HIF-1 directly induces TET3 expression to redistribute 5-hydroxymethylcytosine and induce erythroid gene expression in hypoxia [RNA-seq]
Ontology highlight
ABSTRACT: We performed RNA-sequencing on erythropoietic cells cultured in normoxia or hypoxia at days 3,7, and 10. We found numerous expression changes induced by hypoxia, including the canonical hypoxia targets as well as a number of genes encoding genes involved in epigenetic processes.
Project description:We performed HIF-1alpha ChIP-seq in K562 cells cultured in normoxia vs. hypoxia for 3 days. We identified two new HIF binding sites in TET3 intron 2 that control TET3 expression in hypoxia
Project description:We characterized genome-wide 5-hydroxymerhylcytosine (5-hmC) distribution of CD34+ cells undergoing erythroid differentiation in normoxia vs. hypoxia by chemically labeling 5-hmC followed by pull-down and sequencing (hMe-SEAL). We found that hypoxia increases both the number and the prominance of 5-hmC peaks at each time point, especially at day 7 and 10.
Project description:In mammalian cells, cytosines found within cytosine guanine dinucleotides can be methylated to 5-methylcytosine (5-mC) by DNA methyltransferases and further oxidized by the Ten-eleven translocation dioxygenase (TET) enzymes to 5-hydroxymethylcytosine (5-hmC). We have previously shown that hematopoietic stem and progenitor cells (HSPCs) with TET2 mutations have aberrant 5-hmC distribution and less erythroid differentiation potential. However, these experiments were performed under standard tissue culture conditions with 21% oxygen (O2), whereas HSPCs in human bone marrow reside in ∼1% O2. Therefore, to model human erythropoiesis more accurately, we compared 5-hmC distribution and gene expression in hypoxic vs normoxic conditions. Despite TET enzymes having limited O2 as a substrate in hypoxia, 5-hmC peaks were more numerous and pronounced than in normoxia. Among the TET genes, TET3 was upregulated specifically in hypoxia. We identified 2 HIF-1 binding sites in TET3 by chromatin immunoprecipitation of HIF-1α followed by sequencing, and TET3 upregulation was abrogated with deletion of both sites, indicating that TET3 is a direct HIF-1 target. Finally, we showed that loss of one or both of these HIF-1 binding sites in K562 cells disrupted erythroid differentiation in hypoxia and lowered cell viability. This work provides a molecular link between O2 availability, epigenetic modification of chromatin, and erythroid differentiation.
Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells. 12 samples were analysed comprising 4 experimental conditions (normoxia scr, hypoxia scr, hypoxia siHIF1, hypoxia siHIF2) in triplicate. We made pairwise comparisons between the averages of each triplicate set to normoxia scr using the Partek suite.
Project description:PyMT cells (HIF-1 wild type, WT and HIF-1 knockout, KO) were compared for differential gene expression at normoxia or after exposure to hypoxia, 0.5% oxygen for 6h