Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

Abstract

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.

Figure 1.

Su(Hw) recruits histone acetyltransferase complex SAGA. (A, C, E) The levels of (A) Su(Hw), (C) GCN5 and (E) ADA2b on Su(Hw)-binding sites in control S2 cells (dark bars) and after Su(Hw) knockdown (light bars) as evaluated by ChIP analysis. The results are expressed as the percentage of DNA input. Error bars show standard errors of the means from three replicate experiments. Sites 1A1 and 1A6 were used as a negative control. The Mcp insulator was used as a reference site. (B, D) The levels of (B) Su(Hw) and (D) GCN5 on Su(Hw)-binding sites in wild-type (dark bars) and Su(Hw)^V/E8 mutant pupae (light bars) as evaluated by ChIP analysis. (F) Co-immunoprecipitation of Su(Hw) and GCN5 from Drosophila embryo nuclear extract by rabbit antibodies against each of these proteins; IgG from rabbit pre-immune serum was used as a negative control. Ten percent portions of the input nuclear fraction (in) and immunoprecipitated fraction (ip) were resolved by SDS–PAGE and western blotted with antibodies against Su(Hw) and GCN5.

Figure 2.

Su(Hw) recruits chromatin remodeler Brahma. (A, B) The levels of (A) OSA and (B) BAP111 on Su(Hw)-binding sites in control S2 cell (dark bars) and after Su(Hw) knockdown (light bars) as evaluated by ChIP analysis. Results are expressed as percentage of DNA input. Error bars show standard errors of the means from three replicate experiments. Sites 1A1 and 1A6 were used as a negative control. The Mcp insulator was used as a reference site. (C) The levels of OSA on Su(Hw)-binding sites in wild-type (dark bars) and Su(Hw)^V/E8 mutant pupae (light bars) as evaluated by ChIP analysis. (D) Co-immunoprecipitation of Su(Hw) and OSA from Drosophila embryo nuclear extract by rabbit antibodies against each of these proteins; IgG from rabbit pre-immune serum was used as a negative control. Ten percent portions of the input nuclear fraction (in) and immunoprecipitated fraction (ip) were resolved by SDS–PAGE and western blotted with antibodies against Su(Hw) and OSA. (E, F) Histone H3 levels on Su(Hw)-binding sites in (E) Drosophila S2 cells and (F) pupae as evaluated by ChIP analysis. The dark bars refer to control cells or wild-type pupae; light bars, to Su(Hw)-knockdown cells or Su(Hw)^V/E8 mutant pupae.

Figure 3.

Whole-genome analysis of Su(Hw)-binding sites for association with other factors. (A–D) Plots of average log2 enrichment ratios for indicated factors at positions −5 to +5 kb relative to Su(Hw) peaks: (A) insulator-associated proteins Su(Hw), CP190 and Mod(mdg4) in S2 cells; (B) histones H1, H3 and H4 in S2 cells; (C) Pol II, ORC2 and MCM2-7 in S2 cells; (D) nucleosome exchange profiles: CATCH-IT at 20-, 40- and 60-min time points. (E) The distribution of average AT contents of total ORC and Su(Hw)-binding sites. (F) Plots of average log2 enrichment ratios for Su(Hw) at positions −5 to +5 kb relative to replication origins. Profiles were calculated taking into account either all replication origins (dotted line) or only the origins localized in BLACK and BLUE chromatin (solid line).

Figure 4.

Su(Hw) recruits ORC. (A) The levels of ORC3 on Su(Hw)-binding sites in control S2 cell (dark bars) and after Su(Hw) knockdown (light bars) as evaluated by ChIP analysis. The results are expressed as the percentage of DNA input. Error bars show standard errors of the means from three replicate experiments. Sites 1A1 and 1A6 were used as a negative control. The Mcp insulator was used as a reference site. (B) Co-immunoprecipitation of Su(Hw) and ORC3 from Drosophila embryo nuclear extract by rabbit antibodies against each of these proteins; IgG from rabbit pre-immune serum was used as a negative control. Ten percent portions of the input nuclear fraction (in) and immunoprecipitated fraction (ip) were resolved by SDS–PAGE and western blotted with antibodies against Su(Hw) and ORC3. (C, D) ORC3 and CDC45 levels on Su(Hw)-binding sites in wild-type pupae (dark bars) and Su(Hw)^V/E8 mutant pupae (light bars) as evaluated by ChIP analysis.

Figure 5.

Su(Hw) is sufficient for recruiting SAGA, BAP and ORC. (A) Recruitment of 3 × FLAG-GAL4BD–SuHw (dark bar) or 3 × FLAG-GAL4BD alone (light bar) to 10 × UAS as evaluated by ChIP with an antibody against the FLAG epitope in two S2 cell lines stably expressing those proteins. (B) Recruitment of OSA, GCN5 and ORC3 to 10 × UAS in S2 cell lines stably expressing 3 × FLAG-GAL4BD–SuHw (dark bars) or 3 × FLAG-GAL4BD alone (light bars). (C) Recruitment of OSA, GCN5 and ORC3 to the 4 × Su(Hw)-binding site in transgenic pupae.

Figure 6.

Whole-genome analysis of Su(Hw), CTCF, GAF and BEAF32-binding sites. (A–C) Plots of average log2 enrichment ratios for indicated factors at positions −5 to +5 kb relative to Su(Hw), CTCF, GAF and BEAF32-binding sites. Random sites were used as a negative control. (A) Pol II, ORC2 and MCM2-7 in S2 cells. (B) Histones H1, H3 and H4 in S2 cells. (C) Nucleosome exchange profiles. CATCH-IT profiles at 20-, 40- and 60-min time points.