ChIP-seq of Pbx1, Prep1 and Sp2 in WT MEFs, Sp2ko MEFs and in Sp2ko MEFs ectopically expressing Flag-tagged full length Sp2; and ChIPexo of Sp2 in WT and Sp2ko MEFs
ABSTRACT: We investigated the genomic binding profiles of the transcription factors Pbx1, Prep1, and Sp2 in mouse embryonic fibroblasts using ChIPseq and ChIPexo. Refer to E-MTAB-2970 and E-MTAB-994 for KO and IgG Controls.
Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its clo ...[more]
Project description:A novel missense mutation has been identified in a human pain-insensitive family in the gene ZFHX2. We modelled this mutation in mice by changing the orthologous amino acid and carried out pain behaviour tests in mutant BAC transgenic mice. These showed a phenotype of hyposensitivity to noxious thermal stimuli. In this experiment, we generated a stable cell line expressing the mutant human ZFHX2 protein in order to identify chromatin binding sites. Complementary microarray data from ZFHX2 mutant mice were also deposited at ArrayExpress under accession number E-MTAB-5650 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5650 ).
Project description:Investigation of the binding behaviour of Sp1, Sp2, Sp3 and NF-ya, NF-yb and NF-yc in mouse embryonic fibroblasts and of Sp1, Sp2 and Sp3 in HEK-293 cells reveals distinct binding of the seemingly similar transcription factors Sp1/3 and Sp2.
Project description:Investigation of the co-occurance of the ATP-dependent chromatin remodeler SMARCAD1 and the histone modification H3K9me3 in the genome of PGK12.1 XX ES cells. ChIP-seq of endogenous SMARCAD1 was carried out in control and SMARCAD1-knockdown ES cells using a double crosslinking procedure. Additionally, H3K9me3 ChIP-seq was performed in double-cross linked control PGK12.1 XX cells. Input samples and precipitates with an IgG antibody were sequenced (Illumina HiSeq1500) as controls. Antibodies used were SMARCAD1 PAB15737 (Abnova) and H3K9me3 ab8898 (abcam).
Project description:To understand the role of the SWI/SNF-like ATP-dependent chromatin remodeller SMARCAD1 in pluripotent murine embryonic stem (ES) cells we determined the genome wide binding of triple FLAG tagged SMARCAD1 in PGK12.1 XX ES cells. ChIP was carried out using an antibody against the FLAG-epitope (F1804, Sigma) in double-cross linked chromatin. As controls, input samples and ChIP of PGK12.1 XX ES cells transfected with the empty triple-flag vector were used. The precipitated DNA was subsequently sequenced on an Illumina HiSeq1500.
Project description:In order to identify the effects of starvation on the MEFs wt trascriptome, we performed Affymetrix Gene-Chip hybridization experiments for the starved cells Transcriptome analysis of the starved MEFs wt cells For the analysis on the starved MEFs wt cells, total RNA was extracted; RNA extracted from MEFs wt cells grown in Normal Medium was used as control
Project description:We performed ChIP-seq analyses of Rad2, Mediator (Med17 and Med5) and RNA Polymerase II in Kin28-ts16 mutant, Med17-Q444P and Med17-Q444P/M442L mutants and in an Rpb9 deleted strain.
Project description:C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg-/- MEFs proliferated poorly, entered senescence prematurely, and expressed a pro-inflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β~β) inhibited the proliferation and tumorigenicity of RasV12-transformed NIH3T3 cells, activated SASP gene expression, and recruited the CBP co-activator in a Ras-dependent manner, whereas γ~β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg-/- MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg-/- MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg-/- bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes. Mouse embryonic fibroblasts were isolated at E13.5 and propagated. At passage 3 cells were plated with equal density and collected 48 hours later.
Project description:The ATP dependent chromatin remodeler SMARCAD1 and the co-repressor KAP1 (Trim28/Tif1 beta) interact directly. We wanted to understand the interplay between these two proteins in the context of chromatin; in particular at repetitive sequences. Therefore, we carried out an investigation in E14 ES cells of the genome wide binding behaviour of KAP1 and FLAG-tagged SMARCAD1; both wild-type SMARCAD1 and a catalytically inactive mutant. The E14 cells analysed were either wild-type or depleted for endogenous SMARCAD1 protein. FLAG and KAP1 ChIP was performed on double-cross linked chromatin and sequenced on an Illumina HiSeq1500 with matched input controls. A FLAG antibody precipitation was carried out on cells not expressing FLAG tagged protein as an additional control.