# Diversity and distribution of the subtelomeric Y' elements across Saccharomyces cerevisiae strains

Liébaut Dudragne, Juliana Silva Bernardes, Zhou Xu

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## To cite this version:

Liébaut Dudragne, Juliana Silva Bernardes, Zhou Xu. Diversity and distribution of the subtelomeric Y' elements across Saccharomyces cerevisiae strains. 2025. hal-05371164

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1 **Title:** Diversity and distribution of the subtelomeric Y' elements across *Saccharomyces cerevisiae*

2 strains

3 **Authors:** Liébaut Dudragne¹,², Juliana Silva Bernardes³,*, Zhou Xu¹,²,*

4

5 **Affiliations:**

6 ¹ Sorbonne Université, CNRS, Laboratory of Computational, Quantitative and Synthetic Biology, CQSB, F-

7 75005 Paris, France.

8 ² Sorbonne Université, CNRS, Inserm, Institut de Biologie Paris-Seine, IBPS, F-75005 Paris, France.

9 ³ Sorbonne Université, CNRS, UMR 7144, Adaptation & Diversity in the Marine Environment, Station

10 Biologique de Roscoff (SBR), 29680 Roscoff, France.

11

12 **\*Correspondence:**

13 juliana.silva_bernardes@sorbonne-universite.fr; jusilvabernardes@sb-roscoff.fr

14 zhou.xu@sorbonne-universite.fr

15

16 **Keywords:** subtelomere, telomere, repeated element, Y' element, yeast, long-read sequencing.

17

18 Running title: Repertoire of Y' elements in *Saccharomyces cerevisiae*.
1 Abstract

2 The subtelomeric regions of eukaryotic chromosomes harbor repeated elements that contribute to
3 genomic plasticity and adaptation. In *Saccharomyces cerevisiae*, the Y' elements represent a major class
4 of subtelomeric repeats, yet their diversity and evolutionary dynamics remain incompletely characterized.

5 Here, we analyzed Y' elements across 54 S. cerevisiae strains using high-quality telomere-to-telomere
6 genome assemblies. We detected 893 high-confidence Y' elements, which we classified into 12 major
7 clusters, revealing a broader structural diversity than previously described, including canonical short (~5.2
8 kb) and long (~6.7 kb) elements, intermediate-size classes (mid1 and mid2), and a novel family containing

9 CA-rich repeats. Sequence analyses showed that open reading frames (ORFs), including those encoding
10 the putative Y'-Help1 helicase, are highly conserved within clusters, suggesting selective maintenance of
11 functional sequences. The distribution of Y' elements varied widely across strains and chromosome
12 extremities, with some strains lacking Y' entirely and others, such as the clinical ADI isolate, carrying up to
13 149 copies. Interstitial telomeric sequences (ITS) were variably associated with Y' elements and tandem
14 Y' repeats, potentially facilitating recombination and amplification. Analysis of telomere length data
15 further revealed that the presence of long Y' elements, but not Y' elements from other clusters, at the
16 subtelomere is correlated with shorter telomeres at the same chromosome end. Our results provide the
17 most comprehensive catalog of S. cerevisiae Y' elements to date, uncovering unexpected structural and
18 sequence diversity, and a potentially functional role in telomere length regulation.
# 1 Introduction

Genomic regions located at chromosome extremities are often rich in repeated sequences, including telomeres, the terminal repeats widely studied for their role in genome stability, and subtelomeres, adjacent regions containing diverse families of repeated elements such as transposable elements, satellite sequences, ribosomal DNA and paralogous genes involved in life cycle and adaptive processes (Hocher & Taddei, 2020). Subtelomeres are rapidly evolving regions and display structural and composition diversity even in closely related species or within species, as observed for *Saccharomyces cerevisiae* and *Saccharomyces paradoxus* (Yue et al., 2017). The presence of repeated elements promotes homology-dependent mechanisms, which in turn lead to rearrangements and amplification. In *S. cerevisiae*, the two main types of subtelomeric elements are the Y' elements and the X elements (Chan et al., 1983; Chan & Tye, 1983). While X elements are found at nearly all extremities, Y' elements vary widely in number and organization across subtelomeres and strains, ranging from none to four or more tandem copies often separated by interstitial telomeric sequences (ITS), with some clinical isolates displaying unusually high copy numbers (Chan & Tye, 1983; Heasley & Argueso, 2022; O'Donnell et al., 2023). Y' elements are usually separated into two types based on their lengths: short Y' (~5.2 kb) and long Y' (~6.7 kb), differing by an internal deletion (Chan & Tye, 1983; Louis & Haber, 1992), although rare unusually short or long Y' elements have also been described (Louis & Haber, 1992). Y' elements have been reported to recombine during mitosis as well as in stress conditions, and to do so preferentially within classes, perhaps explaining how these two classes coexist (Louis & Haber, 1990a).

Y' elements of previously studied laboratory strains contain a consensus autonomously replicating sequence (ARS), a stretch of 8-20 tandemly repeated 36-mer sequences, as well as one or two open reading frames (ORFs) (Horowitz & Haber, 1984; Louis & Haber, 1992). The ARS sequence seems to be functional, as it is used in *rif1Δ* cells, but does not seem to be used as an active origin of replication in
1 wild-type cells (Theulot et al., 2025). Y' elements potentially express a DNA helicase called Y'-Help as one
2 of the ORFs contain a motif homologous to eukaryotic helicases and can be transcribed in low amounts
3 (Louis & Haber, 1992; Yamada et al., 1998). Accordingly, most Y' sequence variations were found to be in
4 the first half of the element, leaving the helicase ORF mostly intact (Louis & Haber, 1992). One study
5 provided further insight by demonstrating that the artificially cloned ORF sequence exhibited functional
6 helicase activity in *Escherichia coli* (Yamada et al., 1998). Despite the presence of this potentially functional
7 ORF, a recent work used an engineered yeast strain containing only three chromosomes to remove all Y'
8 elements and found no impact on cell growth, telomere length regulation, or telomere silencing (Hu et
9 al., 2024). However, in a specific mutant context where telomere regulator genes are deleted, i.e. *tel1Δ
10 rif1Δ*, Y' elements exert some effect on telomere length (Craven & Petes, 1999; Sholes et al., 2022).
11 Overall, the physiological functions of the Y' elements remain elusive.

12 In telomerase-negative cells, the recombination-based amplification of Y' elements is the molecular basis
13 of type I post-senescence survivors (Lundblad & Blackburn, 1993; Teng & Zakian, 1999). Y' elements can
14 be amplified 100 folds in these survivors and invade X-only subtelomeres. The other major senescence
15 bypass pathway, called type II, is characterized by massive telomere elongation, but Y' elements are also
16 found to be altered and moderately amplified in the early steps of this process (Churikov et al., 2014;
17 Kockler et al., 2021; Teng & Zakian, 1999). Homologous recombination pathways, including break-induced
18 replication (BIR), are required for Y' acquisition and amplification (Le et al., 1999; Lundblad & Blackburn,
19 1993; Lydeard et al., 2007). In addition, circular DNA molecules derived from Y' elements might also
20 contribute to this process (Larrivée & Wellinger, 2006). The Y'-Help helicase is expressed in type I
21 survivors, suggesting it could play a role in subtelomeric recombination (Yamada et al., 1998). Finally, Y'
22 RNAs can be reversed transcribed by Ty1 retrotransposon to promote recombination in the subtelomeres
23 of telomerase-negative cells (Maxwell et al., 2004).
1 Beyond their structural diversity and recombination properties, Y' and X subtelomeric elements also
2 influence chromatin states at chromosome ends. Both contain domains that modulate the telomeric
3 position effect (TPE) (Fourel et al., 1999). Telomere-proximal portions harbor subtelomeric anti-silencing
4 regions (STARs), enriched in Tbf1- and Reb1-binding motifs, and which act as insulators that block the
5 spread of silenced chromatin. In contrast, more centromere-proximal sequences within X and Y' act as
6 silencing relays, including proto-silencers such as ARS consensus sequences and Abf1-binding sites (Louis,
7 1995). Such a balance between anti-silencing and silencing activities may explain how Y' ORFs, such as the
8 Y'-Help helicase, can be expressed despite the generally repressive telomeric environment (Fourel et al.,
9 1999).

10 Long-read sequencing technologies have recently enabled complete telomere-to-telomere assemblies of
11 S. cerevisiae genomes, allowing the accurate resolution of tandemly repeated Y' elements in both natural
12 isolates and experimental contexts involving telomere deprotection or erosion (Dudragne et al., 2025;
13 Heasley & Argueso, 2022; Kockler et al., 2021; O'Donnell et al., 2023). Here, we analyze 54 haploid S.
14 cerevisiae strains spanning the species' phylogenetic and ecological diversity to investigate Y' element
15 diversity. We describe the most comprehensive repertoire of Y' elements to date, uncover a new class
16 containing CA-rich repeats, and characterize the distribution of distinct Y' types across chromosome ends
17 and strains.

## Results

### The repertoire of Y' elements across 54 S. cerevisiae strains

As existing Y' annotations from the *Saccharomyces cerevisiae* Reference Assembly Panel (ScRAP)
(O'Donnell et al., 2023) were often fragmented, likely owing to the limited diversity of the single reference
1 sequence used for detection, we developed a bottom-up approach to exhaustively identify full-length Y'

2 elements across strains.

3 Given that telomeric and subtelomeric regions are particularly prone to assembly errors, we focused on
4 the haploid genomes of the ScRAP database to avoid haplotype phasing artifacts inherent to diploid
5 assemblies. Oxford Nanopore Technologies (ONT) sequencing reads were remapped to each assembly for
6 manual inspection and correction of chromosome ends, leading to the manual correction of 35 out of
7 1696 ends by clipping misassembled terminal sequences. Three strains were discarded due to poor
8 assembly confidence, yielding 53 high-quality haploid genomes for our analysis, to which we added a long-
9 read assembly of the W303 laboratory strain (Garrido et al., 2025). Despite being sequenced as haploids,
10 many of these genomes originate from diploid parental strains, meaning that our dataset captured the
11 genetic diversity present in both haploid and diploid strains. The 54 strains cover a wide range of the
12 diversity of the ScRAP panel, representing 23 out of the 29 clades (Fig. 1A).

13 We sought to build a more complete Y' reference database for Y' detection. To establish an initial set of
14 high-confidence, full-length Y' sequences, we identified all tandem repeats located in subtelomeric
15 regions and classified as complete Y' elements those repeats that matched at least part of the known
16 reference Y' element and were directly flanked by telomeric sequences. This led to the detection of 101
17 high-confidence Y' sequences, to which we added the Y' elements of the S288C reference genome. We
18 then mapped these sequences to the 54 genomes with BLAT (Kent, 2002) for exhaustive Y' detection. This
19 led to the detection of 919 Y' elements of length >1000 bp, among which 884 elements had a length >3500
20 bp for a total length of 5,263,491 bp and a mean Y' length of 5954 bp, which represents an improvement
21 over the 800 Y' elements for 4,499,540 bp (mean Y' length of 5624 bp) reported in the ScRAP annotations
22 for the same set of strains. Our method thus successfully detected a greater number of Y' elements as
23 well as more contiguous and complete copies. 26 elements were removed from further analyses as their
24 coordinates adjoining the extremities of contigs suggested that the assembly was incomplete and the Y'
1 elements were truncated. Therefore, 893 elements were kept for all downstream analyses (Supp. Data S1

2 and S2).

3

4 Y' elements fall into discrete length classes and repetitions of a 36-bp sequence introduce length

5 variability

6 The lengths of all detected Y' elements followed a mostly bimodal distribution, with one mode peaking at
7 ~5,450 bp and another one at ~6,650 bp, accounting for the two well-known length classes "short" and
8 "long" (Fig. 1B), usually described as being 5.2 and 6.7 kb respectively. In addition, 60 Y' elements were
9 measured at ~7,100 bp which, upon further inquiry, were all found to originate from the ADI strain,
10 collected in a clinical environment. The two main modes exhibited substantial spread, suggesting length
11 heterogeneity within the typical length classes. Since Y' elements have been described as containing a
12 stretch of 36-mer repeats of variable lengths, we asked how much these repeats contributed to the length
13 heterogeneity of Y' elements. The median length of the stretch of 36-mers was around 360 bp (10 repeats)
14 but ranged from 0 bp (no repeat) up to 1.3 kb (36 repeats) (Supp. Fig. S1A). Analysis of the repeat motif
15 confirmed the consensus sequence found by Horowitz & Haber, 1984 (Supp. Fig. S1B). Interestingly, the
16 variability observed among 36-mer sequences was comparable both within individual Y' elements and
17 between different Y' elements (Supp. Fig. S1C), suggesting that sequence degeneration originated in the
18 ancestral Y' element or that the 36-mer repeats constantly undergo diversification at a high rate.

19 Removing the 36-mer stretches from the Y' element sequences reduced the spread around the two main
20 length modes and revealed a new smaller peak below 5 kb (Fig. 1C). Furthermore, a substantial number
21 of elements could still be detected between the "short" and "long" peaks. Together, these findings called
22 for a finer description of Y' element classes.

23
<figure><img src="image_3.png" /><figcaption>Figure 1. Discrete Y' element classes with 36-mer repeat variability.</figcaption></figure>

(A) Phylogenetic tree of the 54 *S. cerevisiae* strains selected for this study. The tree is a subset of the ScRAP tree extracted from O'Donnell et al., 2023. Colors indicate clades as defined in that same study. Y' clusters as defined in the next section appear colored when they are present in the strain.
1 (B) Distribution of the lengths of all detected Y' elements.

2 (C) Same as (B), but after removing the sequence of the 36-mer repeats, revealing sharper peaks as well as new ones.

3

4 Identification of canonical, intermediate-size, and novel CA-rich clusters of Y' elements

5 To describe the diversity of Y' elements and identify conserved domains, we calculated a pairwise
6 similarity matrix between all Y' elements and performed hierarchical clustering, excluding the variable 36-
7 mer repeats which might artificially introduce differences and hinder the analysis (Fig. 2A). It is important
8 to note that the similarity measure, based on per-base match of sequence alignments, is highly sensitive
9 to large insertions and deletions and less sensitive to single nucleotide polymorphisms (SNPs), thus leading
10 to a clustering analysis that prioritizes the structural differences between Y' elements.

11 After optimizing the silhouette score, we identified 46 clusters representing distinct groups of Y' elements
12 (Supp. Data S2): the two largest clusters, comprising 293 and 203 elements, corresponded to the canonical
13 long Y' elements and short Y' elements, respectively. Together, the five largest clusters accounted for 82%
14 (729/893) of all Y' elements. Many clusters were very small, containing less than five elements (n = 27) or
15 just one element.

16 Strikingly, 71 sequences diverged drastically from all others (Fig. 2A, red square at the bottom right of the
17 heatmap). Manual inspection of these elements revealed the presence, in 49 of them, of a stretch of a
18 non-telomeric CA-rich region in the middle of the elements. As for the 36-bp repeats, this CA-rich region
19 was highly variable in length and could artificially introduce noise in our clustering (Supp. Fig. S2A). We
20 therefore removed this region from the Y' elements before running a similar but separate clustering on
21 these 49 sequences alone, resulting in eight clusters (Fig. 2B and Supp. Data S2).
1 To focus on the most represented classes of Y' elements, we dismissed clusters containing fewer than five
2 elements, clusters restricted to a single strain, or clusters with high intracluster heterogeneity. This filter
3 kept 12 clusters containing the vast majority (88%, 790/893) of all Y' elements for further study. Only 49%
4 (24/49) of CA-containing Y' elements were kept, indicating that these sequences formed smaller and more
5 heterogeneous clusters.

6 Using within-cluster multiple sequence alignment, we then retrieved a consensus sequence for each
7 cluster, which we used for further alignment and domain comparison (Fig. 2C). Cluster 6 corresponded to
8 the canonical long Y' element with a consensus sequence of 6,256 bp, and cluster 3 represented the
9 canonical short Y' element with a consensus sequence of 5,088 bp, excluding the 36-mer repeats for both.
10 While the short Y' element differed from the long one by two large deletions of 465 bp and 600 bp, the
11 analysis revealed two clusters (clusters 4 and 5) of Y' elements containing only one or the other deletion,
12 thus delineating two distinct families of intermediate Y's that we named Y'-mid1 and Y'-mid2, respectively.
13 Clusters 11 and 12 lacked the second half present in other clusters. Instead, each presented a unique 1.6-
14 1.8 kb domain, within which were nested the CA-rich repeats. Most other clusters differed from the
15 canonical Y' elements either by having a truncated start or a different end (Fig. 2C), with cluster 7 being
16 almost exclusively composed of Y' from the ADI strain (64/67 elements).
<figure><img src="image_4.png" /><figcaption>**B** Hierachical clustering of CA-rich Y' elements</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**C** Domain structure of clusters</figcaption></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** The 12 main clusters of Y' elements found across *S. cerevisiae* strains.</figcaption></figure>

(A) Clustermap of pairwise sequence similarities between Y' elements, excluding the 36-mer repeats. The hierarchical clustering tree is shown above. Red square highlights highly divergent sequences. Color scale truncated below the 5th percentile.

1

2

3

4
1 (B) Clustermap of pairwise sequence similarities between the 49 CA-repeat-containing Y' elements, computed after removing the

2 CA-rich repeats. The hierarchical clustering tree is shown above.

3 (C) Schematic representation of the 12 main Y' clusters, using colors to show conserved homology domains. The 36-mer repeats

4 and the CA-rich repeats are depicted between brackets with a fixed length.

5 (D) Logo representation of the two types of patterns found in CA-rich sequences. See Supp. Fig. S2B for per-strain variations of

6 pattern 1.

7

8 Diversity of CA-rich sequences

9 We examined the CA-rich regions identified within the 49 Y' elements previously flagged as divergent from
10 canonical sequences. These regions displayed a marked compositional bias, with an average base
11 composition of 45% adenines, 42% cytosines, 7.5% thymines, and 5.5% guanines. This near-equal
12 proportion of adenine and cytosine clearly distinguished them from telomeric CA-rich tracts, which are
13 more enriched in cytosines (62.5% C, 37.5% A).

14 Inspection of the sequences suggested two recurrent types of repeated motifs, which we characterized
15 by generating consensus sequence logos (Fig. 2D and Supp Fig. S2B). Pattern 1 spans 37 bp, closely
16 matching the length of the canonical 36-mer repeats observed in other Y' classes. However, no detectable
17 sequence similarity was found between the CA-rich motifs and the 36-mer repeats, suggesting
18 independent origins. Pattern 2, detected in all but 3 strains (CEI, AKH, CEQ), is more variable in length but
19 contains a highly conserved 29-bp core. Its second half bears similarity to the corresponding region of
20 pattern 1, suggesting that pattern 2 may have evolved from pattern 1, or vice versa. Out of all 33,413
21 bases comprising the extracted CA-rich regions, 19,656 bases (59%) were assigned to pattern 1, and 8,432
22 bases (25%) to pattern 2.
1 These observations indicate that CA-rich sequences represent a distinct and heterogeneous component

2 of Y' elements, with recurrent but diverging motifs that may contribute to structural diversification within

3 this subtelomeric repeat family.

4

5 Distribution of Y' elements across strains and chromosome extremities

6 As previously observed (Louis et al., 1994; Louis & Haber, 1990b; O'Donnell et al., 2023), the number of Y'

7 elements is variable across strains with an average ± standard deviation of 14 ± 9 per strain (Fig. 3A),

8 excluding the ADI outlier displaying 149 Y' elements. In our dataset, strains ADE and CCC were devoid of

9 Y' elements and strains BAK, BAI and CCQ contained only one Y' element, found at chromosome extremity

10 V.L. At the other end of the spectrum, haploid strain ADI presented 149 Y' elements, spread throughout

11 25 chromosome extremities. Across all strains, Y' elements were found at all chromosome extremities

12 (Fig. 3B). Two extremities stood out, with V.L and XIV.R being relatively enriched in Y' elements. Most

13 chromosome extremities were associated with zero or one Y' element (1106/1728 and 513/1728,

14 respectively), while tandem repeats of two or more Y' elements were a relatively rare occurrence (Supp.

15 Fig. S3A).

16 Previous reports suggested that tandem Y' elements at a given chromosome end are homogeneously

17 composed of the same length class (Louis & Haber, 1990b; Yamada et al., 1998). However, our analysis

18 showed that this was not always the case: out of the 109 extremities containing tandem Y' elements, 34

19 contained a combination of short and long Y' elements (clusters 1-3 and clusters 6-10, respectively), out

20 of which 25 contained a combination of canonical short and canonical long Y' elements. Nonetheless,

21 analysis of the conserved regions in Y' clusters, excluding the CA-repeat-containing ones, revealed that

22 sequence diversity is lower within strains than between strains, consistent with partial homogenization

23 of Y' elements within individual genomes (Supp. Fig. S2B).
1 We then explored the distribution of Y' elements per strain according to our clustering analysis (Fig. 3C).

2 Clusters 6 and 3, representing the canonical long and short Y', respectively, were found in the vast majority

3 of strains (45/54 and 30/54, respectively). Notably, in 9 strains (ALH, AVI, BAG, BAI, BAK, BAL, BFP, and

4 CCQ), the only Y' cluster detected corresponded to cluster 6. These strains disproportionately belonged

5 to early divergent lineages (Fig. 1A), and their total number of Y' elements was relatively low. Overall,

6 these results depict a scenario in which the canonical long Y' of cluster 6 represents the ancestral Y'

7 element, from which all other clusters subsequently derived.

8 Cluster 2, in which the Y' elements differ from the short Y' by an additional deletion of ~300 bp on its left

9 side, is highly represented (>90%) in strains BDN, BGN, and BGP, belonging to two closely related clades:

10 clade 7 ("French dairy") and clade 8 ("African beer") (Fig. 1A). Additionally, the vast majority of strains

11 possessing such an element were contained in clades 1-8, suggesting that the emergence of cluster 2 can

12 be traced back to the common ancestor of these clades.

13 Although CA-containing Y' elements seemed to be especially present in clades 14 ("European clinical")

14 and 15 ("Israel evolution canyon"), they were found across the whole phylogenetic tree (Fig. 1A). This

15 suggests that these elements either appeared early in the species and were often lost, or have spread

16 among strains through out-crossing and recombination.
<figure><img src="image_8.png" /><figcaption>Figure 3. Distribution of Y' elements across strains and chromosome extremities.</figcaption></figure>

(A) Distribution of the number of Y' elements per strain.

(B) Fraction of strains exhibiting at least one Y' element at corresponding extremities.

(C) Distribution of the clusters of Y' elements across all strains. Strain ADI is set apart with a different scale.

Open reading frames are highly conserved within clusters and across strains

The presence of ORFs in Y' elements has been known for a long time, but some discrepancies regarding

their length and copy number have been reported between strains and studies (Louis & Haber, 1992;

Yamada et al., 1998). Because long-read sequencing is prone to errors, especially in homopolymers,
frameshifts in ORFs can sometimes occur in the assembled sequences. Indeed, ORFs were observed to frequently shift reading frames in homopolymers in our Y' element sequences (Supp. Fig. S4A), although we could not strictly exclude that these frame shifts represented genuine biological events. We quantified the probability of ORF presence at the cluster level by aggregating, across all Y' elements, the positions classified as belonging to an ORF, regardless of the reading frame (Fig. 4 and Supp. Fig. S4B). The results clearly indicated a well conserved single long ORF region in the canonical long Y' cluster (cluster 6) as well as in the mid-1 Y' cluster (cluster 4), and two smaller well conserved ORF regions in short Y' clusters (clusters 1, 2 and 3). The first half of the CA-repeat-containing Y' elements (clusters 11 and 12) also appeared to contain an ORF, in accordance with their high similarity with Y' elements of other clusters in this region, but in cluster 12, the ORF was split in two parts, whereas in cluster 11, the ORF appeared less conserved, suggestive of a potential evolutionary drift.

<figure><img src="image_9.png" /><figcaption>Figure 4. ORF probability within Y' clusters.</figcaption></figure>
1 Likelihood of a given position in the Y' element consensus sequence of a cluster belonging to an ORF, computed for 6 clusters.

2 Color bars represent cluster domain structures as depicted in Fig. 2C. Vertical black bars in domain structures indicate the position

3 of the 36-mer or CA-rich repeats. See Supp. Fig. S4B for the other clusters.

4

5 **Variability of internal and terminal telomeric sequences associated with Y' elements**

6 Internal telomeric sequences (ITS) can be found centromere-proximal to Y' elements and for tandem Y'

7 repeats, between Y' elements. Through their homology to the terminal telomere sequence, they

8 contribute to subtelomere rearrangements and Y' amplification, notably in telomerase-negative cells. We

9 found that only half of the Y' elements were preceded by an ITS on their centromere-proximal side.

10 Similarly, only 145/234 (62%) tandem repeat junctions presented an ITS in between Y' elements.

11 The ITSs displayed a wide length distribution ranging from 20 bp to ~500 bp and even 628 bp and 957 bp

12 in two instances (Fig. 5A). Overall, the size distributions of ITS between tandem Y' elements and next to

13 isolated Y' elements followed a roughly similar shape, with the exception of more very short ITS detected

14 in the proximity (distance < 100 bp) of isolated Y' elements (Fig. 5A).

15 A recent study reported a subtle but significant influence of Y' elements on terminal telomere length: the

16 telomeres immediately adjacent to Y' elements are shorter than the ones next to subtelomeres devoid of

17 Y' element (Garrido et al., 2025). As this analysis was conducted without distinguishing between Y' types,

18 we reexamined the telomere length data from that study in light of our Y' classification. We found that

19 telomeres adjacent to canonical long Y' elements (cluster 6) showed the largest reduction in telomere

20 length: telomeres at these extremities were 6.4% shorter than extremities devoid of Y' (Fig. 5B), which

21 was significantly more than the impact of short Y' elements. Telomeres associated with canonical short Y'

22 element and other short Y' elements (clusters 2 and 3) or mid-1 Y' elements (cluster 5) were also shorter,

23 but the differences were not individually significant (p = 0.062 and p = 0.304, respectively; Student's t-
1 test). Combining these classes yielded a statistically significant telomere length reduction (p = 0.042). CA-
2 rich Y' elements did not appear to clearly affect telomere length in cis. These results suggest that the
3 region specifically present in canonical long Y' elements might exert some negative effect on cis telomere
4 regulation.

5 As Y' elements have been reported to bind the silencing regulators Tbf1 and Reb1, we examined whether
6 differences in their binding motifs could account for the distinct effects of Y' types on telomere length
7 regulation. However, no differences were observed in the number or position of these binding sites
8 between long and short Y' elements, ruling out a role for these regulators in this phenomenon.

<figure><img src="image_10.png" /><figcaption>Figure 5. Distributions of ITS lengths and telomere lengths associated with Y' elements.</figcaption></figure>

(A) Distribution of ITS lengths for ITS between tandem Y' elements (left panel) and not between tandem Y' elements (right panel), excluding ADI.

(B) Boxplot of mean extremity-specific telomere lengths, normalized by the average telomere length in the corresponding strain, associated with different types of Y' elements. Points outside of the boxplot whiskers are not shown. Red lines indicate mean values. Asterisks show statistical differences with p-values < 0.05 based on two-tailed Student's t-test.
1 Discussion

2 In this study, we systematically analyzed hundreds of subtelomeric Y' elements from strains representing
3 the diversity of S. cerevisiae to characterize the structural variations within this family of elements. While
4 56% of the analyzed Y' elements fell into what has until now been described as the Y'-short and Y'-long
5 classes, the rest encompassed a large spectrum of structural diversity. The newly identified Y' classes
6 differed from the canonical ones mostly owing to deletion of domains at the start or the end of the
7 element, sometimes replaced by other sequences. In addition, two distinct repeated motifs (the 36-mer
8 or the CA-rich ones) are found in the second half of the Y' elements and their variable lengths, up to ~1.2
9 or ~2.1 kb, respectively, add another layer of structural diversity.

10

11 The distribution of the clusters across all studied strains suggests that the canonical long Y' element might
12 have been the ancestral one, from which all others could have derived. The widespread presence of the
13 canonical short Y' element also suggests that it derived early from the canonical long Y' and then amplified.
14 Interestingly, we found clusters (mid1 and mid2) with structures that display one or the other of the two
15 deleted parts that differentiate the canonical short Y' from the canonical long Y'. While any of these two
16 structures could be likely a candidate for an intermediate structure between canonical long Y' and the
17 canonical short Y', they are much less abundant and we cannot exclude that they derived independently
18 from the canonical long Y'.

19

20 Our analyses revealed a new family of CA-repeat-containing Y' elements among the most divergent Y'
21 elements. They are characterized by a CA-rich degenerate repetitive sequence distinct from the 36-mer
22 repeats found in all other Y' elements. These sequences are highly variable in length and composition, and
1 yet display recurrent motifs (patterns 1 and 2) that are conserved across multiple strains. Intriguingly, in
2 the two main clusters where they are found, the CA-rich repeats are nested within different, non-
3 homologous domains, which are also distinct from the domain found in all other clusters of Y' elements,
4 thus begging the question of their molecular origin. The near-equal adenine-cytosine content
5 distinguishes these sequences from canonical telomeric CA-rich tracts and suggests that they are not
6 simply divergent telomeric repeats. The strand orientation of the CA-rich repeats also precludes their use
7 as a seed sequence for new telomere addition, should there be an accidental double-strand break
8 occurring close to them. Although none of the identified motifs match the canonical Rap1 binding
9 consensus (Vignais et al., 1990), their similarity to the consensus still leaves open the possibility that Rap1
could bind them directly, albeit with lower affinity.
10
11

12 The number of Y' elements per strain is widely variable, ranging from 0 to ~150 in our dataset, highlighting
13 the dynamic and heterogeneous nature of subtelomeres even within a species. We suggest that further
14 investigations on the potential physiological functions of Y' elements should take into account this natural
15 variability, and a single laboratory strain, such as the W303 strain included in our study, might not be
16 representative. Additionally, it would be interesting to compare strains with highly amplified Y' elements
17 (e.g. ADI or the strain YJM311 described by Heasley & Argueso, 2022) and telomerase-negative type I post-
senescence survivors, with the underlying idea that extended periods of telomere shortening or transient
18 telomere deprotection might have triggered Y' element amplification (Dudragne et al., 2025; Lundblad &
19 Blackburn, 1993; Teng & Zakian, 1999).
20
21

22 ORFs had previously been identified several times in various Y' elements (Louis & Haber, 1992; Yamada et
23 al., 1998). However, it remained unclear whether they are functional or merely a vestige of their
1 evolutionary history. While their functionality is still not addressed, we showed that ORFs are well
2 conserved across Y' elements, with one ORF being present in "long" clusters, and two ORFs being present
3 in "short" clusters, with the exception of CA-repeat-containing Y' elements showing reduced
4 conservation. This result favors a model in which ORFs are actively conserved within Y' elements
5 throughout evolution within the S. cerevisiae species, suggesting a potentially functional role for Y'
6 element encoded helicases.

7

8 Whether the Y' element might display a function in telomere length regulation has also been elusive
9 (Craven & Petes, 1999; Hu et al., 2024; Sholes et al., 2022). We recently showed that the presence of Y'
10 elements is associated with shorter mean telomere length in cis, after normalization by the strain-specific
11 global mean telomere length (Garrido et al., 2025). Here, we refine this observation by showing that long
12 Y' elements are the main contributor to this association, whereas short or mid1 Y' elements have no
13 association to shorter telomeres or only a minor one when considered together. We thus speculate that
14 the sequence present in long Y' elements but absent in mid1 (and short) Y' elements might recruit a factor
15 that negatively regulates telomere length in cis. Among known factors binding to Y' elements, both Tbf1
16 and Reb1 bind to the terminal part of the Y' element (Fourel et al., 1999) and are not likely candidates.

17

18 Overall, this work characterizes the exhaustive repertoire of the highly variable Y' elements. It uncovers
19 the full spectrum of structural variants, including whole new classes, their distribution across S. cerevisiae
20 strains. Beyond the insights it provides on the evolution and potential functions of Y' elements, this study
21 highlights the diversity and dynamics of subtelomeres, a property common to most eukaryotes.

22
1 Material and Methods

2 Data

3 All data analyzed in this study besides data for W303 strain were downloaded from the European
4 Nucleotide Archive (ENA), from project number PRJEB59869 (O'Donnell et al., 2023). Both reads and
5 assemblies were downloaded, and reads were mapped back on assemblies with Minimap2 (Li, 2018)
6 before visual inspection of chromosome extremities using Tablet (Milne et al., 2013). All chromosome
7 extremities for which there was suspicion of overassembly or misassembly, were trimmed from
8 assemblies. The W303 strain was sequenced and assembled in a previous work (Garrido et al., 2025), and
9 reads and assembly can be found on the ENA under project number PRJEB89249.

10

11 Detection of X elements, Y' elements and telomeres

12 Telomeres were detected on assemblies using Telofinder (O'Donnell et al., 2023)  
13 (https://github.com/GillesFischerSorbonne/telofinder). This detection was used for terminal telomere  
14 detection as well as ITS detection. X elements were detected in the assemblies using LRSDAY 1.7.2 (Yue &  
15 Liti, 2018). For Y' elements, telomeres were first masked from the genomes, then a high-confidence Y'  
16 element database was built by looking for perfect repeats within sliding windows of size 25 kb with a 5 kb  
17 step, over the 100 kb extending from every chromosome ends of every strain. Repeats were detected in  
18 these windows by mapping the window against itself using BLAST and flagging alignments of lengths  
19 comprised between 4.0 and 9.5 kb with an identity percentage of 90 or greater. Y' elements from the  
20 S288C reference genome (version R64-2-1) (Cherry et al., 2012) were used as queries in a BLAST search  
21 across the assemblies, and repeats identified in the previous step that matched at least partially to one of  
22 these reference Y' sequences were classified as Y' elements. To make sure these repeats were detected
1 completely, only repeats directly flanked by telomeric sequences were kept for the high-confidence Y'

2 element database. This database was then used to detect Y' elements across all genomes using BLAT

3 v.37x1, with parameter -maxIntron=1000. Because BLAT tends to overextend alignments over large

4 stretches of non-mapping regions, we trimmed the extremal blocks detected by BLAT if they were smaller

5 than 20 bp and the gap separating them from the next block was greater than 100 bp.

6

7 **36-mer detection and masking**

8 All possible 36-mer sequences derived from the degenerate sequence given by Horowitz & Haber, (1984)

9 were used to calculate a 36-meric alignment score across the Y' elements of the S288C reference genome.

10 At each position, a window of size 36 was extracted, and a score was derived from the Levenshtein

11 distance between the window and the reference 36-mers, calculated as the proportion of matching bases.

12 36-mers were then detected as peaks in the scores, using scipy.signal findPeaks function with parameters

13 min_distance = 30 and threshold = 0.85. Any match of length different than 36 was discarded, and 36-

14 mers identified this way from the reference genome were used as a new 36-mer database to repeat the

15 same process on every Y' element from our 54 strains. Matches of size 36 were used to generate a

16 consensus sequence logo using Python's Logomaker library, and all nucleotides between the first and last

17 detection peak were designated as part of the 36-mer stretch. These regions were then removed from

18 the Y' element sequences for the subsequent analyses.

19

20 **Similarity matrix and hierarchical clustering**

21 Y' elements were aligned using MAFFT v7.490 (Katoh & Standley, 2013) with "-auto" option, in order to

22 retrieve a multiple sequence alignment (MSA). Similarity matrices were then computed from the
1 alignments by extracting pairs of sequences from the MSA and computing the percentage of shared
2 nucleotides over the full length covered by the union of these two sequences. Positions where the two
3 sequences shared a gap were ignored. The similarity matrix was subjected to hierarchical clustering using
4 seaborn "clustermap" function (method = "average"), and clusters were defined by cutting the resulting
5 dendrogram at the chosen cluster count. The optimal number of clusters was defined as the value
6 maximizing the silhouette score across cluster counts from 2 to 100.

7 The second clustering (Fig. 2B), which ran on CA-containing Y' elements after the removal of the CA-rich
8 regions, was used to replace the initial cluster assignation of these elements, without changing the cluster
9 assignation of the other elements.

10

11 ORF detection

12 ORFs were detected as intervals of at least 300 bases between a start codon (ATG) and one of the following
13 stop codons (TAA, TAG or TGA) over the three possible frames. For each element, a masked array
14 indicating the presence of an ORF in any frame was derived. ORF density plots were computed from the
15 masks of all elements of a given cluster, using as a shared coordinate system the coordinates from the
16 MSA of sequences from this cluster.

17

18 Within- vs between-strain diversity of Y' elements

19 Y' elements from clusters 6, 7, 8, 12, 15, 16, 17, 18, 19 and 21, excluding elements from ADI strain, were
20 aligned using MAFFT v7.490 (Katoh & Standley, 2013) with "-auto" option, and the conserved region
21 corresponding to the purple 36-mer-containing region (color from Fig. 2C) was used for diversity analysis.
22 Pairwise distances between elements were then computed from alignment by extracting pairs of
1 sequences from the MSA and computing the percentage of divergent nucleotides. Positions where the

2 two sequences shared a gap were ignored. All pairs of Y' elements present in the same strain were kept

3 for within-strain boxplot, and all pairs of Y' elements containing Y' elements from two different strains

4 were kept for between-strain boxplot.

5

6 Acknowledgement

7 We thank Gilles Fischer for his critical reading of the manuscript and his feedback. We thank Clotilde

8 Garrido and Oana Ilioaia for their technical help and for discussion. Research in ZX's laboratory was

9 supported by Agence Nationale de la Recherche (ANR-24-CE12-7740-01 and ANR-24-CE12-0083-03).

10

11 Author contributions

12 LD: Methodology, Investigation, Conceptualization, Formal Analysis, Writing – Original Draft, Writing –

13 Review & Editing. JSB: Methodology, Investigation, Conceptualization, Formal Analysis, Writing – Original

14 Draft, Writing – Review & Editing. ZX: Conceptualization, Supervision, Writing – Original Draft, Writing –

15 Review & Editing.

16

17 Conflict of interest

18 The authors declare that they have no conflict of interest.

19
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