Discovering Hematopoietic Mechanisms through Genome-wide Analysis of GATA Factor Chromatin Occupancy

Abstract

SUMMARY GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

CDC6 expression is regulated by lineage-specific transcription factor GATA1

“…Also, the surrounding bases appear to follow a preference pattern, and C/G precedes the W, while a G preferentially follows the final A. [21][22][23] Second is that GATA1-regulated region is located at more than 3,000 bp upstream CDC6 promoter and functions as an enhancer when placed with a minimal promoter, in agreement with the observation that 90% of the sites occupied in vivo are located in were found in this 100 bp region, one of them being adjacent to GATA1 binding site (Fig. 3C), in a similar position as in other previously described regulatory regions of GATA1 controlled genes ( Fig.…”

“…41 Also, GATA1 inhibits cell proliferation through control of many genes involved in cell cycle regulation, as revealed by microarray expression analysis 15 and, more significantly, by ChIP-seq approach. 21,22 Among the identified putative cell cycle targets, E2f4 and Nek6, together with CDC6 are under direct control of GATA1. Most significantly, p21 has recently been shown to be directly regulated by GATA1.…”

“…In fact, only a small fraction of the in vitro characterized binding sites are actually occupied, according to various independent genome-wide occupancy analysis. [21][22][23] These reports have defined with further precision GATA1 preferred binding motif and unveiled a rich collection of targets that suggests a more global action of GATA 1 that would affect not only the lineage-specific program, but also cell processes such as signaling and cell cycle control.…”

“…19,20 GATA1 also cooperates with master hematopoietic regulator Scl/Tal1 at composite sites containing (CANNTG)-WGATAR sequences. 21,22 However, simple identification of putative single double, or composite GATA binding sequences, even when phylogenetically conserved, is a poor predictor of their use by GATA1. In fact, only a small fraction of the in vitro characterized binding sites are actually occupied, according to various independent genome-wide occupancy analysis.…”

“…In fact, transcriptional control of most of these lineages genes has been shown to depend on GATA1 activity. 15,21,22,39,40 However, final differentiation and maturation of these cells is accompanied by changes in cell proliferation and therefore in cell cycle status: megakaryocytic maturation is characterized Mutagenesis Kit (Stratagene). The DNA sequence of all mutant and/or tag-fused cDNAs were verified accordingly.…”