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

Vorobyeva NE, Mazina MU, Golovnin AK, Kopytova DV, Gurskiy DY, Nabirochkina EN, Georgieva SG, Georgiev PG, Krasnov AN - Nucleic Acids Res. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Transcriptional Regulation and Chromatin Dynamics, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.

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.

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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.
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gkt297-F1: 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.

Mentions: As shown in our previous study, the ENY2 protein binds to the zinc-finger domain of Su(Hw) and is recruited to the insulator complex (4). As ENY2 is a component of the SAGA complex (5), it was relevant to find out whether there is an association between SAGA and Su(Hw)-dependent insulators. To this end, we tested the presence of the GCN5 and ADA2b subunits of SAGA on Su(Hw)-binding sites by ChIP in Drosophila S2 cells. In these experiments, Su(Hw)-dependent insulators gypsy, 1A2, 50A, 62D, 66 E and 87 E were examined versus the 1A1 and 1A6 sites used as a negative control. The results showed that Su(Hw) and ENY2 readily interacted with these insulators, but did not bind to the 1A1 and 1A6 sites (4). As a reference site, we used the CTCF-dependent insulator Mcp, which does not bind Su(Hw) protein (Figure 1A) and therefore is appropriate as a Su(Hw)-independent control. All tested Su(Hw)-binding regions and the Mcp insulator showed significant enrichment with GCN5 and ADA2b subunits on ChIP with corresponding antibodies, providing evidence for strong binding of the SAGA complex to these regions in S2 cells (Figure 1C and E; dark bars). The binding of SAGA to the Mcp insulator is consistent with the previous finding that CTCF-dependent insulators are enriched with histone acetyltransferase complexes (42).Figure 1.


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

Vorobyeva NE, Mazina MU, Golovnin AK, Kopytova DV, Gurskiy DY, Nabirochkina EN, Georgieva SG, Georgiev PG, Krasnov AN - Nucleic Acids Res. (2013)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3675495&req=5

gkt297-F1: 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.
Mentions: As shown in our previous study, the ENY2 protein binds to the zinc-finger domain of Su(Hw) and is recruited to the insulator complex (4). As ENY2 is a component of the SAGA complex (5), it was relevant to find out whether there is an association between SAGA and Su(Hw)-dependent insulators. To this end, we tested the presence of the GCN5 and ADA2b subunits of SAGA on Su(Hw)-binding sites by ChIP in Drosophila S2 cells. In these experiments, Su(Hw)-dependent insulators gypsy, 1A2, 50A, 62D, 66 E and 87 E were examined versus the 1A1 and 1A6 sites used as a negative control. The results showed that Su(Hw) and ENY2 readily interacted with these insulators, but did not bind to the 1A1 and 1A6 sites (4). As a reference site, we used the CTCF-dependent insulator Mcp, which does not bind Su(Hw) protein (Figure 1A) and therefore is appropriate as a Su(Hw)-independent control. All tested Su(Hw)-binding regions and the Mcp insulator showed significant enrichment with GCN5 and ADA2b subunits on ChIP with corresponding antibodies, providing evidence for strong binding of the SAGA complex to these regions in S2 cells (Figure 1C and E; dark bars). The binding of SAGA to the Mcp insulator is consistent with the previous finding that CTCF-dependent insulators are enriched with histone acetyltransferase complexes (42).Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Transcriptional Regulation and Chromatin Dynamics, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.

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.

Show MeSH
Related in: MedlinePlus