Functional and physical interaction between ASH2 and Sin3A mediated by HCF. (a) ASH2 and Sin3A mutants share a large number of commonly misregulated genes. Each full circle encompasses the total number of genes that are downregulated (left) or upregulated (right) in Sin3A-deficient cells (according to Pile et al. ) and have valid log2 ratios in one or both of the ash2 alleles. The number of genes that are downregulated (green), upregulated (red) or do not display altered expression (yellow) over 1.5 times in one or both of the ash2 alleles is shown in brackets. An asterisk indicates statistically significant overlap: p = 1.06 × 10-4 (left), p = 5.88 × 10-32 (right). (b) Diagram showing Sin3A-HA, HCF-Flag and ASH2-V5 fusion proteins. The number of amino acids and the predicted molecular weight without counting tags are indicated above each construct. (c) HCF interacts with ASH2 and Sin3A in S2 cells. Anti-Flag immunoprecipitations were performed using cells expressing HCF-Flag and ASH2-V5, or ASH2-V5 alone as a negative control, and immunoblotted with anti-V5 (left). Anti-HA immunoprecipitations were performed with S2 cells transfected with Sin3-HA and HCF-Flag, or HCF-Flag alone as a negative control, and immunoblotted with anti-Flag (right). Input lane shows 4% of the total extract volume used for co-immunoprecipitations. (d) HCF interacts with ASH2 and Sin3A in embryos. Co-immunoprecipitation experiments were performed in transgenic embryos overexpressing ASH2-HA (left) or HCF-Flag (right) with daughterless-gal4. wt embryos were used as a negative control. Input lane shows 10% of the total extract volume used for co-immunoprecipitations.