Project description:The MHC class II transactivator (CIITA) is essential for the expression of MHC-II genes. CIITA functions as a transcriptional activator when bound to genes although repressive roles have been reported at some loci in other cell types. To define the role of CIITA in gene regulation in human B cells, we performed ChIP-seq for CIITA using three independent antibodies, the histone marks H3K4me3 and H3K27ac, and ATAC-seq in the Raji human B cell line. Additionally, we profiled H3K27ac and ATAC-seq in the CIITA-null RJ2.2.5 cell line. A core set of CIITA peaks were identified that overlapped in all antibodies. Using the H3K27ac, H3K4me3, and ATAC-seq profiles generated in CIITA+ Raji and CIITA-null RJ2.2.5 cells CIITA dependent chromatin modifications were identified. These data define the CIITA regulated genes in human B cells.
Project description:Bulk RNAseq analysis was done on single cell clones of U2OS cells either expressing exogeonous CIITA or an empty vector control to identify genes regulated by CIITA expression.
Project description:Acute myeloid leukemia cell lines were treated with the hypomethylating agent decitabine and interferon gamma to investigate if these treatments induce HLA II gene expression. Cells carrying either control or CIITA-targeting sgRNAs were used to test the dependence of the HLA II induction on CIITA.
Project description:Osteolytic destruction is a hallmark of multiple myeloma, resulting from activation of osteoclast mediated bone resorption and reduction of osteoblast-mediated bone formation. However, the molecular mechanism underlying the differentiation and activity of osteoclasts and osteoblasts within a myelomatous microenvironment remains unclear. Here, we demonstrate that the osteocyte-expressed major histocompatibility complex class II transactivator (CIITA) contributes to myeloma-induced bone lesions. CIITA upregulates the secretion of osteolytic cytokines from osteocytes through acetylation at histone 3 lysine 14 in the promoter of TNFSF11 (encoding RANKL) and SOST (encoding sclerostin), leading to enhanced osteoclastogenesis and decreased osteoblastogenesis. In turn, myeloma cell–secreted 2-deoxy-D-ribose, the product of thymidine catalyzed by the function of thymidine phosphorylase, upregulates CIITA expression in osteocytes through the STAT1/IRF1 signaling pathway. Our work thus broadens the understanding of myeloma-induced osteolysis and indicates a potential strategy for disrupting tumor-osteocyte interaction to prevent, or treat patients with, myeloma bone disease.
Project description:Description of differentially expressed genes between KMH2 CIITA-BX648577 knockdown cultures and non-silencing controls To determine the change in gene expression after siRNA interference with the fusion transcript CIITA-BX 648577 identified in KMH2 cells.
Project description:Ciita has been suggested to control the expression of a number of genes based on ChIP-Seq or reporter anaysis but in vivo regulation beyong MHC class II has largely not been confirmed. We crossed Ciita knock out mice to Zbtb46 GFP knock-in knock out mice to identify classical dendritic cells in vivo in a Ciita deficient background. Expression microarray analysis of CD24+ and CD172a+ DCs from homeostatic spleen or activated BM Flt3l cultures show a highly resitrcited transcriptional footprint of Ciita. These findings underscore the fact that canonical binding sites active in transient expression assays may not always be functional in vivo. In addition, ChIP-Seq alone is not sufficient to discriminate binding from control of expression of a nearby gene.
Project description:CD4+ T cell responses are crucial for inducing and maintaining effective anti-cancer immunity, and the identification of HLA-II cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of CIITA coupled to a detailed and sensitive mass spectrometry based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17 and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared to IFNɣ treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor-antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.
Project description:Analysis of H3ac, H4ac, STAT1 and IRF1 binding in IFNg treated and untreated HeLa cells for 6 hours was done using 50mer oligonucletide probes at 30bp intervals tiling over non-repetitive 122kb CIITA locus(HG17) Keywords: ChIP-chip