Characterization of a Functional ZBP-89 Binding Site That Mediates Gata1 Gene Expression during Hematopoietic Development

Kinuko Ohneda,Shin'Ya Ohmori,Yasushi Ishijima,Mayu Nakano,Masayuki Yamamoto

doi:10.1074/jbc.m109.026948

Abstract

GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.

Highlights

Four discrete cis-acting regions have been identified as playing essential roles in the activity of the Gata1-HRD
We found that five contiguous deoxyguanosine residues (G5 string) within the GCrich sequence are essential for Gata1 gene reporter expression in vivo
These data indicate that the G5 string is necessary for Gata1 gene expression and binding of ZBP-89 to this region is critical for the G1HE activity in vivo

Summary

A Functional ZBP-89 Binding Site in the Gata1 Gene

A Kruppel-type zinc finger transcription factor ZBP-89 ( referred to as zfp148, BERF-1, and BFCOL1 [13,14,15]) was recently identified as a novel GATA-1-associated transcription factor in both erythroid cells and megakaryocytes [16]. Erythroid and megakaryocyte differentiation is severely attenuated in the absence of ZBP-89 in zebrafish and mouse embryos [16, 18], overlapping with the phenotype of Gata1deficient animals (19 –21) These findings confirmed that ZBP-89 plays essential roles for Gata gene regulation during hematopoietic development. Because ZBP-89, Sp1, and EKLF associate with GATA-1, these factors might be able to act as cofactors and direct DNA binding of these factors might not be necessary To test these possibilities, we examined the GC-rich region in the G1HE by transgenic mouse reporter assays and gel mobility shift assays (EMSAs). We show that the G4 mutation significantly reduced the Gata1HRD reporter activity in fetal liver of transgenic mice These data indicate that the G5 string is necessary for Gata gene expression and binding of ZBP-89 to this region is critical for the G1HE activity in vivo

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION