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.

Transcriptional networks orchestrate complex developmental processes. Such networks are commonly instigated by master regulators of development. Considerable progress has been made in elucidating GATA factor-dependent genetic networks that control blood cell development. GATA-2 is required for the genesis and/or function of hematopoietic stem cells, whereas GATA-1 drives the differentiation of hematopoietic progenitors into a subset of the blood cell lineages. GATA-1 directly represses Gata2 transcription, and this involves GATA-1-mediated displacement of GATA-2 from chromatin, a process termed a GATA switch. GATA switches occur at numerous loci with critical functions, indicating that they are widely utilized developmental control tools.

© F e r r a t a S t o r t i F o u n d a t i o nsequence of primers and probes used in this study are listed in the Online Supplementary Tables. Polymerase chain reaction-based quantitative chromatin immunoprecipitationReal-time PCR-based quantitative chromatin immunoprecipitation (ChIP-qPCR) analysis was conducted essentially as previously described. 22 Electrophoretic mobility shift assayElectrophoretic mobility shift assay (EMSA) was performed using "DIG Gel Shift Kit, 2 nd Generation" (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol. Sequences of oligonucleotides for probes are indicated by the horizontal bar in the relevant figures. Nuclear extracts were prepared, as described previously, 23 from K562 cells or HEK293 human embryonic kidney cells that were transfected with a GATA1-FLAG fusion protein expression vector or its backbone vector. Promoter/enhancer activity assaysEach target DNA fragment was prepared from genomic DNA from normal volunteers (WT) or patients with CSA (referred to as "GGTA" or "delGATA" in each reporter construct) and was cloned into pGL3basic plasmid (Promega Corporation, Madison, WI, USA). The human ALAS2 proximal promoter region (g.4820_5115, between -267 and +29 from the transcription start site) 16,24 was cloned into the multiple-cloning site of pGL3basic [referred to as pGL3-AEpro (-267)]. A single DNA fragment (5.2 kbp), carrying the ALAS2 proximal promoter, first exon, first intron and the untranslated region of the second exon, was subcloned into the multiple cloning site of pGL3basic [referred to as pGL3-AEpro(-267)+intron1]. A DNA fragment containing the GATA1-binding region in the first intron of the ALAS2 gene (corresponding to g.7488_7960), which was defined by ChIP-seq analysis, 22 is referred to as the ChIP-peak. The length of the WT ChIP-peak is 473 bp. In addition, a 130-bp fragment containing ALAS2int1GATA, the consensus sequence for the GATA1-binding site in the ChIP-peak, is referred to as ChIPmini. Several deletion mutants of ChIPmini were prepared using pGL3-AEpro(-267)+ChIPmini(WT) as a template. The pGL3-TKpro plasmid was constructed by cloning herpes simplex virus thymidine kinase promoter into the multiple cloning site of pGL3basic plasmid. Each reporter vector and pEF-RL25 were introduced into K562 cells or HEK293 cells. Luciferase activity was determined using a dualluciferase reporter system (Promega).Disruption of a GATA binding element causes CSA haematologica | 2014; 99 (2) 253 Figure 1. Identification of a functional GATA1 element in the first intron of the ALAS2 gene. (A) Chromatin immunoprecipitation assay. Fragmented genomic DNA segments were immunoprecipitated with anti-GATA1 antibody or control IgG, and then precipitated fragments were quantified using real-time PCR as described in the Online Supplementary Methods. PC or NC indicates positive control or negative control, respectively, for the ChIP assay using anti-GATA1 in K562 cells. 22 One GATA element is present in the proximal promoter region and ...

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element −1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the −1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the −1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed.

SUMMARY GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1-independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.