Key mediators of the enhancer blocker function of Drosophila CTCF

	Functional eukaryotic gene units including all of their regulatory elements are insulated from each other to allow the action of regulatory elements on the appropriate gene. We and others have analyzed the DNA binding factor CTCF, which is the only protein known to mediate a directional blocking activity of enhancer elements in vertebrates (3, 8). In focusing on Drosophila CTCF, we found out that dCTCF mediates enhancer blocking on the Fab-8 insulator and binds to several other known insulator sequences (1, 2, 9). One set of dCTCF binding sites separates the homeotic genes responsible for the segmental identity in flies (2, 5). Deletion of dCTCF causes a defect in abdominal segmentation (homeotic mutation) and lethality (1). Search for co-factors involved in dCTCF function revealed a new, unknown protein (7), several proteins known to mediate chromatin remodelling (4, 5, 6) and chromatin modification (10). These results support a model for insulator function: DNA bound dCTCF recruites several co-factors removing changes in chromatin modification, which may have spread from flanking regions. 1. Mohan, M., Bartkuhn, M., Herold, M., Philippen, A., Heinl, N., Bardenhagen, I., Leers, L., White, R.A.H., Renkawitz-Pohl, R., Saumweber, H. and Renkawitz, R. (2007). The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J 26, 4203-4214. 2. Bartkuhn, M., Straub, T., Herold, M., Herrmann, M., Rathke, C., Saumweber, H., Gilfillan, G.D., Becker, P.B. and Renkawitz, R. (2009). Active promoters and insulators are marked by the centrosomal protein 190. EMBO J. 28, 877-888. 3. Herold, M., Bartkuhn, M. and Renkawitz, R. (2012) CTCF: Insights into insulator function during development. Development 139, 1045-1057. 4. Ahanger, S.H., Günther, K., Weth, O., Bartkuhn, M., Bhonde, R.R., Shouche, Y.S., and Renkawitz, R. (2014). Ectopically tethered CP190 induces large-scale chromatin decondensation. Scientific Reports 4, 3917. 5. Bohla, D., Herold, M, Panzer, I, Buxa, M.K., Ali, T., Demmers, J., Krüger, M., Scharfe, M., Jarek, M., Bartkuhn, M.1, Renkawitz, R. (2014). A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking. PLoS One 9: e107765. 6. Weth, O., Paprotka, C., Günther, K., Schulte, A., Baierl, M., Leers, J., Galjart, N. and Renkawitz, R. (2014). CTCF Induces Histone Variant Incorporation, Erases the H3K27me3 Histone Mark and Opens Chromatin. Nucl. Acids Res. 42, 11941-11951. 7. Maksimenko, O., Bartkuhn, M., Stakhov, V., Herold, M., Zolotarev, N., Jox, T., Buxa, M.K., Kirsch, R., Bonchuk, A., Fedotova, A., Kyrchanova, O., Renkawitz, R., Georgiev, P. (2015) Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin. Genome Res. 25, 89-99. 8. Ali, T., Renkawitz, R. and Bartkuhn, M. (2016). Insulators and domains of gene expression. Curr Opin Genet Dev 37, 17-26. 9. Buxa, M.K., Slotman, J.A., van Royen, M.E., Paul, M.W., Houtsmuller, A.B. and Renkawitz, R. (2016). Insulator speckles associated with long-distance chromatin contacts. Biology Open 5, 1266-1274. 10. Ali, T., Krüger, M., Bhuju, S., Jarek, M., Bartkuhn, M. and Renkawitz, R. (2017). Chromatin binding of Gcn5 in Drosophila is largely mediated by CP190. Nucl. Acids Res. 45, 2384-2395.

Functional eukaryotic gene units including all of their regulatory elements are insulated from each other to allow the action of regulatory elements on the appropriate gene. We and others have analyzed the DNA binding factor CTCF, which is the only protein known to mediate a directional blocking activity of enhancer elements in vertebrates (3, 8). In focusing on Drosophila CTCF, we found out that dCTCF mediates enhancer blocking on the Fab-8 insulator and binds to several other known insulator sequences (1, 2, 9). One set of dCTCF binding sites separates the homeotic genes responsible for the segmental identity in flies (2, 5). Deletion of dCTCF causes a defect in abdominal segmentation (homeotic mutation) and lethality (1).

Search for co-factors involved in dCTCF function revealed a new, unknown protein (7), several proteins known to mediate chromatin remodelling (4, 5, 6) and chromatin modification (10). These results support a model for insulator function: DNA bound dCTCF recruites several co-factors removing changes in chromatin modification, which may have spread from flanking regions.

1. Mohan, M., Bartkuhn, M., Herold, M., Philippen, A., Heinl, N., Bardenhagen, I., Leers, L., White, R.A.H., Renkawitz-Pohl, R., Saumweber, H. and Renkawitz, R. (2007). The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J 26, 4203-4214.

2. Bartkuhn, M., Straub, T., Herold, M., Herrmann, M., Rathke, C., Saumweber, H., Gilfillan, G.D., Becker, P.B. and Renkawitz, R. (2009). Active promoters and insulators are marked by the centrosomal protein 190. EMBO J. 28, 877-888.

3. Herold, M., Bartkuhn, M. and Renkawitz, R. (2012) CTCF: Insights into insulator function during development. Development 139, 1045-1057.

4. Ahanger, S.H., Günther, K., Weth, O., Bartkuhn, M., Bhonde, R.R., Shouche, Y.S., and Renkawitz, R. (2014). Ectopically tethered CP190 induces large-scale chromatin decondensation. Scientific Reports 4, 3917.

5. Bohla, D.*, Herold, M*, Panzer, I, Buxa, M.K., Ali, T., Demmers, J., Krüger, M., Scharfe, M., Jarek, M., Bartkuhn, M.1, Renkawitz, R. (2014). A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking. PLoS One 9: e107765.

6. Weth, O., Paprotka, C., Günther, K., Schulte, A., Baierl, M., Leers, J., Galjart, N. and Renkawitz, R. (2014). CTCF Induces Histone Variant Incorporation, Erases the H3K27me3 Histone Mark and Opens Chromatin. Nucl. Acids Res. 42, 11941-11951.

7. Maksimenko, O.*, Bartkuhn, M.*, Stakhov, V.*, Herold, M.*, Zolotarev, N., Jox, T., Buxa, M.K., Kirsch, R., Bonchuk, A., Fedotova, A., Kyrchanova, O., Renkawitz, R., Georgiev, P. (2015) Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin. Genome Res. 25, 89-99.

8. Ali, T., Renkawitz, R. and Bartkuhn, M. (2016). Insulators and domains of gene expression. Curr Opin Genet Dev 37, 17-26.

9. Buxa, M.K., Slotman, J.A., van Royen, M.E., Paul, M.W., Houtsmuller, A.B. and Renkawitz, R. (2016). Insulator speckles associated with long-distance chromatin contacts. Biology Open 5, 1266-1274.

10. Ali, T., Krüger, M., Bhuju, S., Jarek, M., Bartkuhn, M. and Renkawitz, R. (2017). Chromatin binding of Gcn5 in Drosophila is largely mediated by CP190. Nucl. Acids Res. 45, 2384-2395.

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Key mediators of the enhancer blocker function of Drosophila CTCF