Specificity Protein 1 (Sp1) Consensus Binding Sequences Flank Many Functional GATA-1 Binding Sites and Are Sufficient To Allow GATA-1 Binding to a WGATAR Sequence in Nuclear Chromatin.

GATA-1 is an hematopoietic transcription factor known for its roles in erythropoiesis and erythroid gene expression. In vitro, GATA-1 readily binds to WGATAR and closely related sequences. However, in vivo, GATA-1 appears to bind only a small subset of potential sites - those associated with gene regulatory elements. It is currently unclear what allows stable binding of GATA-1 at these sites in nuclear chromatin. We hypothesized that the binding of factors adjacent to WGATAR sites may “open” the local chromatin region to allow GATA-1 access to binding sites, while other factors may stabilize GATA-1 binding. To begin to test this model, we examined 11 well-characterized regulatory elements which are known to bind GATA-1 to determine whether there were any common associated binding sites. To do this we used the Transcription Element Search Software (TESS). These sites were from the human β-globin LCR HSs 2–4, human and mouse β-globin promoters, human γ-globin, NF-E2, EKLF and SCl/TALI (proximal and distal) promoters and the chicken 3′ β-globin enhancer. This analysis revealed that in each case, Sp1 consensus binding sites were found flanking the WGATAR elements. While this association between Sp1 and GATA binding motifs has been noted before, the reason for this association has not been determined. On average, the 5′ Sp1 motifs were located 18 +/− 8 bp upstream of the WGATAR elements, and the 3′ Sp1 motifs were located 30 +/− 17 bp downstream. To determine whether these flanking Sp1 elements were sufficient to allow GATA-1 binding in chromatin, we developed a new approach based on the Bouhassira lab’s recombinase mediated cassette exchange (RMCE) system. This system allows us to design oligonucleotides up to ~120 bp in length containing clusters of test binding sites and then insert them as single copies into a defined genomic location in MEL cells. ChIP experiments, analyzed by quantitative RT-PCR, can then be used to characterize factor binding and local histone modification. The positive internal control for GATA-1 binding and histone H3 acetylation was the −2.8G region of the mGATA-1 promoter. The negative control was the murine necdin gene promoter. As an additional positive control, we inserted the 100 bp LCR HS4 core element and found, as expected, that it was able to mediate GATA-1 binding. We then inserted 3 different GATA-1 binding elements and two derivative sites which have been shown to bind GATA-1 with high affinity in vitro. None of these sites alone was able to bind GATA-1 in our test system. We next placed one of the high affinity sites, a GATA-PAL motif from the murine gata-1 gene promoter, between two exact Sp1 consensus sequences (GGGGCGGGG) located 18 bp upstream and 30 bp downstream of the WGATAR element. When tested in our system, the flanking Sp1 elements were sufficient to allow GATA-1 binding to the test site at levels equivalent to those seen for the positive control site. As expected, no GATA-1 bound when the Sp1 sites were used alone, but H3 histones in the region did become hyperacetylated, indicating that factors interacting with the Sp1 direct localized histone acetylation and mediate GATA-1 binding. These results begin to provide a mechanism for GATA-1 targeting to sites in nuclear chromatin and provide an explanation for the observed association of WGATAR and Sp1 binding elements within erythroid gene regulatory elements.