Protein Landscape atDrosophila melanogasterTelomere-Associated Sequence Repeats

Abstract

eThe specific set of proteins bound at each genomic locus contributes decisively to regulatory processes and to the identity of a cell. Understanding of the function of a particular locus requires the knowledge of what factors interact with that locus and how the protein composition changes in different cell types or during the response to internal and external signals. Proteomic analysis of isolated chromatin segments (PICh) was developed as a tool to target, purify, and identify proteins associated with a defined locus and was shown to allow the purification of human telomeric chromatin. Here we have developed this method to identify proteins that interact with the Drosophila telomere-associated sequence (TAS) repeats. Several of the purified factors were validated as novel TAS-bound proteins by chromatin immunoprecipitation, and the Brahma complex was confirmed as a dominant modifier of telomeric position effect through the use of a genetic test. These results offer information on the efficacy of applying the PICh protocol to loci with sequence more complex than that found at human telomeres and identify proteins that bind to the TAS repeats, which might contribute to TAS biology and chromatin silencing. P roteomic analysis of isolated chromatin segments (PICh) was developed as an unbiased method of identifying proteins that physically interact with a specific locus in the genome (11), and this was done by using the telomeres of mammalian cells as a target. Telomeres are found in multiple copies of a simple repeat sequence and so do not offer the same challenge to the use of PICh as other genomic loci do. In this work, we applied PICh to the telomere-associated sequence (TAS) repeats of Drosophila to demonstrate the efficacy of the technology and to learn about the biology of these repeats.TAS repeats are found in the subtelomeric region of chromosomes 2, 3, and X and nucleate a particular kind of heterochromatin which is responsible for the telomeric position effect (TPE; for a review, see reference 37). As also seen with pericentromeric heterochromatin-mediated position effect variegation (PEV), reporter genes inserted at the TAS repeats, or between the TAS repeats and the telomeric retrotransposon (HTT) arrays, variegate. The extent of gene silencing depends on the size, and hence the strength of the transcription activating effect, of the HTT array and the transcription-repressing activity of the TAS repeats (28). Interestingly, though, most modifiers of PEV [Mod(PEV)] have no effect on TPE; in fact, very few modifiers of TPE [Mod(TPE)] have been unambiguously described so far. Among these are the Polycomb group (PcG) genes, which in some studies have been proposed to act as dominant suppressors of TPE [Su(TPE)] and whose encoded proteins have been found to be located at the telomeric regions of polytene chromosomes (6). These findings indicate that TPE is a distinctive class of chromatin silencing which shares mechanistic features with both pericentromeric heterochromatin and PcG-mediated silencing of de...

Targeting of P-Element Reporters to Heterochromatic Domains by Transposable Element 1360 in Drosophila melanogaster

“…Interestingly, some classical heterochromatic marks are also present: H3K9me3 and HP1a are enriched at the TAS in third instar larvae. These findings are supported by a recent proteomic analysis of isolated chromatin segments, which found SU(VAR)3-9 (one of the three H3K9 methyltransferases) along with TRL and PC (Polycomb system components) associated with the TAS sequences (Antao et al 2012). As noted above, PEV studies have found that inserts into the TAS are generally unaffected by mutations in HP1a [i.e.…”

Stacking the DEK: From chromatin topology to cancer stem cells

“…150,151 Interestingly, in a recent screen Drosophila Dek was identified as a novel factor that could bind to telomere-associated sequence (TAS) repeats. 152 Although definitive studies have not been performed to elucidate the molecular functions of DEK specifically in stem and progenitor cells, growing evidence that changes in DEK expression impact several crucial pathways is suggestive that DEK, too, may be fundamentally important for the viability and function of the stem and/or progenitor cell populations. Combined, DEK either regulates or is regulated by several signaling pathways and transcription factors implicated in stem cell proliferation, differentiation and self-renewal, potentially through its roles in regulating chromatin topology (Fig.…”

The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity

“…Association of DEK with telomeres has been reported in Drosophila (Antao et al 2012). However, it is important to note that not all telomeres contain DEK in mouse ESCs.…”