In vivo chromatin organization on native yeast telomeric regions is independent of a cis-telomere loopback conformation

Background DNA packaging into chromatin regulates all DNA-related processes and at chromosomal ends could affect 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 unmodified subtelomere–telomere junctions in Saccharomyces cerevisiae. Results By ChEC experiments and indirect end-labeling, we characterized nucleosome positioning as well as specialized 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 definition of the chromatin found at chromosome ends in budding yeast. This definition, 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 telomeric 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.


Fig. S1
Chromatin organization of terminal Y' elements. a Features identified on terminal portions of Y' elements, starting at the conserved XhoI site. Sequences from all Y' chromosomal ends (17/32) were analyzed and potential binding sites for Tbf1 and Reb1 proteins determined. Position of ACS is included. *, incomplete sequencing data available at the very distal tip, the end of sequencing data is indicated by the end of the horizontal line. b Distances between MNase-sensitive sites identified in Fig. 1d. A size greater than 146 bp is compatible with positioning of a nucleosome, indicated by a dotted line. c, d Efficiency of cleavage by MNase-fused proteins on whole genomic DNA. Ethidium bromide staining of XhoI digested genomic DNA -related to Fig. 1c in c; and to Fig. 1b in d. Time of MNase activity in minutes and MNase-fused proteins are indicated on top of gels. Mono-, di-, tri-, tetra-refer to DNA sizes consistent with mono-, di-tri-and tetranucleosomes. M: DNA size marker.

Fig. S2
Chromatin organization of the terminal TEL03L X element. Top: schematic drawing of XY' telomeres (left) and X-only telomeres (right) with positions of XhoI sites. The TG-repeat specific probe used is represented by a solid black line. TRF: Terminal Restriction Fragment. Bottom, autoradiograms of the blots shown in Figs. 1b and 1c as re-hybridized to the TG-repeat specific probe.

Fig. S3
Chromatin organization of the terminal TEL06R X element. a Features identified on all terminal X elements, with respect to their start site. Sequences from X elements of X-only chromosomal ends were analyzed and potential binding sites of Tbf1 and Reb1 determined. Position of ACS is included. The end of X element is indicated by the end of the horizontal line. b Analysis of distances between MNase-sensitive sites. Size greater than 146 bp is compatible with positioning of a nucleosome, displayed by a dotted line. c related to Fig. 3c and 2b, plot profile of indicated strains. Y axis: Distance (cm), X axis: Intensity signal by pixels (% of total signal). (M) profiles are marker lanes.

Fig. S4
Chromatin organization of subtelomeric elements is independent of telomere length. a Related to Fig. 4a; b related to Fig. 4b.; plot profiles of signal intensities obtained with YKU80 and yku80Δ strains. Y axis: Distance (cm), X axis: Intensity signal by pixels (% of total signal). Telomeric area analyzed is indicated on top. Note that the shift to smaller size of the full TRF peak in the yku80Δ cells (see gray lines vs black lines) is fully expected as it corresponds to the short telomeric repeat tracts left in yku80Δ cells.

Fig. S5
Chromatin organization of X element from XY' chromosomal ends. a Features identified on all X-elements of XY' junctions. Location of the ACS is included. The end of the X element is indicated by the end of the horizontal line. b Comparison of cut-sites is plotted with respect to a terminal X (TEL03L) versus an X of an XY' junction (TEL05R). Location of features (ACS, Abf1, Rap1, Tbf1 and Reb1 potential binding sites) are included. c Schematic drawing of TEL16R with the position of BamHI and XhoI sites. TEL16R specific probe used in d is represented by a solid black line. AF: Analyzed Fragment. d In vivo ChEC experiments with GBD-MN, NLS-MN and MN-RAP1 analyzed on X element from TEL16R. Southern blot with BamHI and XhoI-digested genomic DNA hybridized to the TEL16R specific probe. Time of MNase activity in minutes is indicated on top of gels. Arrowheads with solid line indicate detectable cutting common to all MNase-fused proteins. e Location of MNase-sensitive sites on the TEL16R XY' junction. Position of features (ACS, Abf1, Rap1, Tbf1 and Reb1 potential binding sites) is included.

XY' junctions Terminal Xs
Distance from XhoI site