Abstract
Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.
Highlights
Eukaryotic genomes are composed of cytologically and functionally distinct chromatin domains called heterochromatin and euchromatin (Heitz, 1928)
Heterochromatin is primarily comprised of simple repetitive DNA sequences (Peacock et al, 1978) and transposons (Carlson and Brutlag, 1978), this domain is necessary for organismal functions, including pericentromeric sister chromatid cohesion (Bernard et al, 2001), achiasmate chromosome pairing and segregation in male and female meiosis (Dernburg et al, 1996; Karpen et al, 1996; McKee and Karpen, 1990), and genome integrity (Peng and Karpen, 2009)
Consistent with expectations for heterochromatic proteins, these HP1 interacting proteins (HPips) were enriched for gene ontology (GO) categories that include ’chromocenter’, ’chromatin organization’, ’chromatin assembly or disassembly’ and ’posttranscriptional regulation of gene expression’ (Supplementary file 1)
Summary
Eukaryotic genomes are composed of cytologically and functionally distinct chromatin domains called heterochromatin and euchromatin (Heitz, 1928). Heterochromatin is primarily comprised of simple repetitive DNA sequences (Peacock et al, 1978) and transposons (Carlson and Brutlag, 1978), this domain is necessary for organismal functions, including pericentromeric sister chromatid cohesion (Bernard et al, 2001), achiasmate chromosome pairing and segregation in male and female meiosis (Dernburg et al, 1996; Karpen et al, 1996; McKee and Karpen, 1990), and genome integrity (Peng and Karpen, 2009). To understand how HP1 is able to regulate diverse cellular and organismal functions (Grewal and Jia, 2007), researchers have affinity purified HP1 in human tissue culture lines (Rosnoblet et al, 2011; Lechner et al, 2005), S. pombe (Motamedi et al, 2008) and D. melanogaster (Ryu et al, 2014; Alekseyenko et al, 2014) and identified >100 putative HP1 interacting proteins (HPips) by mass spectrometry. Biochemical (i.e. salt fractionation and size exclusion chromatography) and cytological (i.e. fluorescence correlation spectroscopy and fluorescence recovery after photobleaching) experiments
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