Inheritance of gene expression patterns through multiple rounds of cell division is crucial for the normal development of multi-cellular organisms and is mediated by epigenetic mechanisms. Many epigenetic mechanisms are believed to involve heritable changes to chromatin structure. This includes maintenance of transcriptional repression by Polycomb Group (PcG) proteins. It is unknown how PcG-dependent repression is maintained during or re-established after mitosis, a process that involves many physical and biochemical changes to chromatin. Understanding the behavior of PcG proteins during mitosis is key to answering this question: if PcG proteins remain bound in mitosis they may constitute the memory themselves, or else transcriptional memory must reside elsewhere, such as in the altered chromatin structures induced by PcG proteins. PcG protein association with chromosomes in mitosis in Drosophila S2 cells was examined by immunofluorescence and cellular fractionation. PcG proteins are associated with mitotic chromosomes, which is consistent with a role in carrying information about transcriptional repression through mitosis. Localization of PcG proteins to specific sites in the genome was assessed by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChIP- SEQ) on mitotic cells. A method for isolating pure populations of mitotic cells was developed to access PcG protein localization in mitosis unambiguously. PcG proteins were not detected at well-characterized PcG targets including Hox genes on mitotic chromosomes, but a covalent modification of histone H3 associated with PcG- dependent repression, trimethylation of lysine 27 (H3K27me3), is retained at these sites. Two PcG proteins Posterior Sex Combs (PSC) and Polyhomeotic (PH) remain at about 10% of their interphase binding sites in mitosis. PSC and PH are preferentially retained in mitosis at sites that overlap recently described borders of chromatin domains (1), including sites that overlap domain borders flanking Hox gene clusters. These persistent binding sites may serve to nucleate re-establishment of PcG binding at target genes upon mitotic exit, perhaps with assistance of H3K27me3. Thus PcG proteins may form part of the transcriptional memory carried through mitosis, but perhaps not by persistent association at the targets of repression. Retention of elements at chromatin boundaries in mitosis may serve as a general mechanism for epigenetic memory.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/9548617 |
| Date | 21 June 2013 |
| Creators | Follmer, Nicole Elizabeth |
| Contributors | Francis, Nicole J. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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