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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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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).
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gkt297-F3: 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).

Mentions: To gain an insight into Su(Hw)-associated chromatin organization in general, we developed a software allowing analysis of ChIP-Seq and ChIP-chip data on any set of genomic positions. We identified the exact positions of 3120 Su(Hw) peaks in the genome and then calculated the average genomic distributions of different factors in the regions between −5 and +5 kb relative to a Su(Hw) peak (see ‘Materials and Methods’ section). This approach allowed us to visualize ChIP-Seq data and calculate the average level of the factor of interest on Su(Hw) peaks relative to surrounding sequences (the background level). To substantiate this approach, we calculated average profiles of CP190 and Mod(mdg4) proteins, which are known to strongly associate with Su(Hw) (3,49). Distinct peaks of CP190 and Mod(mdg4) proved to coincide with Su(Hw) genomic positions, confirming the strong association between these proteins (Figure 3A). As a negative control, we calculated the profile of Pol II around Su(Hw)-binding sites and revealed no colocalization of Pol II with Su(Hw) genomic positions (Figure 3C). This result is consistent with the fact that promoter association has been reported for Class I insulator proteins, but not for Su(Hw) (48,50). In addition, we performed ChIP with an antibody against Pol II in S2 cell and observed no enrichment in Pol II on Su(Hw)-binding sites (data not shown). At the next step, we considered genome-wide data on chromatin structure at Su(Hw) peaks. As shown previously, binding sites for insulator-associated proteins are regions of reduced nucleosome density, although nucleosome depletion on Su(Hw)-binding sites is not as significant as on CTCF sites (48). Analyzing new high-quality ChIP-Seq data on histones H1, H3 and H4 in S2 cells, we found that Su(Hw)-binding sites are regions with a low nucleosome density (Figure 3B).Figure 3.


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)

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

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

gkt297-F3: 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).
Mentions: To gain an insight into Su(Hw)-associated chromatin organization in general, we developed a software allowing analysis of ChIP-Seq and ChIP-chip data on any set of genomic positions. We identified the exact positions of 3120 Su(Hw) peaks in the genome and then calculated the average genomic distributions of different factors in the regions between −5 and +5 kb relative to a Su(Hw) peak (see ‘Materials and Methods’ section). This approach allowed us to visualize ChIP-Seq data and calculate the average level of the factor of interest on Su(Hw) peaks relative to surrounding sequences (the background level). To substantiate this approach, we calculated average profiles of CP190 and Mod(mdg4) proteins, which are known to strongly associate with Su(Hw) (3,49). Distinct peaks of CP190 and Mod(mdg4) proved to coincide with Su(Hw) genomic positions, confirming the strong association between these proteins (Figure 3A). As a negative control, we calculated the profile of Pol II around Su(Hw)-binding sites and revealed no colocalization of Pol II with Su(Hw) genomic positions (Figure 3C). This result is consistent with the fact that promoter association has been reported for Class I insulator proteins, but not for Su(Hw) (48,50). In addition, we performed ChIP with an antibody against Pol II in S2 cell and observed no enrichment in Pol II on Su(Hw)-binding sites (data not shown). At the next step, we considered genome-wide data on chromatin structure at Su(Hw) peaks. As shown previously, binding sites for insulator-associated proteins are regions of reduced nucleosome density, although nucleosome depletion on Su(Hw)-binding sites is not as significant as on CTCF sites (48). Analyzing new high-quality ChIP-Seq data on histones H1, H3 and H4 in S2 cells, we found that Su(Hw)-binding sites are regions with a low nucleosome density (Figure 3B).Figure 3.

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