Functional characterization of ATP-dependent chromatin remodelers of the CHD family of Drosophila

Members of the CHD family (Chromodomain-Helicase-DNA binding) of ATP-dependent chromatin remodelers play key roles at different steps of the transcription cycle. They are essential in regulation of developmental and differentiation programs in multicellular organisms. However, the complexity of thes...

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Bibliographic Details
Main Author: Murawska, Magdalena
Contributors: Brehm, Alexander (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2011
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Summary:Members of the CHD family (Chromodomain-Helicase-DNA binding) of ATP-dependent chromatin remodelers play key roles at different steps of the transcription cycle. They are essential in regulation of developmental and differentiation programs in multicellular organisms. However, the complexity of these remodelers makes it difficult to study them in higher eukaryotes. In this study, advantage was taken of Drosophila melanogaster as a model organism, which possesses only four CHD family members. In the first part of this study, a novel chromatin remodeler, dCHD3, has been characterized biochemically and functionally. dCHD3 is highly similar to dMi-2 and consequently it possesses similar enzymatic activities in vitro. dCHD3 is a highly active, nucleosome stimulated ATP-dependent chromatin remodeler which slides mononucleosomes in vitro. The chromodomains of dCHD3 seem to be important for substrate recognition and for the remodeling activity of this enzyme. Despite the similarities, dCHD3 and dMi-2 differ significantly in other aspects. In contrast to dMi-2, dCHD3 exists as a monomer in vivo and it is not associated with deacetylase activity. Moreover, dCHD3 expression is restricted to early developmental stages and certain tissues. Finally, dCHD3 cannot compensate for the loss of dMi-2 which suggests that they are not functionally redundant. In the second part of this work, a role of dMi-2 in active transcription has been studied. dMi-2 has been implicated in transcriptional repression as a part of dNuRD or dMec complexes. This study shows that dMi-2 colocalizes with active regions on polytene chromosomes and it is recruited to heat shock genes. Both, reduction of dMi-2 expression in flies or ectopic expression of a catalytically inactive mutant, impair heat shock gene response. Interestingly, 3’ end processing and splicing of some of these genes is affected. In agreement with this, dMi-2 binds to nascent hsp gene transcripts upon heat shock induction. Consequently, these results suggest a role of dMi-2 catalytic activity in co-transcriptional RNA processing. Study of the recruitment mechanism of dMi-2 to heat shock genes suggests that it occurs in a poly(ADP-ribose) dependent manner. Several results support this hypothesis. First, dMi-2 recruitment to hsp70 gene is reduced upon PARP inhibition. Second, dMi-2 binds PAR polymers directly in vitro and several dMi-2 regions, which bind PAR independently in vitro, have been identified. Third, a dMi-2 mutant unable to bind PAR does not localise to active heat shock loci in vivo. Moreover, RNA and PAR compete for dMi-2 binding in vitro suggesting a two-step process for dMi-2 association with active heat shock genes. First, dMi-2 is recruited to the locus via PAR binding followed by association with nascent RNA transcripts. Collectively, these studies suggest, that stress-induced chromatin modification by PARP serves as a scaffold for rapid recruitment of factors that are required for quick and efficient transcriptional response.
DOI:https://doi.org/10.17192/z2011.0570