Dokument

Summary:
Regulation of chromatin in the cell is achieved by protein complexes that are assembled in a combinatorial fashion. These complexes often contain different isoforms of the same subunit, thus, greatly increasing the number of related, but still diverse complexes. We are just beginning to unravel the rules that dictate isoform-specific complex assembly and the contributions that distinct subunit isoforms make to complex function. CoREST has been identified as a subunit of complexes that typically combine several histone modifying activities. For example, it directly binds to HDAC1 and KDM1A/ LSD1 and enhances their activity on nucleosome substrates. However, the CoREST interactome has not been systematically characterised. CoREST complexes are considered to generate transcriptionally repressive chromatin structures during development. Drosophila expresses two isoforms of CoREST, dCoREST-L and dCoREST-M. In this study analysis of Drosophila macrophage-like S2 cell nuclear extracts by size exclusion chromatography has suggested that the two dCoREST isoforms reside in different complexes. Additionally, the dCoREST interactome was determined by affinity chromatography and mass spectrometry identifying subunits of both the LINT and the dLSD1/dCoREST complexes. Notably, whereas both dCoREST-L and dCoREST-M bound the LINT complex subunits, only dCoREST-L was capable of interacting with dLSD1 in vitro and in vivo, providing support for isoform-specific complex formation. Proteomic analysis also identified the histone methyltransferase dG9a as a novel dCoREST interactor. dG9a itself did not bind dLSD1 or the LINT complex subunits suggesting it forms an independent assembly with dCoREST. These proteomic data indicate that at least three distinct dCoREST complexes exist: LINT, dLSD1/dCoREST and dG9a/dCoREST. Genome-wide experiments established that dCoREST complexes associate with chromatin predominantly at promoters. To assess the relative contributions of the three dCoREST complexes to gene regulation and to wing and male germ cell development, RNA interference approaches targeting individual complex subunits were used. RNA- seq revealed that the LINT complex, but not dLSD1/dCoREST or dG9a/dCoREST, is a major regulator of transcription in S2 cells. In particular, LINT repressed a set of malignant brain tumour (MBTS) genes. Conversely, the dLSD1/dCoREST complex was essential for spermatogenesis, whereas the LINT and the dG9a/dCoREST complexes appeared dispensable. Indeed, RNA-seq analysis of dissected testes demonstrated that dLSD1/dCoREST is required to repress the inappropriate expression of cell-type- specific genes during spermatogenesis. These results define isoform-specific dCoREST complexes and suggest that distinct dCoREST complexes are differentially utilised to maintain appropriate transcriptional programmes in different cell types.

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