Protein Landscape at Drosophila melanogaster Telomere-Associated Sequence Repeats

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2012; 32(12):2170-82. DOI: 10.1128/MCB.00010-12
Source: PubMed


The 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.

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    • "It is clear that the proteome of telomeres is complex and includes a variety of DNA and RNAbinding and other proteins (Mason et al. 2008; Raffa et al. 2011; Antao et al. 2012; Takacs et al. 2012). Although hnRNPs have been noted to be present at Drosophila telomeres in earlier studies, present study directly establishes for the first time a role of Hrb87F in maintenance of telomeres in Drosophila. "
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    DESCRIPTION: Abstract Unlike the telomerase dependent mammalian telomeres, HeT-A, TART and TAHRE (HTT) retroposon arrays regulate Drosophila telomere length. Cap prevents telomeric associations (TA) and telomeric fusions (TF). Our results suggest important roles of Hrb87F in telomeric HTT array and cap maintenance in Drosophila. All chromosome arms, except 2L, in Df(3R)Hrb87F homozygotes (Hrb87F-null) displayed significantly elongated telomeres with amplified HTT arrays and high TA, all of which resolved without damage. Presence of FLAG-Hrb87F on cap and sub-telomeric regions following hsFLAG-Hrb87F transgene expression in Df(3R)Hrb87F homozygotes suppressed TA without affecting telomere length. A normal X-chromosome telomere expanded within five generations in Hrb87F-null background and displayed high TA, but not when hsFLAG-Hrb87F was co-expressed. Tel1/Gaiano line or HP1 loss of function mutant derived expanded telomeres carry Hrb87F on cap and HTT arrays while Hrb87F-null telomeres have HP1 and HOAP on caps and expanded HTT arrays. ISWI, seen only on cap on normal telomeres, was abundant on Hrb87F-null expanded HTT arrays. Extended telomeres derived from Tel1 (Gaiano) or HP1-null mutation background interact with those from Hrb87F-null since the end association frequency was negligible in Df(3R)Hrb87F/+ nuclei but increased significantly in co-presence of Tel1 or HP1-null based expanded telomere/s. Together these suggest complex interactions between members of the proteome of telomere so that absence of any key member leads to telomere expansion and/or enhanced TA/TF. HTT expansion in Hrb87F-null condition is not developmental but a germline event presumably because absence of Hrb87F in germline may deregulate HTT retroposition/replication leading to telomere elongation.
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    • "We also show that the absence of DEK results in a loss of chromatin repression at telomeres, which may be attributed to a lack of ATRX-mediated H3.3 loading (Goldberg et al. 2010). 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. "
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    ABSTRACT: Histone variant H3.3 is deposited in chromatin at active sites, telomeres and pericentric heterochromatin by distinct chaperone, but the mechanisms of regulation and coordination of chaperone-mediated H3.3 loading remain largely unknown. We show here that the chromatin-associated oncoprotein DEK regulates differential HIRA- and DAAX/ATRX-dependent distribution of H3.3 on chromosomes in somatic cells and in embryonic stem cells. Live cell imaging studies show that non-nucleosomal H3.3 normally destined to PML nuclear bodies is re-routed to chromatin after depletion of DEK. This results in HIRA-dependent wide-spread chromatin deposition of H3.3, and H3.3 incorporation in foci of heterochromatin, in process requiring the DAXX/ATRX complex. In embryonic stem cells, loss of DEK leads to displacement of PML bodies and ATRX from telomeres, redistribution of H3.3 from telomeres to chromosome arms and pericentric heterochromatin, induction of a fragile telomere phenotype and telomere dysfunction. Our results indicate that DEK is required for proper loading of ATRX and H3.3 on telomeres and for telomeric chromatin architecture. We propose that DEK acts as a 'gate-keeper' of chromatin, controlling chromatin integrity by restricting broad access to H3.3 by dedicated chaperones. Our results also suggest that telomere stability relies on mechanisms ensuring proper histone supply and routing.
    Genome Research 07/2014; 24(10). DOI:10.1101/gr.173831.114 · 14.63 Impact Factor
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    • "The TPE assays with the JIL-1 mutant alleles have shown the same result for both TAS domains, TAS-L (2L, 3L telomeres, line 39C-5) and TAS-R (2R, 3R and XL telomeres, line 39C-27) [19], demonstrated by the increase in eye color of the resulting descendants (Figure 4A). These results suggest that the protective boundary built by JIL-1 also exists in the other Drosophila telomeres. "
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    ABSTRACT: In Drosophila, the non-LTR retrotransposons HeT-A, TART and TAHRE build a head-to-tail array of repetitions that constitute the telomere domain by targeted transposition at the end of the chromosome whenever needed. As a consequence, Drosophila telomeres have the peculiarity to harbor the genes in charge of telomere elongation. Understanding telomere expression is important in Drosophila since telomere homeostasis depends in part on the expression of this genomic compartment. We have recently shown that the essential kinase JIL-1 is the first positive regulator of the telomere retrotransposons. JIL-1 mediates chromatin changes at the promoter of the HeT-A retrotransposon that are necessary to obtain wild type levels of expression of these telomere transposons. With the present study, we show how JIL-1 is also needed for the expression of a reporter gene embedded in the telomere domain. Our analysis, using different reporter lines from the telomere and subtelomere domains of different chromosomes, indicates that JIL-1 likely acts protecting the telomere domain from the spreading of repressive chromatin from the adjacent subtelomere domain. Moreover, the analysis of the 4R telomere suggests a slightly different chromatin structure at this telomere. In summary, our results strongly suggest that the action of JIL-1 depends on which telomere domain, which chromosome and which promoter is embedded in the telomere chromatin.
    PLoS ONE 11/2013; 8(11):e81543. DOI:10.1371/journal.pone.0081543 · 3.23 Impact Factor
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