Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus

Abstract

The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at ؉9.5 kb. Despite indistinguishable GATA-1 occupancy at ؊77 kb and ؉9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the ؊77 kb region. The ؊77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1-and GATA-2-expressing cells. By contrast, the ؉9.5 kb region exhibited considerably stronger enhancer activity in GATA-2-than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1-or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the ؊77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

A network including TGFβ/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells

“…Based on our findings that TGFβ has a direct effect on Gata2 through Smad4 ( Fig 4A) we observed direct binding of Smad4, to the -77kb upstream enhancer of the Gata2 gene 39,40 (Fig 4G), strengthening the observations from our gene expression analysis. We could also detect binding of Smad4 to a Smad7 intronic region (identified from our ChIP-Seq data) suggesting Smad7 as a direct target of TGFβ signaling in these cells (Fig 4G).…”

“…We verified these observations using qPCR of Gata2-bound chromatin in both Lhx2 and primary cells. The known -77kb Gata2 enhancer 39,40 as well as the Smad7 intronic region and the p57 promoter region identified from the ChIP-Seq were all enriched by GATA2 immunoprecipitation in Lhx2 cells (Fig 4C-D). Binding to the p57 promoter was further confirmed in a second multipotent cell line (Fig S1) -as well as in primary cells (Fig 4E-F) where the minimal amount of material needed for reliable ChIP-PCR results limited us to the use of LSK cells.…”

Mechanism governing a stem cell-generating cis -regulatory element

“…A quantitative ChIP assay was conducted as described previously (8). Cells cross-linked with 1% formaldehyde were sonicated to yield DNA with an average size of 200-700 bp and immunoprecipitated with specific antibodies [rabbit anti-GATA-1 (68), rabbit anti-GATA-2 (68), and rabbit anti-TAL1 (11)], LDB1 (N18, sc11198), and BRG1 (ab110641).…”

BRG1 requirement for long-range interaction of a locus control region with a downstream promoter

“…3C analysis was conducted as described (15,45). A 190-kb BAC (RP23-370E12) clone containing sequences from Ϫ100 to ϩ92 kb of the murine ␤-globin locus was used to assess primer .…”

“…ER-GATA-1 induces a loop at c-Kit, which correlates with repression (43), whereas it represses Gata2 (44) without disrupting a Gata2 loop (45). c-Kit and Gata2 mRNAs are expressed in WT and Brg1-mutant fetal livers, with expression being Ϸ2-fold higher in Brg1-mutant fetal liver (Fig.…”