In vitro und in vivo Analyse von DOMINO, einem ATP-verbrauchenden Histon-Austausch-Faktor aus der Familie der "split"-ATPasen
Final Report Abstract
The initial goal of the project was to characterize in vitro and in vivo the poorly understood split ATPase Domino B from Drosophila melanogaster. Developmental processes such as cell proliferation, cell fate determination and cell differentiation as well as cell interactions and movement depend on selective gene expression and hence on a dynamic nature of chromatin, which is regulated by epigenetic programs. One molecular mechanism to change gene expression is ATP-dependent chromatin remodeling, which can change the contact between histones and DNA, catalyze nucleosome repositioning or eviction and replace histones with their variants. A known chromatin remodeling factor that is involved in histone exchange is the highly conserved ATPase Domino A of the TIP60 complex. Its other isoform Domino B is hitherto less characterized and just beginning to be uncovered. Previous studies found this enzyme involved in essential processes during development of flies and mammals. But the biological function and the precise mechanism of Domino B is poorly understood. In my PhD thesis, I have analyzed its expression pattern, characterized some associated factors in a putative novel complex and explored its potential functions. Domino belongs to the SWR1-type chromatin remodelers and contains the characteristic bipartite ATPase domain. By fractionation of Drosophila embryo nuclear extracts a so far unappreciated diversity of nucleosome remodeling complexes emerged. In distinct fractions, Domino B was associated with known TIP60 subunits and to our surprise, with ACF1 and ISWI. Both factors belong to the ACF/CHRAC complexes which change chromatin through nucleosome sliding. The physical interaction of Domino B, ACF1 and ISThe initial goal of the project was to characterize in vitro and in vivo the poorly understood split ATPase Domino B from Drosophila melanogaster. Developmental processes such as cell proliferation, cell fate determination and cell differentiation as well as cell interactions and movement depend on selective gene expression and hence on a dynamic nature of chromatin, which is regulated by epigenetic programs. One molecular mechanism to change gene expression is ATP-dependent chromatin remodeling, which can change the contact between histones and DNA, catalyze nucleosome repositioning or eviction and replace histones with their variants. A known chromatin remodeling factor that is involved in histone exchange is the highly conserved ATPase Domino A of the TIP60 complex. Its other isoform Domino B is hitherto less characterized and just beginning to be uncovered. Previous studies found this enzyme involved in essential processes during development of flies and mammals. But the biological function and the precise mechanism of Domino B is poorly understood. In my PhD thesis, I have analyzed its expression pattern, characterized some associated factors in a putative novel complex and explored its potential functions. Domino belongs to the SWR1-type chromatin remodelers and contains the characteristic bipartite ATPase domain. By fractionation of Drosophila embryo nuclear extracts a so far unappreciated diversity of nucleosome remodeling complexes emerged. In distinct fractions, Domino B was associated with known TIP60 subunits and to our surprise, with ACF1 and ISWI. Both factors belong to the ACF/CHRAC complexes which change chromatin through nucleosome sliding. The physical interaction of Domino B, ACF1 and ISWI is specific to earliest stages of embryonic development in Drosophila, since all of them were predominantly detected in pre-blastodermal embryos and were abolished during further stages. To analyze Domino B in vitro as well as in vivo, three different expression systems (in E.coli, SF9 cells and D. melanogaster) were established. In vitro, recombinant ACF1 and ISWI bound directly to Domino B and its split ATPase domain was mapped as the binding region to ACF1. These findings indicate a novel putative remodeling complex consisting among others of Domino B, ACF1 and ISWI, which we referred to as ACDC (ACF-Domino containing) complex. The functional meaning of Domino B and ACDC were characterized in in vivo experiments studying loss- and gain-of-function phenotypes in Drosophila. Domino B is involved in cell fate determination, cell differentiation and cell cycle related processes in specific cell types. Remarkably, phenotypic abnormalities of Domino B correspond to them of ACF1 indicating their functional relationship in vivo. A co-expression of both factors during early developmental stages resulted in synergistic effects and synthetic lethality. A putative ATPase deficient form of Domino B (Domino B KR) could rescue observed synthetic lethal phenotypes of ACDC. Synthetic actions of Domino B and ACF1 are restricted to oogenesis and early embryogenesis in agreement with their association in ovaries and pre-blastodermal embryos. In conclusion, the results of this work show that Domino B is involved in cell differentiation and cell cycle related processes in Drosophila. A novel physical and functional interaction between Domino B and ACF that was unappreciated so far was determined during oogenesis and early embryogenesis. This reveals a novel type of complex combining two distinct remodeling mechanisms, nucleosome sliding and histone variant exchange.
Publications
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Developmental role for ACF1‐containing nucleosome remodellers in chromatin organization. Development 2010; 137, 3513‐3522
Mariacristina Chioda, Sandra Vengadasalam, Elisabeth Kremmer, Anton Eberharter and Peter B. Becker
