3. The expression of controls the Gata3-dependent proliferation of Th2 cells. (mRNA in Th1 WT Th2 WT … To identify Gata3-bindng sites around the locus we performed a chromatin immunoprecipitation assay followed by Asaraldehyde (Asaronaldehyde) a massive parallel sequencing (ChIP-Seq) analysis using 3xFlag-Gata3-expressing Th2 clone cells (D10G4.1). Statistics of the tags generated for the experiment are summarized in Fig. S3loci) was confirmed (Fig. S3 and locus (Intron2 and +7.5-kb regions) (Fig. S3locus (Fig. 3was observed in primary Th2 cells compared with Th1 and Th17 cells in the previously reported ChIP-seq analysis for endogenous Gata3 (Fig. S3G3BS (+7 261 ~ +7 760 (Fig. S4) was placed at the 5′-end of the promoter (?500) and luciferase reporter assays were performed (Fig. 3promoter whereas insertion of a G3BS with three mutations at the GATA consensus binding sequence did not show any effects (Fig. 3and Fig. S4). These results indicate that Gata3 binds directly to the locus and represses the mRNA expression of Expression Rescued the Impaired Proliferation of mRNA expression in and Expression. The GATA family transcriptional factors (Gata1 to -6) typically bind to a consensus motif (A/T)GATA(A/G) and regulate the specification and differentiation of numerous tissues. All GATA family members share two highly conserved C2H2-type zinc fingers both of which are involved in DNA binding and protein-protein interactions (22 23 Two transactivation domains Asaraldehyde (Asaronaldehyde) are also known Asaraldehyde (Asaronaldehyde) to Asaraldehyde (Asaronaldehyde) be important for the function of Gata3 (24). We examined which domains of Gata3 were important for the binding to Ruvbl2. Flag-tagged wild-type or deletion mutants of Gata3 (as depicted in Fig. S5 and and was up-regulated in the Gata3 or Ruvbl2 knockdown 68-41 cells (Fig. S5expression whereas the dTA mutant did not repress the expression of expression. Ruvbl2 Is Necessary for the Recruitment of Gata3 to the Locus in Th2 Cells. To further investigate the molecular requirements for the Gata3-mediated repression of expression in primary T cells we used differentiating Th2 cells from whereas the dTA mutant did not show any effect in the G3BS region was significantly compromised (Fig. 4G3BS region was impaired in Ruvbl2 KD Th2 cells (Fig. 4G3BS region in Th2 cells. Taken together these results suggest that the association of Ruvbl2 with Gata3 is required for the binding of Gata3 to the G3BS region. Fig. 4. Ruvbl2 is necessary for the recruitment of Gata3 at the locus in developing Th2 cells. (Locus Induced by the Expression of Gata3 and Ruvbl2. We previously reported that the polycomb group (PcG) gene product Bmi1 associates with Gata3 and controls the stability of the Gata3 protein in Th2 cells (25). In addition Ruvbl2 was previously shown to be a component of the PcG complex in (26 27 Thus we next investigated the histone modifications at the locus particularly.