Effect of Hls5 on FOG-1 and GATA-1. (A) Schematic representation of Hls5 and FOG-1 with various domains displayed. The boxed area of FOG-1 bound Hls5 in the yeast 2-hybrid screen. (B) Hls5 bound FOG-1 through its B box, coiled coil domains, as determined by yeast 2 hybrid analysis. (C) Hls5 bound full-length FOG-1 in in vitro pull down assays. COS cells were transfected with Flag-tagged FOG-1 and lysates were immunoprecipitated with anti-Flag antibodies in the presence of [³⁵S]-labeled Hls5. (D) COS cells were transfected M1α reporter together with various combinations of Vector, GATA-1, FOG-1 and Hls5 (200 ng each). Constant DNA levels were maintained by varying the amount of pcDNA3; Hls5 did not affect expression of GATA-1 or FOG-1 in these transient transfection experiments. Luciferase activity was determined using the Dual-Luciferase system (Promega, Madison, WI) after 36 hours, normalized to the activity of a Renilla reporter plasmid (pRL-TK), and are shown relative to GATA-1 activation (100). Error bars represent SD (n = 3). (E) COS cells were transiently transfected with Hls5 alone (top panel), or Hls5 and GATA-1 together (bottom panel). Hls5 and GATA-1 were visualized using anti-myc and anti-GATA-1 antibodies, and confocal microscopy. DNA was identified by Hoescht staining. Images were acquired as in Figure 1C. (F) The GATA-1 and Hls5 interaction was demonstrated by transfecting COS cells with myc-tagged Hls5 and GATA-1, followed by immunoprecipitation (IP) and immunoblotting (IB). Vertical lines have been inserted to indicate a repositioned gel lane. (G) Yeast 2-hybrid analyses demonstrated that the B Box, Coiled Coil domains of Hls5 were required for GATA-1 binding. (H) Yeast 2-hybrid assays showed that the GATA-1 N-terminal finger domain was required for binding to Hls5. (I) Hls5 alone (200–500 ng) was able to repress GATA-1 transcriptional activity of the M1α promoter, as described in panel D. Error bars represent SD (n = 3). (J) Electrophoretic mobility shift assay using a radiolabeled GATA-1 oligonucleotide and J2E cell nuclear extracts revealed a GATA-1/DNA complex, which decreased with increasing amounts of purified Hls5. (K) Chromatin immunoprecipitation assay of GATA-1 recruitment to the β globin promoter in J2E (vector-alone) and J-Hls5 cells. GATA-1 binding, relative to input DNA, was expressed as a percentage of J2E (vector-alone) cells. Similar data were obtained for the HS2 site.