Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

Abstract

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

Global transcriptome and chromatin occupancy analysis reveal the short isoform of GATA1 is deficient for erythroid specification and gene expression

“…We also observed strong enrichment of binding motifs for ETS family transcription factors, which are known to co-operate with GATA1 in megakaryocytic differentiation (60% and 52%). 4,43 We then mapped each peak location to the closest transcription start site and assigned the peaks to the categories: upstream (2-100kb), promoters (0-2kb), intragenic, downstream (0-100kb), and gene desert (>100kb). This analysis revealed that the GATA1 peaks were significantly enriched at promoters and intragenic regions and significantly depleted in gene desert regions compared to randomly distributed genomic regions, consistent with the expected locations for gene regulatory regions ( Figure 3E).…”

“…As a master transcriptional regulator, GATA1 directs global gene expression to specify terminal erythroid or megakaryocytic fate. [1][2][3][4][5] It accomplishes this by both activating lineage specifying genes and repressing progenitor maintenance genes through a variety of gene regulatory mechanisms that depend on context-dependent co-factor interactions. 6 Importantly, GATA1 interacts with and controls the expression of many other lineage-specifying transcription factors to co-ordinately repress factors promoting other cell fates while activating those of megakaryocytes and erythrocytes.…”

Maps of open chromatin highlight cell type–restricted patterns of regulatory sequence variation at hematological trait loci

“…After antibody incubation, 500 mL washing buffer PBE buffer (PBS [Sigma-Aldrich] at pH 7.2, 2 mM EDTA [Sigma-Aldrich], and 0.5% BSA [Sigma-Aldrich]), and 5 mg/mL 7-amino actinomycin D (7-AAD; Invitrogen) were added. Flow cytometric analysis of MKs and EBs was performed according to the method previously described (Macaulay et al 2007;Tijssen et al 2011) using the following antibodies: human anti-CD41a-APC clone HIP8, anti-CD42a-FITC clone ALMA.16, anti-CD235a-FITC clone GA-R2 (HIR2), and anti-CD34-PE clone 581 (BD Biosciences). All samples were analyzed on the CyAn ADP 9-Color flow cytometer using the software Summit v4.3.02 (Beckman Coulter).…”

“…Supplemental Table 2 and Supplemental Table 3 give an overview of the peak data sets. ChIP-seq data sets in primary MKs were obtained from Tijssen et al (2011) (GEO accession no. GSE24674).…”

Pathogenesis and management of inherited thrombocytopenias: rationale for the use of thrombopoietin-receptor agonists

“…The main transcription factors that regulate Mk differentiation and maturation are RUNX1, FLI1, GATA-1, GATA-2, NF-E2, and TAL1/SCL. These factors act synergistically, often bound in complexes, for the coordinated activation and repression of genes required for development of mature Mks [15]. Mk development is also regulated by a complex cross-talk between Mk and cells, cytokines, and extra-cellular matrix (ECM) proteins of the surrounding bone marrow (BM) microenvironments [13].…”