Project description:In our model, p53 is acting through recruitment of SIRT1 to reduce histone acetylation. A common myeloid cell line THP-1 are p53 mutant cells that have a significant deletion in p53 coding sequence leading to loss of p53 expression.We immunoprecipitated chromatin in THP1 cells using antibodies against H3K27ac and SIRT1 to identify the bingding peak in NEAT1 gene promoter to confirm P53' function
Project description:ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands including cholesterol. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocyte and endothelial cell lines, THP1 and HUVEC, respectively. ChIP-on-chip analysis of ROR-alpha demonstrated that ROR-alpha may target more than 3000 genes in monocytic and endothelial cells.
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and the NPC-associated THSC/TREX-2 complex. Here we show that yeast sac3∆ and thp1∆ cells accumulate genome-wide replication obstacles as determined by the distribution of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Sac3 and its interacting partner Thp1 are preferentially bound in wild-type cells.
Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and the NPC-associated THSC/TREX-2 complex. Here we show that yeast sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells accumulate genome-wide replication obstacles as determined by the distribution of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Sac3 and its interacting partner Thp1 are preferentially bound in wild-type cells. ChIP-chip studies were perfomed with antibodies against Flag-tagged Thp1 and Sac3 proteins in wild-type cells of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells that were compared with Rrm3 in wild-type cells from Santos-Pereira et al., 2013 (accession number GSE50185).