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In Vivo Chromatin Organization on Native Yeast Telomeric Regions Is Independent of a Cis‑Telomere Loopback Conformation Emeline Pasquier and Raymund J

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In Vivo Chromatin Organization on Native Yeast Telomeric Regions Is Independent of a Cis‑Telomere Loopback Conformation Emeline Pasquier and Raymund J Pasquier and Wellinger Epigenetics & Chromatin (2020) 13:23 Epigenetics & Chromatin https://doi.org/10.1186/s13072-020-00344-w RESEARCH Open Access In vivo chromatin organization on native yeast telomeric regions is independent of a cis-telomere loopback conformation Emeline Pasquier and Raymund J. Wellinger* Abstract Background: DNA packaging into chromatin regulates all DNA-related processes and at chromosomal ends could afect both essential functions of telomeres: protection against DNA damage response and telomere replication. Despite this primordial role of chromatin, little is known about chromatin organization, and in particular about nucleosome positioning on unmodifed subtelomere–telomere junctions in Saccharomyces cerevisiae. Results: By ChEC experiments and indirect end-labeling, we characterized nucleosome positioning as well as spe- cialized protein–DNA associations on most subtelomere–telomere junctions present in budding yeast. The results show that there is a relatively large nucleosome-free region at chromosome ends. Despite the absence of sequence homologies between the two major classes of subtelomere–telomere junctions (i.e.: Y’-telomeres and X-telomeres), all analyzed subtelomere–telomere junctions show a terminal nucleosome-free region just distally from the known Rap1-covered telomeric repeats. Moreover, previous evidence suggested a telomeric chromatin fold-back structure onto subtelomeric areas that supposedly was implicated in chromosome end protection. The in vivo ChEC method used herein in conjunction with several proteins in a natural context revealed no evidence for such structures in bulk chromatin. Conclusions: Our study allows a structural defnition of the chromatin found at chromosome ends in budding yeast. This defnition, derived with direct in vivo approaches, includes a terminal area that is free of nucleosomes, certain positioned nucleosomes and conserved DNA-bound protein complexes. This organization of subtelomeric and telo- meric areas however does not include a telomeric cis-loopback conformation. We propose that the observations on such fold-back structures may report rare and/or transient associations and not stable or constitutive structures. Keywords: Telomeric chromatin, Telomere, Subtelomere Background protection of natural DNA ends and avoid them being Telomeres, the ends of linear chromosomes, are special- recognized as sites of damage [1]. Te telomere proximal ized nucleoprotein structures primordial for genome area, also called subtelomeric area, usually is gene-poor stability. Generally composed of short, tandem repeats and enriched in complex repeated elements of various of DNA sequences that end in a single-strand extension structures [2]. Tis overall organization of the terminal of the 3′-end, telomeres and associated proteins ensure areas of chromosomes is highly conserved in eukaryotes, from yeast to mammals. Saccharomyces cerevisiae tel- omeres comprise of 300 75 bp of heterogenous telom- *Correspondence: [email protected] ± Department of Microbiology and Infectious Diseases, Faculty of Medicine eric repeats (abbreviated (TG2-3(TG)1-6)n, hereafter also and Health Sciences, Université de Sherbrooke, Cancer Research Pavilion, referred to as TG-repeats) and recruit numerous proteins Rm 3025, 3201, rue Jean-Mignault, Sherbrooke, QC J1E 4K8, Canada [3]. Tis terminal repeat-containing area appears devoid © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pasquier and Wellinger Epigenetics & Chromatin (2020) 13:23 Page 2 of 22 of nucleosomes and instead is covered by multiple Rap1 and X elements [29, 31, 32]. SIR-bound chromatin at molecules that recruit Rif1, Rif2 and the SIR complex subtelomeres or at mating-type loci is refractory to (Sir2, Sir3, Sir4) [3–5]. transcription, and also to other DNA-related processes Yeast subtelomeric repeat elements, i.e., X elements like replication origin fring or DNA repair [33]. Upon and Y’ elements, infuence several aspects of telomere recruitment of Sir2 via the concerted action of DNA- functions such as telomere length regulation and tran- bound Rap1, the ORC complex and Abf1, it deacetylates scriptional repression induced by telomeres, also known histones of nearby nucleosomes allowing Sir3 and Sir4 as Telomere Position Efect (TPE) [6–8]. Y’ elements exist binding to hypoacetylated histones, eventually leading to in two forms (Y’ long and Y’ short) and are found in 1 SIR complex propagation to adjacent chromatin [33]. On to 4 copies at approximately half of chromosomal ends the other hand, at chromosome ends, bound Tbf1 and [9, 10]. Unlike Y’ elements, X elements are found at all Reb1 at subtelomeres appear to counteract this SIR com- chromosomal ends and are much more heterogenous in plex spreading [34]. length and sequence [10]. On chromosomal ends with Compelling results suggest that due to multiple inter- both the X and Y’ elements, the X element is found on actions between themselves and histones, the SIR pro- the centromeric side of the Y’ element and in certain teins may facilitate a chromatin folding, resulting in instances, there are several telomeric repeats at the X–Y’ specifc high-order structures. ChIP analyses on cross- junction [3]. Despite the fact that there is no detectable linked chromatin showed that Rap1 is not only bound to sequence homology between X and Y’ elements, most the terminal telomeric repeats, as expected, but it unex- subtelomere–telomere junctions share binding sites for pectedly is also associated with subtelomeric sites rela- several DNA-binding proteins such as the ORC complex, tively far from bona fde Rap1-binding sites [35, 36]. Te Tbf1 and Reb1 [2, 11–13]. Te ORC complex functions in results also showed that this association is SIR depend- replication origin fring and silencing, and binds DNA at ent. Moreover, experiments using a reporter construct, ARS consensus sequences (ACS) [14–17]. Tbf1 and Reb1 in which an Upstream Activating Sequence (UAS, or proteins are sequence-specifc DNA-binding factors with enhancer) was positioned downstream of the coding numerous genomic-binding locations in vivo, mainly in region, allowed the detection of an SIR-dependent tran- gene promoter regions [18–22]. It is thought that they act scription of the reporter [37]. Tese fndings lead to the on chromatin organization by allowing proper nucleo- suggestion that terminal telomeric repeats fold back onto some positioning on either side of their binding location subtelomeric areas, hence allowing the UAS to relocate [18, 23, 24]. In addition to those binding sites above, X upstream of the gene and activate transcription of it [37]. elements also harbour an Abf1 binding site [2]. Like Te SIR-dependent transcription of the reporter gene Reb1, Tbf1 and Rap1, Abf1 is a General Regulatory Factor with the downstream UAS is only genetically detectable (GRF), an abundant and essential protein with numerous when the construct is inserted close to telomeric repeats DNA-binding sites at gene promoters [23, 24]. and does not occur when it is inserted elsewhere in the On subtelomeric areas, chromatin organization such genome [37]. Te contacts of terminal Rap1 proteins with as nucleosome positioning has been investigated in small subtelomeric areas can be explained with a similar logic and large scale. However, direct analyses for nucleosome [35, 36] and this cis-telomere fold-back model therefore positioning over X elements via MNase experiments so is also congruent with the Rap1 enrichment over X ele- far have been reported only for one unmodifed chro- ments, even in an X–Y’ context where the X-element is mosomal end (TEL03L) [25]. Other analyses concerned separated from the physical end by more than 5 kb [29]. modifed chromosomal ends with the URA3 or ADE2 Finally, this high-order structure at telomeres is thought genes inserted upstream of the X element [26]. How- to participate in TPE and the protection of chromosomal ever, it has become clear that these marker insertions and ends from ectopic recombination [8, 38–40]. analyses for TPE for example may be difcult [27]. Also, Here, we used the in vivo ChEC method to characterize marker insertion at any location in the genome can afect subtelomeric chromatin organization in its native state, the transcriptional potential of an area surrounding the without any inserted sequence or marker gene nearby. marker and hence the chromatin confguration may not We analyzed a number of distinct subtelomere–telomere refect that of the native state [28]. Genome wide analy- junctions and report a detailed account of similarities ses of nucleosome positioning suggested a low content of and diferences in chromatin organization between Y’- nucleosomes across the X elements [29], whereas a par- telomeres and two X-telomere junctions (TEL03L and allel study suggested positioned nucleosomes over those TEL06R). Our detailed molecular analyses on X–Y’ junc- same areas [30]. tions that are far from the physical chromosome end on A particularity of subtelomeric chromatin is that the which they reside are incompatible with the classical view SIR complex appears enriched over telomeric repeats of the cis-telomere fold-back model.
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