Transcriptome analysis of naïve or stimulated WT and TRIM28 KO CD4 T cells (RNA-seq)
ABSTRACT: Critical role for TRIM28 and HP1b/g in the epigenetic control of T cell metabolic reprogramming and effector differentiation The study explores the role of gene silencing by TRIM28, an adaptor protein for histone binding modules , in CD4 T cell function. Overall design: Littermate and TRIM28KO naïve CD4 T cells.
Project description:Critical role for TRIM28 and HP1b/g in the epigenetic control of T cell metabolic reprogramming and effector differentiation The study explores the role of gene silencing by TRIM28, an adaptor protein for histone binding modules , in CD4 T cell function. Overall design: Littermate and TRIM28KO CD4 T cells, naïve or stimulated for indicated times with anti-CD3 (plate-bound) and anti-CD28 (soluble).
Project description:Critical role for TRIM28 and HP1b/g in the epigenetic control of T cell metabolic reprogramming and effector differentiation The study explores the role of gene silencing by TRIM28, an adaptor protein for histone binding modules , in CD4 T cell function. Overall design: Littermate and TRIM28KO CD4 T cells were sorted by flow cytometry, naïve or stimulated for 72h with anti-CD3 (plate-bound) and anti-CD28 (soluble) in Th0, Th1, Th2, Th17 or Treg conditions.
Project description:Critical role for TRIM28 and HP1b/g in the epigenetic control of T cell metabolic reprogramming and effector differentiation Overall design: The study explores the role of gene silencing by TRIM28, an adaptor protein for histone binding modules , in CD4 T cell function.
Project description:Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ? and ? isoforms (HP1?/?, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1?/? control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.
Project description:Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs.
Project description:The tripartite motif-containing protein 28 (TRIM28) is a transcription corepressor, interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of Trim28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. To understand the molecular mechanism of Trim28-mediated adipogenesis, the RNA-seq was performed to find out the possible Trim28-regulated genes. Dlk1 (delta-like homolog 1) was increased in Trim28 knockdown 3T3-L1 cells both untreated and induced to differentiation. Dlk1 is an imprinted gene and known as an inhibitor of adipogenesis. Further knockdown of Dlk1 in Trim28 knockdown 3T3-L1 would rescue cell differentiation. The epigenetic analysis showed that DNA methylation of Dlk1 promoter and differentially methylated regions (DMRs) was not altered significantly in Trim28 knockdown cells. However, compared to control cells, the histone methylation on the Dlk1 promoter was increased at H3K4 and decreased at H3K27 in Trim28 knockdown cells. Finally, we found Trim28 might be recruited by transcription factor E2f1 to regulate Dlk1 expression. The results imply Trim28-Dlk1 axis is critical for adipogenesis.
Project description:Since the first discovery in 1996, the engagement of TRIM28 in distinct aspects of cellular biology has been extensively studied resulting in identification of a complex nature of TRIM28 protein. In this review, we summarize core biological functions of TRIM28 that emerge from TRIM28 multi-domain structure and possessed enzymatic activities. Moreover, we will discuss whether the complexity of TRIM28 engagement in cancer biology makes TRIM28 a possible candidate for targeted anti-cancer therapy. Briefly, we will demonstrate the role of TRIM28 in regulation of target gene transcription, response to DNA damage, downregulation of p53 activity, stimulation of epithelial-to-mesenchymal transition, stemness sustainability, induction of autophagy and regulation of retrotransposition, to provide the answer whether TRIM28 functions as a stimulator or inhibitor of tumorigenesis. To date, number of studies demonstrate significant upregulation of TRIM28 expression in cancer tissues which correlates with worse overall patient survival, suggesting that TRIM28 supports cancer progression. Here, we present distinct aspects of TRIM28 involvement in regulation of cancer cell homeostasis which collectively imply pro-tumorigenic character of TRIM28. Thorough analyses are further needed to verify whether TRIM28 possess the potential to become a new anti-cancer target.
Project description:This study explores the genomic alterations that contribute to the formation of a unique subset of low-risk, epithelial differentiated, favorable histology Wilms tumors (WT), tumors that have been characterized by their expression of post-induction renal developmental genes (Subset 1 WT). We demonstrate copy neutral loss of heterozygosity involving 19q13.32-q13.43, unaccompanied by evidence for imprinting by DNA methylation. We further identified loss-of-function somatic mutations in TRIM28 (also known as KAP1), located at 19q13, in 8/9 Subset 1 tumors analyzed. An additional germline TRIM28 mutation was identified in one patient. Retrospective evaluation of previously analyzed WT outside of Subset 1 identified an additional tumor with anaplasia and both TRIM28 and TP53 mutations. A major function of TRIM28 is the repression of endogenous retroviruses early in development. We depleted TRIM28 in HEK293 cells, which resulted in increased expression of endogenous retroviruses, a finding also demonstrated in TRIM28-mutant WT. TRIM28 has been shown by others to be active during early renal development, and to interact with WTX, another gene recurrently mutated in WT. Our findings suggest that inactivation of TRIM28 early in renal development contributes to the formation of this unique subset of FHWTs, although the precise manner in which TRIM28 impacts both normal renal development and oncogenesis remains elusive.
Project description:A microarray study was performed in unstimulated and TCR-stimulated CD4 + T cells and Treg in wild type and conditional Trim28 KO mice to identify genes that are regulated by Trim28. These experiments constitute a portion of the study described below: Paper Abstract: Peripheral T cell activation and differentiation into specialized effectors are regulated by TCR- and cytokine-mediated signals that induce clonal expansion and unique transcriptional factors. These processes may include active chromatin modification by nuclear factors. In search of such molecules, we found Trim28, a component of large nuclear chromatin-regulatory complex is tightly controlled upon TCR stimulation at the level of phosphorylation, and examined global impact of Trim28 loss in especially CD4+ T cells, by generating T cell-specific conditional Trim28 KO mice (CKO). CD4+ T cells from CKO mice showed defective IL-2 production and T cell proliferation associated with defective upregulation of cell-cycle associated proteins. Accordingly, young CKO showed T-lymphopenia. Surprisingly, Trim28 CKO mice eventually accumulated auto-reactive memory-phenotype T cells that produced inflammatory IL-17. CKO mice are also susceptible to induced auto-inflammatory disease with TH-17 dominant immune response. Loss of Trim28 showed aberrant accumulation of TH-17 and FoxP3+ T cells, two key T cells in inflammation vs. tolerance. We found CKO T cells showed a cell-extrinsic promotion of TH-17 and FoxP3+ T cell development by a mechanism involving overproduction of TGF-beta. Our study revealed unexpected roles of Trim28, a global chromatin regulator in both T cell activation and tolerance. Trim28 conditional KO mice and age-matched control mice were sacrificed, and neive CD4+ T cells (CD4+CD62+CD25-) and Treg (CD4+CD62+CD25+) were sorted. Stimulation of naive T cells was done with anti-CD3 and anti-CD28 for 13 hours. We collected quadruplicates for each group.
Project description:T-cell receptor-? (TCR?) rearrangement in CD4(+)CD8(+) double-positive immature thymocytes is a prerequisite for production of ?? T cells and invariant natural killer T cells. This developmental event is regulated by the TCR? enhancer (E?), which induces chromatin modification and recruitment of the recombination-activating proteins Rag1 and Rag2. However, the molecular mechanism underlying the activation and long-range action of E? remains incompletely understood. We show here that the chromatin-modifying factor TRIM28 is highly expressed in double-positive thymocytes and persistently phosphorylated at serine 473. TRIM28 binds to E? and induces histone 3 lysine 4 trimethylation in the E? and distant regions of the TCR? locus, coupled with recruitment of Rag proteins. T-cell-conditional ablation of TRIM28 impaired TCR? gene rearrangement and compromised the development of ?? T cells and invariant natural killer T cells. These findings establish TRIM28 as a unique regulator of thymocyte development and highlight an epigenetic mechanism involving TRIM28-mediated active chromatin modification in the TCR? locus.