The Binding of GATA-1 to a β-Globin LCR HS Core Element in Nuclear Chromatin Requires Multiple Associated Factor Binding Sites.
Authors: Lowrey Christopher H. , Ackley Catherine , Ermentrout Mitchell 
Published: April 2020

Abstract
The GATA-1 transcription factor has been shown to interact in vitro with many gene regulatory elements including promoters, enhancers, silencers and locus control region elements. The GATA-1 consensus binding sequence is generally considered to be WGATAR, yet it can also bind to other related sequences that do not perfectly fit this consensus. This raises the question of whether all potential GATA-1 binding sites identified by in vitro DNA binding assays or sequence analysis are actually bound by GATA-1 in nuclear chromatin. We hypothesized that it was unlikely that all, or even most, predicted GATA-1 sites were bound in vivo. If correct, this would imply that additional mechanisms, beyond DNA sequence, are required to target GATA-1 to specific genetic regulatory elements. To address this hypothesis we first performed GATA-1 ChIP assays in MEL cells at 10 WGATAR sites which spanned the mouse β-globin locus but were not associated with known regulatory elements. Positive controls for GATA-1 binding were the core region of LCR HS2 and the -2.8 mGATA-1 gene upstream enhancer. Negative controls included two regions from the β-globin locus which do not contain WGATAR elements and a WGATAR element from the proximal mGATA-1 gene promoter previously shown by the Bresnick lab to not bind GATA-1. While enrichment of the positive control sites using anti-GATA-1 antibody was 10-20-fold more than with IgG (p&lt;0.001), binding at none of the 10 test WGATAR sites was enriched. This result implied that conditions other than the presence of the WGATAR consensus sequence are required to specify GATA-1 binding in nuclear chromatin. To better understand this process we decided to more closely evaluate the binding of GATA-1 to β-globin LCR HS core elements. We first tested the idea that GATA-1 was able to bind these sites due to high-level affinity based on the presence of tandem-inverted WGATAR elements and the known ability of GATA-1 molecules to self-interact. However, when tested in gel shift assays, GATA-1 binding to the LCR HS core elements had a very rapid off-rate which was much faster than a known high-affinity control site from the murine α-globin promoter. This led us to next test the idea that surrounding factor binding sites were required for GATA-1 binding to the LCR HS cores. Using human LCR HS4 as a model, we systematically assembled combinations of binding sites (3 KLF sites and 1 NF-E2 site) known to flank the tandem-inverted WGATAR elements and tested these artificial HS cores for GATA-1 binding after integration into MEL cell chromatin. The wild-type HS4 core served as a positive control and a fragment of luciferase cDNA served as a negative control for GATA-1 binding. Results for each cell line were normalized to the internal positive control -2.8 mGATA-1 gene site. Our experiments showed that only when all known flanking sites were present was GATA-1 binding detected. Similarly, DNase HS formation and histone H3 acetylation only occurred when all of the surrounding sites were present. Finally, we used ChIP to show that the assembled complex, with all sites present, includes BRG1 a component of the swi/snf nucleosome remodeling complex. These results indicate that GATA-1 targeting to the LCR HS cores involves complex, multi-protein interactions including histone acetylation and nucleosome displacement. Similar mechanisms are likely to be involved in targeting this and other transcription factors to functionally important sites in the genome.