Paradoxical delay of senescence upon depletion of BRCA2 in telomerase‐deficient worms

BRCA2 is a multifunctional tumor suppressor involved in homologous recombination (HR), mitotic checkpoint regulation, and telomere homeostasis. Absence of Brca2 in mice results in progressive shortening of telomeres and senescence, yet cells are prone to neoplastic transformation with elongated telomeres, suggesting that BRCA2 has positive and negative effects on telomere length regulation along the path to tumorigenesis. Using Caenorhabditis elegans as a model, we show here that depletion of BRC‐2, an ortholog of BRCA2, paradoxically delays senescence in telomerase‐deficient mutant worms. Telomerase‐deficient worms (trt‐1) exhibit early replication senescence due to short telomeres. It should be noted that worms mutated in brc‐2 are not viable as well due to massive genotoxic insults. However, when BRC‐2 is depleted by RNA interference in trt‐1 mutant worms, the number of generations is unexpectedly increased with telomere length maintained, compared to telomerase mutants. Interestingly, depletion of other HR genes such as rad‐51 and rad‐54 exhibited similar effects. In worms doubly deficient of telomerase and brc‐2, rad‐51, or rad‐54, extra telomeric C‐circles were generated, suggesting that abrogation of HR induces an alteration in telomere environment favorable to illegitimate telomere maintenance when telomerase is absent. Collectively, absence of BRC‐2 in telomerase‐deficient background first leads to telomere shortening, followed by an induction of an as‐yet‐unknown telomere maintenance pathway, resulting in delay of senescence. The results have implications in the understanding of dysfunctional BRCA2‐associated tumorigenesis.

BRCA2 is a multifunctional tumor suppressor involved in homologous recombination (HR), mitotic checkpoint regulation, and telomere homeostasis. Absence of Brca2 in mice results in progressive shortening of telomeres and senescence, yet cells are prone to neoplastic transformation with elongated telomeres, suggesting that BRCA2 has positive and negative effects on telomere length regulation along the path to tumorigenesis. Using Caenorhabditis elegans as a model, we show here that depletion of BRC-2, an ortholog of BRCA2, paradoxically delays senescence in telomerase-deficient mutant worms. Telomerase-deficient worms (trt-1) exhibit early replication senescence due to short telomeres. It should be noted that worms mutated in brc-2 are not viable as well due to massive genotoxic insults. However, when BRC-2 is depleted by RNA interference in trt-1 mutant worms, the number of generations is unexpectedly increased with telomere length maintained, compared to telomerase mutants. Interestingly, depletion of other HR genes such as rad-51 and rad-54 exhibited similar effects. In worms doubly deficient of telomerase and brc-2, rad-51, or rad-54, extra telomeric C-circles were generated, suggesting that abrogation of HR induces an alteration in telomere environment favorable to illegitimate telomere maintenance when telomerase is absent. Collectively, absence of BRC-2 in telomerase-deficient background first leads to telomere shortening, followed by an induction of an as-yet-unknown telomere maintenance pathway, resulting in delay of senescence. The results have implications in the understanding of dysfunctional BRCA2-associated tumorigenesis.
The linearity of eukaryotic chromosomes poses many problems, including replication and the protection of chromosome ends against nucleolytic attacks. During neoplastic transformation, telomerase is reactivated to overcome the end replication problem. This allows cancer cells to replicate their DNA indefinitely avoiding telomere shortening. About 10% of cancer cells, however, do not possess telomerase activity. Instead, they utilize DNA recombination to elongate telomeres and are thus classified as ALT cancers [1]. The precise mechanism of how ALT-type tumorigenesis is induced, and how it is suppressed in normal cells remains unresolved.
BRCA2 is a multifunctional tumor suppressor with crucial activities in maintaining genome integrity throughout the cell cycle [2]. In S/G2 phase, BRCA2 is critically required for double-strand break (DSB) repair, homologous recombination (HR), by regulating Rad51 filament formation [3,4]. In HR, BRCA2 is responsible for proper loading of Rad51 to the damaged sites [5]. Subsequently, Rad51 filament invades into the undamaged homologous strand, followed by D-loop formation and DNA synthesis in an error-free manner.
BRCA2 is also involved in telomere replication homeostasis as it loads Rad51 for efficient telomere replication and telomere capping [6]. In addition, we previously showed that BRCA2 inhibits MRE11mediated resection of stalled replication forks at the lagging strand telomere synthesis [7]. Therefore, loss of BRCA2 results in progressive shortening of telomeres and growth arrest [6,7]. As the shortening of telomeres triggered by BRCA2 deficiency is independent from telomerase activity and is incompatible with the fact that dysfunctional BRCA2 leads to tumorigenesis, we asked whether BRCA2 depletion has any possibility of inducing ALT.
Here, we show that abrogation of HR, particularly BRC-2, in telomerase-null (trt-1) worms overcomes the early cessation provoked by telomerase deficiency, and this phenomenon is associated with telomere length maintenance. Chromosome fusions were observed, suggesting that telomere uncapping occurred prior to the induction of illegitimate telomere length maintaining mechanism. Furthermore, extra telomeric C-circles were generated upon depletion of BRC-2 in trt-1 mutants, suggesting that depletion of BRC-2 can lead to an induction of one form of alternative telomere maintenance. However, BRC-2 depletion in trt-1 mutant worms only partially overcame senescence of trt-1 mutants, suggesting that additional mutation may be required for full activation of ALT induction. Taken together, our study suggests that absence of BRC-2 first results in telomere shortening then induce an illegitimate telomere maintenance mechanism. Our study implies that absence of BRCA2 embraces the possibility to instigate an unidentified ALT pathway in tumorigenesis.

Materials and methods
Caenorhabditis elegans and feeding RNAi assay trt-1 (ok410) worms were grown on RNAi-expressing lawns of HT115 (DE3) bacteria provided by Dr. Julie Ahringer (Gurdon Institute, Cambridge, UK). Twenty worms at the L3 or L4 stage were passaged every week. Each passage corresponds to two generations. More than 12 sets in each group were tested.

Terminal restriction fragment analysis
Genomic DNA was digested with MboI/AluI (NEB) and separated on a 0.7% agarose gel at 5 VÁcm À1 at 4°C and subjected to Southern blotting. After transfer, the DNA was fixed on the membrane by UV cross-linking, prehybridized with DIG Easy Hyb (Roche, Indianapolis, IN, USA) at 62°C for 1 h, and then hybridized with telomere-specific DIG-labeled probes at 62°C for 3 h. The blot was washed and the detection was done by exposure to Las-3000 Imaging System (Fuji Film, Tokyo, Japan) using a DIG Luminescent Detection Kit (Roche).

Telomere florescent in situ hybridization (FISH)
Gonads from 24 h post-L4 adults were dissected and fixed in 2% paraformaldehyde. Slides were frozen in liquid nitrogen, then immersed in methanol for 10 min at À20°C, followed by rehydration in 29 SSC containing 0.1% Tween-20. PNA-(GCCTAA) 3 probe was applied in hybridization buffer (29 SSC and 50% formamide). The slides were washed three times with 50% formamide/29 SSC, then counterstained with DAPI and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting media. Images were acquired with DeltaVision (Applied Precision, Issaquah, WA, USA), equipped with a 1009 objective lens (Olympus, Tokyo, Japan).
Amplication was conducted using the following oligonucleotide sequences.

C-circle assay
We basically followed the protocol described by Henson et al. [9]. CC assay was performed with increasing amounts of genomic DNA with same amount of Φ29 DNA polymerase in the reaction. Negative control without Φ29 DNA polymerase was always included. N2 WT control was also employed.

Results and Discussion
Caenorhabditis elegans possesses BRC-2 (CeBRC-2), the ortholog of BRCA2, which is only one-tenth the size of human BRCA2. BRC-2 in worms is also essential in DNA repair through regulating RAD-51 filament formation in HR, like its ortholog BRCA2 [10].
To assess the physiological outcome of BRCA2 deficiency in telomerase-deficient situation, we utilized the telomerase-mutant worm, trt-1 and its properties [12]. Without telomerase, worms progressively cease to lay eggs around generation 20, accompanied by progressive shortening of telomeres. We hypothesized that by assessing the continuation of generations, one could predict if BRC-2 depletion synergizes or suppresses further shortening of telomeres. If BRC-2 depletion synergistically shortens the telomere length, the generation number will be fewer than 20. On the other hand, if depletion of BRC-2 extends the generations, one could interpret that BRC-2 may be a suppressor of ALT (alternative lengthening of telomeres). As the generation duration of worms is markedly shorter compared to mammals, this assay is feasible and robust.
When BRC-2 was depleted from trt-1 mutants via feeding RNAi method, the generation number was extended to~35 (Fig. 1A and Table 1, trt-1;brc-2), but not beyond, while trt-1 mutant worms ceased to lay eggs around generation 21 to 23 (trt-1; L4440). The brood size of the worms declined in the initial stage after brc-2 depletion (first decline around generation 18), then the generation continued with 67% survival until complete cessation of laying eggs (Table 1). It should be emphasized again that worms with null mutation of brc-2 alone are not viable and do not lay eggs because BRC-2 is critical in meiotic recombination and repair [10]. It is interesting that depletion of BRC-2 in trt-1 mutants paradoxically rescues the generations.
Depletion of rad-51 or rad-54, factors that are essential in HR pathway and in telomere length maintenance [13], was also tested. The levels of knockdown, measured by quantitative RT-PCR, were similar (Fig. 1B). The result showed that depletion of HR genes also extended the generations (Fig. 1A and Table 1), suggesting that abrogation of HR in the absence of telomerase may have induced an  alternative repair pathway. In C. elegans, BRC-2 substitutes Rad52 that is involved in single-strand annealing (SSA) pathway [11]. Therefore, the results collectively suggest that depletion of BRC-2 in telomerase-deficient worms instigated a repair pathway that excludes HR and Rad52-mediated recombination as well. As depletion of BRC-2 results in progressive shortening of lagging strand telomere synthesis in mammals [7], the paradoxical delay of senescence upon BRC-2 depletion suggests that dysfunctional BRC-2 combined with telomerase deficiency leads to a change in telomere environment favorable for illegitimate induction of telomere length maintenance mechanism. Pot-1 is essential in telomere capping: it binds to single-stranded telomeric DNA and protects telomeres from recombination [14]. CeOB2/Pot-1 depletion in C. elegans results in increased heterogeneity in telomere length [15] and abundant single-stranded C-rich telomere circles [16], a phenotype similar to that seen in ALT [17]. Depletion of POT-1 in trt-1 mutants also extended the generations (Fig. 1A and Table 1), but not completely overcame senescence. This result implies that before the induction of any kind of ALT, telomere uncapping precedes.
To assess whether the delay of senescence was related to the telomere length, we measured the telomere lengths by telomere-FISH (T-FISH) in germ cells of various generations. The result was in agreement with the survival studies that there were no telomerepositive germ cells from trt-1 after F18 (Fig. 1C). In comparison, brc-2 or rad-51 RNAi in trt-1 mutants exhibited telomere-positive nuclei beyond F18: up to F38 in brc-2 RNAi worms and F32 in rad-51 RNAifed trt-1 worms, respectively (Fig. 1C). Interestingly, in F32 of trt-1; brc-2 RNAi worms, 65.9% of germ cells (n = 41) exhibited larger and brighter T-FISH signals, even compared to F14 of trt-1 or trt-1; brc-2 RNAi worms (Fig. 1C, F32). These bigger T-FISH spots were not observed in F14. These results suggest that some telomeres were elongated after critical shortening (Fig. 1C, bottom). In the same cell, there were chromosomes with very short telomeres, displaying faint T-FISH signals as well.
Next, we measured telomere lengths by Southern blot-based terminal restriction fragment (TRF) analysis. In F23, the telomere lengths of trt-1; brc-2 RNAi worms were more heterogeneous and some were even longer ( Fig. 2A, brc-2 RNAi, marked with an asterisk), compared to the telomerase-mutant worms (trt-1; L4440). In F33, telomere lengths of brc-2or rad-51depleted trt-1 mutants were longer, compared to F16 of trt-1 control RNAi worms (Fig. 2B). These data suggested that the telomeres were first shortened, consistent with the result from mouse [7], then were elongated, or at least protected from further shortening, in later generations. Telomere length measurement was further assessed by PCR-based STELA [8]; the average telomere lengths in trt-1 worms depleted of brc-2 or rad51 were markedly longer, compared to control trt-1 worms (Fig. 2C).
Then we asked whether there was any chromosome alteration in F19, just before the cessation of trt-1 mutant worms. As chromosome structures are most visible in germ cells in diakinesis [18], chromosomes in this stage were compared (Fig. 3A). Wild-type N2 displayed six chromosomes (Fig. 3B, gray bar), whereas trt-1 mutants displayed mostly three chromosomes (Fig. 3B, empty bar), suggestive of the end-end fusions provoked by telomere shortening and erosion [19]. In the same generation (F19), depletion of brc-2 or rad-51 in trt-1 mutants also displayed chromosome fusions (Fig. 3B, dashed and black bars), indicating that telomere erosions occurred prior to an induction of alternative telomere repair pathway responsible for the delay of senescence.
As senescence was delayed and telomere shortening was prevented in worms depleted of brc-2 in trt-1 mutants, we asked whether there was any mark for ALT. Partially single-stranded telomeric C-circle (CCCTAA)(n) DNA are the hallmark of ALT in many Telomerase-deficient C. elegans (trt-1) were depleted of the indicated RNA by feeding RNAi method. Surviving worms were counted and replated every week. Initial numbers of worms plated and the surviving worms in indicated generations are shown. Numbers in the bracket refer to the percentage of survival from initial plating.
human cancers [9] and had been detected in worms as well [16]. When F17 trt-1 mutant worms were subjected to the C-Circle (CC) assay, approximately fourfold increase of C-circle abundance was detected in trt-1 mutants depleted of the three HR genes (Fig. 3C,D). The result suggests an interesting possibility that an as-yet-unidentified ALT can be induced by the depletion of HR in telomerase-deficient cells. As trt-1; brc-2 RNAi worms did not survive beyond F38, a secondary mutation may be required for the full activation of ALT pathway after BRC-2 depletion. These serial mutations may reflect a path to ALT-type tumorigenesis. Interestingly, telomere sister chromatid exchange (T-SCE), an evidence for telomeric recombination, was increased in BRCA2-mutated human cancers [20]. How extra telomeric C-circles are generated after depletion of HR warrants future investigations. A report from a small number of cell lines showed that depletion of BRCA2 results in two different outcomes: in non-ALT cells, depletion of BRCA2 leads to the increase of telomere recombination, whereas BRCA2 depletion in ALT cells resulted in the reduction of T-SCE [21]. However, as one ALT cell line used in the analysis, U2OS, cannot represent all ALT pathways, the information cannot be generalized. Here, we are suggesting the path and possibilities of BRC-2 depletion in inducing illegitimate telomere maintenance pathway, whereas the study from U2OS cell is the assay done at the established cell line. Moreover, U2OS is BRCA2-positive and elongates telomeres via Rad51-dependent recombination [22]. Depleting BRCA2 in U2OS cells is likely to lead to low viability, because its survival depends on Rad51mediated telomeric recombination that critically requires BRCA2. Therefore, the report is not in discrepancy with our results. Here, we have showed with Partial overcome of senescence after the depletion of BRC-2 in trt-1 mutant worms is accompanied by telomere length elongation. (A) TRF analysis of genomic DNA extracted from indicated worms at generation 23 (F23). Capillary transferred digested DNA were hybridized with digoxigenin (DIG)-labeled telomeric probes and detected with anti-DIG (right). GCCTAA probe was hybridized. Ethidium bromide-stained DNA gel before transfer is shown at left and the molecular weight of DNA is marked. Telomeres longer than WT N2 in trt-1; brc-2 RNAi is marked with an asterisk. Note that the long smear is not due to degradation but reflects the heterogeneity in telomere length. (B) TRF analysis of brc-2 or rad-51 RNAi in trt-1 mutants in F33, compared to control RNAi in F16. (C) STELA analysis [8] for telomere length measurement. Amplified DNA was separated and detected with an end-labeled 32 P-(GCCTAA) 4  compelling lines of evidences that HR deficiency in telomerase-deficient situation can overcome the senescence. The results shown here suggest that there are many different paths to ALT induction, and that HR deficiency can instigate a novel ALT pathway. As BRCA2 is the tumor suppressor, frequently mutated in human cancers, these results have clinical implications.
To assess the effect of telomere length on senescence, we measured the continuation of generations in worms. In fact, this assay measures the continuance of meiotic recombination, which is distinct from mitosis. Worms depleted of BRC-2 resulted in a delay of replicative senescence in trt-1 mutants, measured by the ability to lay eggs. However, they eventually ceased egg laying (Fig. 1). This result is somewhat different from reported ALT induction where more than 200 generations can be continued in the absence of telomerase [18]. BRC-2 deficiency leads to accumulation of massive DNA adducts on top of telomere erosions, and therefore the failure to propagate beyond F35 may be due to apoptosis. It should be reemphasized that worms deficient in brc-2 are not viable [10]. Hence, adopting additional mutation in genes such as p53 may fully induce ALT. Indeed, p53 inactivation frequently accompanies neoplastic transformation of Brca2-deficient mice [23], consistent with the notion that some familial breast cancers with BRCA2 mutation in Icelandic population harbor mutation of p53 as well [24]. In summary, BRCA2 suppresses the induction of illegitimate telomere elongation pathway. We do not think that all BRCA2deficient cancers exhibit ALT characteristics. Instead, BRCA2-deficient cells will have very different outcomes depending on whether telomerase is on or off: when telomerase is on, BRCA2-deficiency will not induce a telomere elongation repair pathway. Taken together, assessing telomere status in BRCA2-deficient tumors may be helpful in the choice of adequate therapy.