Role of multi zinc finger proteins in the dynamic change of the nuclear architecture during cell cycle and differentiation
Final Report Abstract
CTCF is an essential organizer of chromatin. This CRP has studied the function of this protein in mammals and drosophila. We consider our in vivo studies in two model organisms on the (dynamic) behaviour of CTCF as the most original scientific contributions, as these have altered our views on how this protein exerts its function. I will list some of the important results here. Our studies have shown that CTCF is essential for early development in mouse and flies. In both organisms we further show that CTCF is required in a dose-dependent manner. Specific inactivation of mouse CTCF in early double-negative thymocytes results in the accumulation of immature single positive (ISP) cells. In wild type mice these ß-selected T cells are relatively large and actively cycling, and CTCF levels positively correlate with ISP cell size. In CTCF-negative mice, however, ISP cells are significantly smaller and they are blocked in the cell cycle, due to highly elevated levels of the cyclin-CDK-inhibitors p21 and p27. These results show that CTCF controls cell cycle progression of ß-selected T-cells in vivo by regulating cell size and, in conjunction, p21 expression. Interestingly, mouse and drosophila CTCF are not required for cell division and survival in all cell types. These data suggest that redundant mechanisms exist to circumvent lethality in CTCF-deleted cells. In cultured T cells such a mechanism is induced when cells are stimulated with phorbol ester and a calcium ionophore. The molecular details of such "redundant" systems remain unknown. In mammals there is one obvious "redundancy" candidate, called CTCFL or BORIS. This protein is highly similar to CTCF in the zinc finger domain and can bind the same consensus site. However, no rescue of CTCF-deficient ES cells with a cDNA encoding CTCFL is observed. By contrast, these ES cells are rescued with GFP-CTCF and CTCF-GFP, showing "proof of principle" of the rescue protocol. We therefore propose that CTCF and CTCFL have different functions. Others have shown that CTCFL is mainly expressed in the male germ line. We have generated CTCFL knockout mice, which are viable. Recent data revealed that not all male homozygous CTCFL knockout mice show a phenotype: some mice are completely fertile whereas others rapidly become infertile. Affected males display testicular degeneration due to a massive death of germ cells. Thus, even though CTCFL is normally only expressed in germ cells, its function is still redundant. Using GFP-CTCF and GFP-CTCFL knock-in mice and in vivo imaging procedures, we have found that both proteins are expressed in a differing manner during spermatogenesis. CTCF and CTCFL are co-expressed in spermatocytes. FRAP studies suggest that both proteins are tightly bound to DNA. We favour the hypothesis that they compete for specific binding sites (for example the rDNA repeats) during spermatogenesis. By purifying biotinylated CTCF (expressed from a knock-in allele) under relatively mild conditions, we were able to identify (by mass spectrometry) novel CTCF-interacting partners, including a nucleolar protein. These data imply involvement of CTCF in nbosome biogenesis and in nbosomal DNA (rDNA) transcription. As the nucleolar factor also interacted with CTCFL, we focussed our attention on this interaction, and on a possible binding of CTCF to the rDNA repeat. We indeed found a novel and conserved CTCF binding site in the rDNA locus. Using wild type and CTCF knockout cells we subsequently showed that CTCF organizes chromatin in a similar manner at RNA polymerase I- and II-dependent promoters, among others by regulating the "deposition" of a variant histone and of histone modifications. In this sense drosophila and mouse CTCF are similar. A conserved feature of an "insulator" protein such as CTCF therefore appears to be the ability to locally define the epigenetic state of chromatin.
Publications
-
(2005). CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin. Embo J 24, 3291-3300
Burke, L.J., Zhang, R., Bartkuhn, M., Tiwari, V.K., Tavoosidana, G., Kurukuti, S., Weth, C, Leers, J., Galjart, N., Ohlsson, R., and Renkawitz, R.
-
(2006). CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. Genes & development 20, 2349-2354
Splinter, E., Heath, H., Kooren, J., Palstra, R.J., Klous, P., Grosveld, F., Galjart, N., and de Laat, W.
-
(2006). Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis. Molecular and cellular biology 26, 976- 989
Wesoly, J., Agarwal, S., Sigurdsson, S., Bussen, W., Van Komen, S., Qin, J., van Steeg, H., van Benthem, J., Wassenaar, E., Baarends, W.M., Ghazvini, M., Tafel, AA., Heath, H., Galjart, N., Essers, J., Grootegoed, J.A., Arnheim, N., Bezzubova, O., Buerstedde, J.M., Sung, P., and Kanaar, R.
-
(2007). CTCF Genomic Binding Sites in Drosophila and the Organisation of the Bithorax Complex. PLoS genetics 3, e112
Holohan, E.E., Kwong, C., Adryan, B., Bartkuhn, M., Herold, M., Renkawitz, R., Russell, S., and White, R.
-
(2007). The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. The EMBO journal 26, 4203-4214
Mohan, M., Bartkuhn, M., Herold, M., Philippen, A., Heinl, N., Bardenhagen, I., Leers, J., White, R.A., Renkawitz-Pohl, R., Saumweber, H., and Renkawitz, R.
-
(2007). Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila. Journal of cell science 120, 1689-1700
Rathke, C., Baarends, W.M., Jayaramaiah-Raja, S., Bartkuhn, M., Renkawitz, R., and Renkawitz-Pohl, R.
