Project description:We used ChIP-Seq to map GABP-alpha binding sites in human hematopoietic progenitor cells (HPCs). Coupled with functional assays using GABP-alpha deficient mouse model and bioinformatics analysis, we systematically determined a transcriptional module controlled by GABP in HPCs. Examination of the role of GABP in hematopoietic stem cells
Project description:We used ChIP-Seq to map GABP-alpha binding sites in human hematopoietic progenitor cells (HPCs). Coupled with functional assays using GABP-alpha deficient mouse model and bioinformatics analysis, we systematically determined a transcriptional module controlled by GABP in HPCs.
Project description:Alpha lipoic acid is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors. Here, we provide new evidence of alpha lipoic acid in promoting erythroid differentiation by targeting transcription factor ELK1 in CD34+CD371– hematopoietic stem progenitor cells. Over expression of both L-ELK1 and S-ELK1 greatly inhibit erythroid cell differentiation, but not knocking down of ELK1. Thus, RNAseq of CD34+CD123+CD38+CD371– HSPCs is performed to dissect the molecular mechanism of ELK1 in blocking erythrocyte differentiation.
Project description:Ets family transcription factor GA-binding protein (GABP) regulates gene expression in CD4 and CD8 T cells. We used microarray to examine genes differentially expressed in GABP-sufficient (WT) and GABP-deficient (KO) CD4 and CD8 T cells
Project description:Epigenetic processes regulate hematopoietic stem cell homeostasis. The recent discovery of 5-hydroxymethylcytosine (5hmC) provides new insights into the epigenetic regulation of gene expression during development. We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize the genome-wide distribution of 5hmC in human CD34+ hematopoietic stem/progenitor cells, lymphocytes, monocytes and granulocytes. We show that 5hmC levels decrease during cell differentiation and that 5hmC is associated with H3K4me1 and H3K27ac histone modifications, indicative of active enhancers. In CD34+ cells, the presence of 5hmC at presumed active enhancers correlates with increased binding of RUNX1 and FLI1, two transcription factors essential for hematopoiesis. Moreover, in progenitor cells, 5hmC poises the expression of transcription factors regulating hematopoietic lineage commitment, such as RUNX1, FOXO1 and C/EBP-alpha. Our study provides the first comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and of its potential role in transcriptional network regulation during hematopoiesis.
Project description:The mammalian blood system is a hierarchically structured tissue. Hematopoietic stem cells (HSC) reside at the top of this hierarchy and the HSC-derived progenitor cells (HPC) have capacities for both differentiation and self-renewal. Systematic study of the regulatory mechanisms of HSC self-renewal and differentiation is fundamentally important for understanding hematopoiesis and for manipulating HSCs for therapeutic purposes. Herein, the mechanisms regulating HSC biology were studied on a genome-wide scale by integrating epigenomic, transcriptomic, proteomic, and protein-protein interaction data. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent hematopoietic progenitor cell line, EML (Erythroid, Myeloid, and Lymphoid). In the present study, we report binding maps for three additional TFs (SOX4, STAT3, and GABP) that were developed by chromatin immunoprecipitation (ChIP)-Sequencing. Assay for Transposase Accessible Chromatin (ATAC)-Sequencing was applied to globally identify the open chromatin regions associated with TF binding in the self-renewing subpopulation lin-CD34+.Mass spectrometry (MS) was also used to identify proteins and assess differences in their relative abundances. We found that MAPK (Mitogen-activated protein kinase) pathway and JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway components were highly enriched among the binding targets of these TFs in CD34+ cells, and that the TGF-β/SMAD signaling pathway comprised a sub-network of molecules that were differentially expressed between the lin-CD34+ and partially differentiated lin-CD34- cellular states. The present study integrates regulatory information at multiple levels to paint for the first time a more comprehensive picture of the mechanisms underlying the decision between HPC self-renewal and differentiation. We anticipate that this work will provide valuable clues for understanding the molecular switch that controls cell fate decisions between self-renewal or differentiation and blood formation.
Project description:Human embryonic stem cells (hESCs) are a powerful tool for modeling regenerative therapy. To search for the genes that promote hematopoietic development from human pluripotent stem cell, we overexpressed a list of hematopoietic regulator genes in human pluripotent stem cell-derived CD34+CD43- endothelial cells (ECs) enriched in hemogenic endothelium. Among genes tested, only SOX17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45-/low cells expressing a hemogenic endothelial maker VE-cadherin. SOX17 was highly expressed in CD34+CD43- ECs but at a low level in CD34+CD43+CD45- pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. SOX17-overexpressing cells formed sphere-like colonies and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies upon inactivation of SOX17. Global gene expression analyses revealed that the CD34+CD43+CD45-/low cells expanded upon overexpression of SOX17 are hemogenic endothelium-like cells developmentally placed between ECs and pre-HPCs. Of interest, SOX17 also reprogrammed both pre-HPCs and HPCs into hemogenic endothelium-like cells. Genome-wide mapping of SOX17 revealed that SOX17 directly activates transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation. Depletion of SOX17 in CD34+CD43- ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a critical role in priming hemogenic potential in ECs, thereby regulates hematopoietic development from hESCs. This SuperSeries is composed of the SubSeries listed below.
Project description:We knocked out different exons corresponding to different sections of GABP, a transcription factor known to bind to the telomerase reverse transcriptase (TERT) promoter with the G228 mutation. We investigated the effects of the knockouts in the regulation of TERT expression and other subunits of GABP. GABP genomic binding sites were determined through chromatin immunoprecipitation sequencing (ChIP-seq), and gene expression was determined through total RNA sequencing (RNA-seq). Combining data pertaining to GABP binding sites and gene expression provided insight into the molecular mechanisms of maintaining the length of telomeres in cancers.