Discussion 101

Since finished sequences from the telomere-containing BAC and fosmid clones we identified, isolated, and initially characterized (see Supplementary Figure 4.7 and Supplementary Table 4.2) have already been incorporated into the current mouse assembly (Build 38/mm10), we used a mouse assembly very similar to that build as our reference genome. We appended two segments of subtelomeric DNA, one at 4q (for which we had identified the remaining q-arm subtelomere sequence) and the other a representative sequence from the highly similar p-arm subtelomeres (Kalitsis et al., 2006) in order to include all telomere-adjacent sequence for our analysis. By trimming the sequenced distal telomere repeats and the placeholder NNNNs representing the unsequenced telomere tracts at mouse telomeres, we provided a consistent coordinate system for mouse subtelomere annotations, which were thus set relative to the base at the start of each terminal repeat tract and oriented from the most distal base towards the centromere. This allowed subtelomere sequence features near individual telomeres to be analyzed and visualized on the mouse browser relative to the same position at the start of each terminal repeat tract.

Our analysis indicates that human and mouse subtelomeres evolved via distinct mechanisms. Large, highly similar and identically oriented SREs that predominate at human subtelomeres are absent in the mouse. Instead, we observe smaller, more ancient and more randomly oriented SREs in the mouse. The current model for explaining human SRE structure - translocations involving the tips of chromosomes, followed by transmission of unbalanced chromosomal complements to offspring [321] – cannot explain SRE structure in mouse. While human SRE structure and chromosomal distribution evolved very recently and in fact is highly polymorphic in the population, mouse SREs are more divergent and, if they originally evolved in a manner similar to human SREs, their sequence organization has subsequently been disrupted by recombination or repair events resulting in locally inverted duplications. Since subtelomere

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available on the subtelomere structure of multiple mouse species; this information would be quite valuable in understanding how these regions evolved and whether the unusually long telomere repeat tracts in mice played a role in suppressing recent subtelomeric duplication events or perhaps promoting inversions.

A relatively large fraction of mouse subtelomeres lack SREs entirely (3q, 5q, 6q, 7q, 12q, 13q, 14q, 15q), and for some of these subtelomeres known genes extend to very near the start of the terminal (TTAGGG) tracts (e.g., wntless homolog (Wls) at 3q, bicaudal D homolog 1 (Bicd1) at 6q, MAS-related GPR, member D (Mrgprd) at 7q, transmembrane protein 196 (Tmem196) at 12q, and 2-cell-stage, variable group, member 3 (Tcstv3) at 13q). It is notable that each of these telomere-adjacent transcripts are oriented telomerically. 18q, which contains a relatively small SRE region, has recently been described as a major site for TERRA transcription in mouse (de Silanes et al., 2014). The position of the putative upstream promoter sites (A2 and A3, Figure 4.6) correspond to RNAPolII peaks in our analysis, whereas the downstream B3, C2, and D promoter regions overlap with properly oriented RNAseq read enrichments in the ES datasets but not the immortalized lymphocyte CH12 dataset (Figure 4.6). While TERRA molecules derived from non- 18q telomeres were not found (with the possible exception of 9q; de Silanes et al, 2014), other subtelomeres were neither exhaustively sampled, nor sampled in multiple cell types by de Salines et al. (2014); it is therefore possible that some of the existing telomerically oriented transcripts could read through subtelomeres into telomeres to form TERRAs, as was suggested by recent TERRA-enriched RNAseq mappings to human subtelomeres that overlap with the WASH transcript family [335].

Strikingly, a mouse repeat element of unknown origin (MurSatRep1) which is known to be enormously enriched in mouse lincRNAs where it is almost always transcribed in the sense orientation [338] is very highly enriched in mouse SREs (>200 fold) as well as in subtelomeric SDs (98-fold). Of the 30 MurSatRep1 clusters we found in mouse subtelomeres, 19 overlap with RNAseq enrichments in the sense orientation. The 18q TERRA locus includes one of these overlap sites; additional telomerically oriented sites within 20 kb of the (TTAGGG)n tract are

found at 8q, 13q, 17q (Figures 4.1 and 4.2), as well as at 10q and 6q (Supplementary Figure 4.8, (http://vader.wistar.upenn.edu/mousesubtel). Each of these subtelomeric sites corresponds to a lincRNA potentially capable of extending into the telomere (TTAGGG)n tract, (thus giving rise to TERRAs), and merits further analysis.

Waves of retrotransposon expansions have remodelled genome organization and CTCF binding in murine lineages [347]; these dramatic evolutionary changes, along with the very different segmental duplication evolution trajectories taken at mouse vs human subtelomeres, may together account for the completely different CTCF and cohesin binding site organization at mouse and human subtelomeres. Our results showing a nearly complete lack of CTCF/cohesin near mouse telomeres and no association with SRE or ITS boundaries indicate radically different mechanisms for TERRA regulation, telomere protection and potential cis-regulation of telomere lengths in mouse relative to human. Given the differences in telomere lengths, overall lifespan, susceptibility to cancer, and other telomere-associated properties in the two species, this is perhaps not surprising. It may be one of many functional consequences of the accelerated evolution of subtelomeric genome regions, where a wide variety of lineage-specific and species- specific functionalities have arisen [302,303,348,349].