Project description:Pax5, a transcription factor, is expressed exclusively in pro B cells. To understand the genes regulated by Pax5 in epithelial cells, we overexpressed Pax5 in lung cancer cell line A549 and examined the change of transcription profile using Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray (Agilent Technologies).
Project description:To investigate the genes regulated by the transcription factor GFI1 in lung cancer cells, we overexpressed GFI1-Flag in A549 cells and performed ChIP-Seq with anti-Flag affinity gel.
Project description:The aim of this study is to identify SMAD3 binding targets affected by the TGFb1/SMAD3 signal transduction on a genome-wide scale Formaldehyde cross-linked, sonicated chromatin was prepared from non-stimulated A549 cell line and stimulated with TGFb1. Chromatin immunoprecipated with anti-SMAD3 antibody is labeled with Cy5 and mock IP (with anti-FLAG antibody) is labeled with Cy3 and co-hybridized on Agilent Human Promoter Set arrays. Two biological replicates were performed.
Project description:Hypomorphic mutations of the transcription factor PAX5 occur in one third of B-progenitor acute lymphoblastic leukemias (B-ALLs). To identify PAX5-regulated genes in B-ALL, here we employ inducible expression of PAX5 in a human B-ALL cell line (REH) that harbors a loss-of-function mutation in PAX5. In this model, inducing PAX5 expression is associated with competitive disadvantage.
Project description:Heterochromatin has a high density of DNA and low rates of gene transcription.H3k9me3 is a conserved histone modification, and is best known for its role in constitutive heterochromatin formation.H1155, which is a neuroendocrine large cell lung cancer cell line, has a dense nucleus and a high level of occupancy of H3K9me3. PAX5 is a neurogenesis and B lymphocyte development transcription factor, and endogenous expressed in neuroendocrine carcinoma, including H1155 cell line. To assess whether PAX5 promotes heterochromatin formation in H1155, we knock out PAX5 in H1155 cell (KO-PAX5) by CRISPR/Cas9 and use H3K9me3 to performed ChIP-seq.
Project description:PAx5 is indispensible for the committment if early lymphoid progenitors to the B cell lineage as well as for the development and maintenance of B cells. To better understand the functional importance of Pax5 in the later stages of B cell development and investigate the targets of Pax5 regulation, we established a novel Pax5 deficient DT40 B cell line.
Project description:In lymphomas derived from mature B cells the expression of the transcription factor PAX5 is maintained whereas classical Hodgkin lymphoma displays significantly reduced PAX5 expression despite its derivation from mature B cells. To elucidate the functional role of PAX5 in classical Hodgkin lymphoma, we re-established the PAX5 expression in the Hodgkin cell line L428 with and without epigenetic modulation. To this end, we stably transfected the Hodgkin cell line L428 with an inducible PAX5 expression construct. Although the overexpressed PAX5 was transcriptionally active as demonstrated by synthetic reporter constructs, no induction of the B-cell phenotype was achieved. PAX5 chromatin immunoprecipitation with subsequent next generation sequencing in B-cell lines and the PAX5 overexpressing L428 cell line showed different binding patterns. Since epigenetic restrictions might affect PAX5 binding, combined DNA demethylation and histone acetylation was performed. However, no re-expression of B-cell genes was observed also under these conditions. Thus, PAX5 is not sufficient for the re-activation of the B-cell program in Hodgkin cells despite epigenetic opening of the chromatin. This clearly indicates that the repression of the B-cell identity of the Hodgkin cells is caused and secured by complex molecular mechanisms. Analysis of genome-wide PAX5 binding sites in B-cell lines (Raji, Namalwa) and the PAX5-producing Hodgkin cell line L428-PAX5 by ChIP-Seq
Project description:Hypomorphic mutations of the transcription factor PAX5 occur in one third of B-progenitor acute lymphoblastic leukemias (B-ALLs). To identify PAX5-regulated genes in B-ALL, here we employ inducible expression of PAX5 in a human B-ALL cell line (REH) that harbors a loss-of-function mutation in PAX5. In this model, inducing PAX5 expression is associated with competitive disadvantage. Comparison of REH cell lines with Dox-inducible expression of PAX5-IRES-GFP, or control GFP alone. GFP positive cells were isolated by FACS.
