H1 physically interacts with STAT92E. Protein-protein interactions between purified STAT92E and H1 were examined in vitro by GST pull-down and ChIP. (A) GST and GST fusion proteins with full-length H1, HP1 or H2A were expressed and purified from E. coli and analyzed by GST pull-down with baculovirus-expressed purified recombinant STAT92E-His6. The pull-down samples were examined by SDS-PAGE and Coomassie staining (top) or immunoblotting with anti-His6 antibody (bottom). As a control, 10% of the input STAT92E-His6 was examined. (B) Binding of STAT92E and Su(var)3-9 to reconstituted chromatin was analyzed by in vitro ChIP. Oligonucleosomes were reconstituted on supercoiled plasmid DNA with purified native core histones, with (H1+, dark-gray bars) or without (H1–, light gray bars) purified native H1. Non-sequence specific binding to the plasmid (DNA, white bars) was also examined. His6-tagged recombinant proteins were incubated with chromatin/DNA templates, cross-linked, immunoprecipitated with anti-His6 antibody and occupancy was measured by real-time PCR of a fragment of the plasmid. The occupancy of proteins relative to input was normalized to occupancy on naked DNA and plotted. The presence of H1 in chromatin templates strongly stimulates binding of both STAT92E and Su(var)3-9. All ChIP experiments were performed in duplicate, and each biological sample was analyzed by PCR in triplicate. Error bars represent standard deviation of six experimental points. (C) GST and GST fusion proteins with full-length H1, H1 N-terminal domain (H1-N, amino acid residues 1–40), the globular domain (H1-G, residues 41–119) and the C-terminal domain (H1-C, 120–256) were expressed and purified from E. coli and used in GST pull-down experiments with baculovirus-expressed purified recombinant STAT92E-His6. The pull-down samples were examined as in (A). Full-length polypeptides of GST fusion proteins are indicated by open triangles. STAT92E associates with GST fusions of H1 and H1-C but does not interact with GST or GST fusions of H1-N and H1-G.