Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

Abstract

During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

In response to various assaults to DNA, cells have evolved a plethora of DNA damage repair proteins to sense and repair lesions on DNA, particularly DNA double-strand break (DSB). The most faithful DNA repair mechanism is homologous recombination (HR), which uses an intact copy from the sister chromatid as a template for repair. Interestingly, despite the hazardous nature of DSBs, programmed DNA DSBs are generated in vivo and are repaired to facilitate certain developmental processes. During germ cell development, programmed DSBs are generated in meiotic prophase and repaired through homologous recombination. Different from the repair in somatic cells, homologous recombination in meiotic prophase prefers homologous chromosomes over sister chromatids as templates for DSB repair. This unique feature allows the exchange of parental genetic information, an important source of genetic diversity for the offspring, via crossovers between homologous chromosomes. Moreover, crossovers are required for proper chromosome segregation during meiotic metaphase I. Therefore, DSB generation and repair through homologous recombination are critical for germ cell development. Studies in the past decade have greatly advanced our understanding of how DSBs are generated and repaired during meiotic prophase and have shed light on the unique regulation of homologous recombination at this stage, which has been nicely reviewed.^1,2 In this review, we will briefly summarize current understanding of DSB repair in meiotic prophase and focus on the unique challenges to male sex chromosomes during this process.

Crossovers are Generated During Meiotic Prophase

Although faithful passage of genetic information from parents to offspring maintains genetic stability, genetic recombination is important for the course of evolution in the whole population. Genetic recombination occurs in meiotic prophase, during which exchange of genetic information between homologous chromosomes leads to the inheritance of mixed genetic information from both parents by haploid gametes (sperm and oocytes) produced through meiotic reductive divisions. Meiotic recombination is generated by a series of highly orchestrated processes including production of programmed DSBs and generation of crossovers through DSB repair. Crossovers are formed at least once between each pair of homologous chromosomes, which is known as the obligatory chiasma. Crossovers directly lead to the exchange of genetic information between homologous chromosomes. In addition, crossovers connect homologous chromosomes and facilitate their proper orientation at the meiotic spindle during the first metaphase. Although most mammalian male sex chromosomes (X and Y) share little homology, crossovers are generated between their subtelomere pseudoautosomal regions (discussed in detail below). Failure to generate crossovers generally arrests meiosis before chromosome segregation and subsequently induces apoptosis. However, there are some exception for some mammalian species, whose male sex chromosomes never recombine.^3,4

Crossovers are Generated by Homologous Recombination Repair of DNA Double-Strand Breaks

The first step of meiotic recombination is production of a massive number of DSBs throughout the chromosomes during early meiotic prophase by SPO11, a topoisomerase II-like endonuclease. It is during the DSB repair that crossovers are formed between homologous chromosomes (Fig. 1a). Thus, the SPO11-dependent DSB is a prerequisite for generating crossover. Consistently, crossovers could not be observed in germ cells from Spo11 knockout mice, which are infertile due to the arrest of germ cells before meiotic division.^5,6 To ensure genetic integrity during reproduction, these SPO11-dependent DSBs have to be repaired in a timely and accurate manner. Extensive studies in somatic cells have revealed that non-homologous end joining (NHEJ) and homologous recombination (HR) are 2 common pathways for repairing DSBs. In meiotic cells, the error-prone NHEJ is suppressed, which is probably due to loss of expression of Ku70, the key protein for NHEJ.^7,8 The error-free HR is used for repair of SPO11-induced DSBs. HR uses an intact copy of DNA as the template for the high fidelity repair, which avoids generation of genetic mutations during DSB repair in meiotic cells. In somatic cells, HR usually uses sister chromatids as templates for repair, whereas sister chromatids are not favored in meiotic cells. Instead, homologous chromosomes are preferable templates during HR in meiotic cells. Although the detailed mechanism of this inter-homolog bias in mammalian cells remains elusive, chromosome synapsis mediated by synaptonemal complex might play an active role. Synaptonemal complex is a multi-protein structure that contains axial/lateral elements and central elements.⁹ During chromosome synapsis, axial elements are first developed along chromosome axis of sister chromatids. Central elements then connect axial elements between homologous chromosomes and promote their synapsis. When homologous chromosomes are fully synapsed, axial elements are termed lateral elements. It is possible that synapsis shortens the distance between homologous chromosomes and allows them to be used as templates for DNA repair. It has also been suggested that components of the axial elements of synaptonemal complex including SYCP2 and SYCP3 might regulate the activities of recombination proteins to favor inter-homolog recombination.¹⁰ Recent studies suggest that HORMA domain-containing (HORMAD) proteins regulate the interaction between homologous chromosomes,^11-15 which might also facilitate the usage of homologous chromosomes as templates for DNA repair and contribute to the inter-homolog bias in mammals.

Figure 1.

DNA double-strand break repair is prolonged in male sex chromosomes. (A) DNA double-strand breaks are generated in both autosomes and male sex chromosomes at leptotene. Synaptonemal complexes are assembled during the synapsis of homologous chromosomes. From zygotene to mid-pachytene, DNA breaks are repaired in autosomes and crossovers are formed. DNA repair on male sex chromosomes is not completed until late diplotene. (B) Synaptonemal complexes are assembled along autosomes, but only at the subtelomere pseudoautosomal regions of male sex chromosomes. Crossovers are not shown here for simplicity.

HR repair initiates by CTIP, MRE11 and EXO1-mediated bidirectional resection that generates a long stretch of single-stranded DNA (ssDNA) with free 3’ ends.^16,17 After DNA end resection, ssDNA is quickly coated with ssDNA-binding protein RPA, which is subsequently replaced by RAD51 and/or its meiosis-specific homolog DMC1 that promote ssDNA invasion into the homologous chromosomes.¹⁸ Depending on subsequent choices of pathways, the HR can proceed through synthesis-dependent strand annealing (SDSA) that generates gene conversion products without crossovers between homologous chromosomes, or through a process that produce double-Holliday junctions that are either dissolved to generate non-crossover or resolved in 2 different ways to give rise to crossover or non-crossover, or through a process that produce single-Holliday junctions that are resolved to generate crossovers.¹⁸ Interestingly, although SPO11 induces DSBs throughout all chromosomes during meiotic prophase, only one or 2 crossovers are generated between each chromosome pair in mice. This phenomenon suggests that the choices of HR pathways and the resolution of Holliday junctions are tightly regulated to control crossover.¹⁹ Recently studies in C. elegans have shown that the synaptonemal complex is important for regulating the number of crossovers per chromosome,²⁰ but its implication in mice or other species remains to be established.

Crossovers are Generated Between Pseudoautosomal Regions of Male Sex Chromosomes

Crossover occurs during both oogenesis in females and spermatogenesis in males. But a unique challenge exists for males as male sex chromosomes (X and Y) share little homology except for short subtelomere pseudoautosomal regions (PAR). Due to the requirement for significant homology between chromosomes, synapsis and crossover in male sex chromosomes are restricted to PAR²¹ (Fig. 1A and B). Importantly, one crossover is always generated within PAR, suggesting that a yet unknown mechanism ensures an obligate crossover within these regions. Although PAR are short, they have relatively longer chromosome axes than autosomes, suggesting that DNA in PAR region is packaged into many short chromosome loops,²² which might increase the chance of DSB formation within this region. It is also reported that X and Y pair late, and an isoform of SPO11 (SPO11α) specifically induces late DSBs in PAR to promote crossover in this region.²² Interestingly, DSB numbers increases on unsynapsed X chromosomes in late prophase and X chromosome is prone to pair with non-homologous partners upon DSB reduction, indicating the presence of a feedback control of DSB and homolog interaction in mice,²³ which is also observed in other organisms.^24-26

Asynapsis Triggers Prolonged DSB Repair in Male Sex Chromosomes

Although the majority of male sex chromosomes remain unsynapsed with no additional crossover generated, SPO11-dependent DSBs still occur in these regions. Due to the absence of homology, DNA breaks in these unsynapsed regions of male sex chromosomes cannot be repaired in the same way as those that are repaired on autosomes. Moreover, the kinetics of DSB repair in male sex chromosomes is different from that in autosomes (Fig. 1A). SPO11 induces DSBs in all chromosomes during leptotene, which triggers a global DNA damage response. H2AX phosphorylation at Ser 139 (aka γH2AX), a surrogate marker for DSBs,²⁷ is observed in both autosomes and sex chromosomes starting from leptotene.²⁸ When meiotic cells proceed into late pachytene, most DNA breaks on autosomes have been repaired and crossovers have been generated. Following the completion of DSB repair, γH2AX signals largely disappear from autosomes. However, DNA repair is not finished in sex chromosomes and γH2AX signals remain in sex chromosomes²⁸ along with many other DNA damage response proteins.²⁹ At late pachytene, male sex chromosomes are isolated in a subnuclear structure known as the XY body. DNA damage response proteins remain in the XY body till late diplotene, suggesting that DSB repair is significantly delayed in male sex chromosomes.

The major difference between male sex chromosomes and autosomes during meiotic prophase is that male sex chromosomes are largely unsynapsed, which might be the cause of prolonged DNA damage response on them. Supporting this idea, prolonged DNA damage response is not found on female sex chromosomes that are homologous and fully synapsed during oogenesis. The kinetics of DSB repair in 2 X chromosome is similar to that in autosomes, and crossovers are generated over the entire female sex chromosomes. Moreover, in male mice with an additional Y chromosome (XYY), the 2 Y chromosomes are fully synapsed. DSBs repair in Y chromosomes occurs at a similar rate as that in autosomes. However, the X chromosome is covered by γH2AX,³⁰ indicating the prolonged DSB repair in the unsynapsed X chromosome. In XO females with only one X chromosome, γH2AX again accumulates on the unsynapsed X chromosome if it is not self-synapsed at pachytene during oogenesis,^30,31 suggesting that asynapsis-induced prolonged DNA damage response in sex chromosomes can happen in both sexes. Chromosome asynapsis can also occur in a pair of autosomes that carry 2 similar but non-identical chromosomal translocations. During late pachytene, γH2AX is enriched in these unsynapsed regions, again indicating the prolonged DNA damage response,^30,31 In the Down syndrome mouse models that carry an additional chromosome or contain extended chromosome regions comprising most of human chromosome 21, the extra chromosome area is unsynapsed and is coated by γH2AX at late pachytene.³² Moreover, in mice with disrupted synaptonemal complexes, all chromosomes have defective synapsis and are coated with γH2AX at pachytene.³³ Collectively, these observations suggest that asynapsis is the cause of the prolonged DNA damage response in male sex chromosomes as well as other abnormal chromosomes.

DSB Repair in Male Sex Chromosomes

Since most regions of male sex chromosomes are unsynapsed, it is likely that an alternative repair mechanism occurs, which causes the prolonged DSB repair. The recruitment of HR machineries such as the key recombinase RAD51 to male sex chromosomes suggests that the repair is still through HR.³⁴ During leptotene and zygotene, RAD51 coats both autosomes and male sex chromosomes, suggesting that HR repair starts at the same time at DSBs in both autosomes and male sex chromosomes. When meiotic cells progress to pachytene, RAD51 is released from autosomes, suggesting the completion of HR repair in autosomes. However, RAD51 is still retained in male sex chromosomes during pachytene, indicating that the HR machinery is still in the process of either homology search or stand invasion. When cells progress to diplotene, RAD51 starts to disappear from male sex chromosomes, suggesting that the DNA breaks are repaired. During the DSB repair in autosomes, homologous chromosomes are used as the template for HR, and the usage of sister chromatids has been restrained. However, for repair in male sex chromosomes, only sister chromatids are available as templates for HR. Thus, it is likely that failure to find homologous templates by the HR machinery during leptotene and zygotene leads to prolonged DSB repair in X and Y chromosomes. Although it is not clear how the breaks in male sex chromosomes are finally repaired during pachytene and diplotene, it is very likely that the restrain on sister chromatids has been finally lifted and HR is eventually completed using sister chromatids as templates. Consistent with the elusive usage of sister chromatids as templates for DSB repair in male sex chromosomes, sister chromatid exchange predominantly occurs on sex chromosomes in male meiotic cells.³⁵ Besides HR, NHEJ is another DSB repair mechanism for somatic cells. However, NHEJ easily induces genetic alterations that may affect genetic integrity for the next generation. Thus, it is unlikely NHEJ plays a major role in repairing DSBs in male meiotic sex chromosomes.

Difference Between DNA Damage Response Proteins Localized at Chromosome Axis and Loops

As a result of prolonged DSB repair in male sex chromosomes, many DNA damage response proteins accumulate in the XY body during pachytene.²⁹ Since pachytene lasts for several days during mouse postnatal development,³⁶ meiotic cells in this stage could be easily identified, which allows to study the prolonged DNA damage response in the XY body. Interestingly, DNA damage response proteins localize at 2 different regions inside the XY body.²⁹ While some proteins cover the entire XY body, others exclusively localize at chromosome axes associated with axial elements of synaptonemal complex, such as SYCP3 (Fig. 2). The best examples are 2 key DNA damage response proteins: RAD51 co-localizes with SYCP3 at chromosome axes; but γH2AX coats the entire XY body. These observations suggest that meiotic chromosomes are organized in a unique way and different DNA damage response proteins localize at different chromosomal structures. It has been proposed that meiotic chromosomes form loops that are aligned at chromosome axes, where axial element of synaptonemal complex SYCP3 is localized.³⁷ According to this model, RAD51 is restricted at chromosome axes, while γH2AX spreads to all chromosome loops. This phenomenon is reminiscent of the localizations of these 2 proteins during DSB repair in somatic cells. In response to DSBs in somatic cells, RAD51 coats ssDNA generated by 5’ to 3’ resection that extends less than a few kilobases away from the DSBs,³⁸ and promotes stand invasion during HR repair. Meanwhile, the DNA damage signaling spreads much farther away from the actual DSBs. Due to DNA break end resection and strand invasion, nucleosomal histones are evicted at the site of actual HR repair. Instead, γH2AX spreads up to a megabase away from the DSBs, which facilitates the recruitment of HR machinery to DSBs.³⁹ The restricted localization of RAD51 and the spread of γH2AX in the XY body suggest that the DNA damage response in the XY body is similar to that in somatic cells. It is likely that the DSBs localize at chromosome axes while the DNA damage response signals spread over to chromosome loops. If this is the case, how could DSBs specifically localize at chromosome axes? It has been suggest that DSBs are generated at hot spots on chromosome loops.⁴⁰ It is possible that the breaks are tethered to chromosome axes following chromatin remodeling.

Figure 2.

DNA damage response proteins localize at 2 different regions in the XY body. Male sex chromosomes form a unique chromatin structure known as the XY body at pachytene. Some DNA damage response proteins exclusively localize at chromosome axes; some cover the entire XY body; and some localize at both regions.

Among many DNA damage response proteins found in the XY body, only a few of them localize exclusively at unsynapsed axes as RAD51 does. A closer examination of these proteins shows that most of them directly participate in HR repair. For example, CTIP initiates DNA end resection at DSBs, the first step of HR;¹⁷ RPA binds the ssDNA after end resection and facilitates the exchange of RAD51 onto the ssDNA.^41,42 RAD51 is the key recombinase for HR by promoting strand invasion.¹⁸ The BRCA1-A complex, a multi-protein complex including RAP80, Abraxas/CCDC98, BRCC36, BRCC45, and MERIT40,⁴³ targets BRCA1 to DSBs and facilitates the loading of RAD51 to DSBs.^44,45 In contrast, most proteins that spread to chromosome loops do not directly participate in HR repair. Instead, these proteins are mainly involved in DNA damage-induced signal transduction. For example, γH2AX-MDC1-RNF8 is a well characterized phosphorylation-dependent DNA damage signaling pathway in response to DSBs in somatic cells^46,47 and all 3 proteins spread over chromosome loops.²⁹ Consistently, RNF8-depedent histone ubiquitination as well as downstream partners such as RAD18 and 53BP1 are also found in chromosome loops.²⁹ Other proteins implicated in DNA damage signaling also cover the entire sex chromosome, such as MCPH1⁴⁸ and sumoylated protein.⁴⁹ In addition, ATR and TopBP1 are found in both unsynapsed axes and chromosome loops.^50,51 It is likely that these 2 proteins relay the DNA damage signals generated from the DSBs at chromosome axis to adjacent chromosome loops.⁵²

Although the spread of DNA damage response in meiotic cells is similar to that in somatic cells, the response spreads much wider than that in somatic cells. DNA damage response spreads to less than a few hundred kilobases away from DSBs in somatic cells.³⁹ However, DNA damage response proteins, such as γH2AX, cover the entire sex chromosomes in pachytene. This suggests that much more chromosome areas are affected by DNA damage signaling in meiotic prophase. It is possible that the organization of meiotic chromosomes into loops and axes facilitates the coverage of DNA damage signals. It is also possible that delayed DSB repair in the sex chromosomes promotes the spread of DNA damage response proteins to the entire XY body.

Meiotic Sex Chromosome Inactivation as a DNA Damage Response

Coincident with the presence of numerous DNA damage response proteins, the transcription of the sex chromosomes but not autosomes is suppressed at mid-pachytene in the XY body, which is also known as meiotic sex chromosome inactivation (MSCI).⁵³ During this process, transcription machineries such as RNA polymerase II are excluded from the XY body. Transcription repressive marks such as di/tri-methylated lysine 9 of histone H3 and heterochromatin protein 1 (HP1β and HP1γ) accumulate in the XY body.⁵⁴ MSCI is important for the integrity of the XY body and the survival of meiotic cells. If MSCI fails, the sex chromosomes fall apart and apoptosis occurs in late-pachytene cells.^51,55 It has been suggested that MSCI is important for suppressing toxic genes Zfy1 and Zfy2, whose expression during pachytene leads to apoptosis.⁵⁶ However, these 2 genes are unlikely to trigger MSCI. MSCI is a special form of meiotic silencing of unsynapsed chromosomes (MSUC) that involve any unsynapsed chromosomes regardless if they carry these genes.⁵⁷ In meiotic cells that contain unsynapsed chromosomes other than the Y chromosome, such as those from XYY mice, XO mice, and those mice with non-identical translocations, the unsynapsed chromosomes are all silenced.^30,31 Therefore, chromosome asynapsis might induce transcriptional silencing.

How is MSCI achieved? The coincidence between prolonged DNA damage response and transcriptional silencing suggests that there is a possible link between these 2 events. In fact, similar association has been observed during DNA damage response in somatic cells. It has been shown that DSBs induce transcription silencing in chromosome regions adjacent to the actual breaks. Concomitantly, DNA damage response proteins that spread away from DNA breaks are important for transcriptional silencing of these regions.⁵⁸ Similar mechanism might function in the XY body as well. As discussed before, DNA breaks likely occur at the unsynapsed axes of sex chromosomes, and the DNA damage response spreads beyond the actual DNA breaks to chromosome loops. It is likely that the extended DNA damage response on the chromosome loops triggers MSCI in the XY body, which prevents generation of truncated transcripts at DNA breaks. This DNA damage-induced transcriptional silencing should underlie the mechanism for MSUC as well.

It is noteworthy that in SPO11-deficient spermatocytes or those with catalytic-inactive SPO11, no SPO11-dependent DNA breaks are generated. Meiosis is arrested and chromosome synapsis is severely affected.^5,6,59,60 Interestingly, pseudo XY bodies are formed. They are covered by DNA damage response proteins, where SPO11-independent DNA breaks are believed to occur,⁵⁹ and are transcriptionally silenced.^59,61,62 Pseudo XY bodies and DNA damage response proteins only cover part of the unsynapsed chromosomes. For unsynapsed chromosome regions that are not covered by DNA damage response proteins, transcription is not repressed. Although it is still not clear how pseudo XY body are formed, and whether or not DNA breaks are present in them, unsynapsed chromosome alone is clearly insufficient for triggering MSUC. These observations suggest that DNA damage response is the bona fide cause of initiation of MSUC, including MSCI in the XY body.

DNA Damage Response Pathways in the XY Body

Accumulating evidence suggests that DNA damage response in the XY body is similar to that in somatic cells. The DNA damage response pathways in somatic cells have been extensively characterized (Fig 3A).⁶³ Following DSBs, histone H2AX adjacent to DSBs is quickly phosphorylated by a group of PI3-like kinases including ATM, ATR, and DNAPK. The phosphorylated H2AX recruits MDC1 and subsequently RNF8, an E3 ubiquitin ligase. RNF8, together with its downstream ubiquitin ligase RNF168, ubiquitinates histone H2AX/H2A, and initiates a cascade of ubiquitin-dependent signaling pathways that recruits a numbers of proteins to DNA damage sites such as RAD18, 53BP1, and BRCA1 for DSB repair. Meanwhile, the resection of DNA break ends occurs to generate a long stretch of ssDNA that is coated with ssDNA binding protein RPA. In the presence of other DNA damage response proteins, RAD51 replaces RPA on ssDNA and promotes strand invasion and HR repair. Moreover, ATR and its activator TopBP1 participate in a unique pathway that is important for the HR repair during replication stress.⁶⁴

Figure 3.

Different regulation of DSB repair pathways in somatic cells and XY body. (A) In response to double-strand breaks after ionizing radiation in somatic cells, histone H2AX is phosphorylated redundantly by ATM, ATR, and DNAPK and recruits MDC1 and RNF8. RNF8 and its partner RNF168 promote protein ubiquitination on histone H2AX, H2A, and other substrates. The ubiquitin signals recruits a numbers of proteins to DSB sites including RAD18, 53BP1, and BRCA1-A complex, which facilitates the loading of RAD51 to replace RPA on single-stranded DNA. RAD51 then promotes strand invasion and HR repair. ATR and its activator TopBP1 participate in a unique pathway that is important for the HR repair during replication stress. Proteins not discussed in this review are omitted for simplicity. (B) In the XY body, H2AX is mainly phosphorylated by ATR. H2AX-MDC1-RNF8-dependent protein ubiquitination regulates the recruitment of RAD18 and 53BP1 to chromosome loops, but not the recruitment of BRCA1-A complex and RAD51 at chromosome axes.

Recent analyses of gene knockout mice have provided important information on the regulation of DNA damage response in the XY body (Fig 3B). As mentioned before, DNA damage response proteins localize at 2 different regions in the XY body: DSB repair proteins mainly localize at unsynapsed axes while DNA damage signaling proteins spread over to chromosome loops.²⁹ Previous studies have shown that the H2AX-MDC1-RNF8 pathway is important for stabilizing DNA repair proteins, such as BRCA1-A complex and RAD51, at DSBs in somatic cells. Interestingly, this pathway is dispensable for the recruitment of DSB repair proteins to the unsynapsed axes of sex chromosomes in the XY body. In mice deficient for H2AX, MDC1, or RNF8, most DSB repair proteins still localize at unsynapsed axes.²⁹ This suggests that the localization of DSB repair proteins is uniquely regulated in the XY body. The regulation is poorly understood, in part because of embryonic lethality of mice lacking DNA repair proteins, such as RPA1,⁶⁵ TopBP1,⁶⁶ BRCA1,^67-69 CTIP,⁷⁰ or RAD51.^71,72 Studies using conditional ATR knockout mice have shown that ATR is the master regulator of the localization of DNA repair proteins at the unsynapsed axes.⁵² Previous studies have suggested that HORMAD proteins are also important for proper localization of DNA repair proteins at unsynapsed axes.^11-15 Although the localization of HORMAD proteins is unaltered, the phosphorylation of HORMAD proteins is suppressed when ATR is depleted.⁵² This observation suggests that some DNA repair proteins might be recruited to unsynapsed axes through recognition of HORMAD phosphorylation. Interestingly, it has been shown that BRCA1 also regulates ATR's localization at unsynapsed axes.⁷³ Therefore, there are possible intercalated regulations among these DNA repair proteins at unsynapsed axes.

In addition, ATR relays the DNA damage-induced signals from unsynapsed axes to chromatin loops through phosphorylation of H2AX and MDC1. ATR is required for spreading of γH2AX and MDC1 to chromosome loops as well as for initiating MSCI.⁵² H2AX and MDC1 are important for the integrity of the XY body. In mice deficient for H2AX or MDC1, male meiotic cells arrest in mid-pachytene. MSCI is disrupted and intact XY body is not observed.^51,55 DNA damage response proteins fail to spread to chromatin loops in these cells, suggesting that these 2 proteins relay the signals from ATR and establish the DNA damage response on chromosome loops.

RNF8 is a protein immediately downstream of H2AX-MDC1 signaling pathways in somatic cells.^46,47 Surprisingly, RNF8 is dispensable for meiotic progression since haploid round spermatids are readily observed.⁷⁴ XY body is intact in Rnf8 knockout meiotic cells and MSCI is maintained. The intact XY body in RNF8 knockout meiotic cells allows study of DNA damage signaling on chromosome loops in these cells. Interestingly, RNF8-dependent histone ubiquitination, together with downstream recruitment of 53BP1 and RAD18, is absent in the XY body.²⁹ This suggests that histone ubiquitination pathways are dispensable for DNA damage signaling and MSCI in the XY body. Consistently, germ cells from mice deficient for 53BP1 or RAD18, the downstream partners of RNF8, progress through meiosis normally.^75-77 If not RNF8-dependent protein ubiquitination, what downstream proteins of ATR-γH2AX-MDC1 pathways are important for DNA damage signaling on chromosome loops? Unlike in somatic cells,⁷⁸ protein sumoylation in the XY body does not require RNF8.²⁹ Although it is reported that protein sumoylation precedes γH2AX in the XY body,⁷⁹ studies have also shown that protein sumoylation in the XY body requires MDC1.⁵¹ Therefore protein sumoylation might be a candidate mechanism responsible for DNA damage signaling and establishment of MSCI in the XY body.⁸⁰

It seems that ATR is the key kinase required for recruitment of proteins to unsynapsed axes and spread of DNA damage response to chromosome loops, and DNAPK or ATM are not redundant with ATR during this process. Although activated DNAPK, the key protein for NHEJ, may be present on unsynapsed axes,²⁹ it is obvious that it is not required for progression through meiotic prophase since Dnapk knockout mice are fully fertile.^81,82 ATM is another key kinase during DSB repair in somatic cells and activated ATM is present on unsynapsed axes.²⁹ ATM is essential for global H2AX phosphorylation during leptotene.⁶¹ It also controls the number of DNA breaks generated by SPO11 and regulates the crossover between homologous chromosomes.^83,84 Meiotic cells without ATM are arrested in mid-pachytene before the XY body is formed.^85,86 Interestingly, Spo11 heterozygosity rescues the prophase arrest of Atm knockout meiotic cells.^61,84 Although X and Y synapsis through PAR are defective in these cells, the XY body forms normally. DNA damage response proteins are recruited as usual and MSCI is established. These observations suggest that ATM is not required for DNA damage signaling in the XY body.

Collectively, studies of DNA damage signaling in the XY body have led to a 2-step recruitment model (Fig. 4). The localization of DNA repair proteins suggests that DNA breaks localize at the unsynapsed axes of sex chromosomes. The DNA repair proteins are recruited in an ATR-dependent manner. The DNA damage response then spreads beyond the actual DNA breaks to chromosome loops in a way that relies on ATR-γH2AX-MDC1 signaling but independent of RNF8-dependent protein ubiquitination. The DNA damage response that spread on chromatin loops silences the transcription of sex chromosomes, leading to MSCI.

Figure 4.

A two-step model for localizations of DNA damage response proteins in the XY body. DNA breaks are likely localized at chromosome axes. The DNA repair proteins are recruited to chromosome axes in an ATR-dependent manner. The DNA damage response then spreads beyond the actual DNA breaks to chromosome loops in a way that relies on ATR-γH2AX-MDC1 signaling but independent of RNF8-dependent protein ubiquitination. The DNA damage response that spread on chromatin loops silences the transcription of sex chromosomes, leading to MSCI.

Concluding Remarks

Homologous recombination repair of DSBs mediates the generation of crossover and the exchange of genetic information between parental chromosomes during meiotic prophase. The absence of homology between sex chromosomes poses a unique challenge for males at this stage. Studies over the past decade have gradually uncovered how DSBs are repaired and how crossovers are generated in male sex chromosomes. Asynapsis between male sex chromosomes leads to prolonged DSB repair, which is likely to complete eventually through homologous recombination using sister chromatids as templates. As a result of prolonged DSB repair, male sex chromosomes form a unique chromatin structure known as the XY body, which is covered by many DNA damage response proteins. Concomitantly, transcription in male sex chromosome is suppressed, which is likely to be caused by prolonged DSB repair as well. Therefore, studies on transcription repression in the XY body will reveal how gene transcription is regulated during DNA damage response in general. Moreover, different localizations of DNA damage response proteins in the XY body suggest that at least 2 sets of DNA damage response including DNA repair at DSBs and adjacent chromatin remodeling orchestrate the maintenance of genomic stability in the XY body. In fact, the XY body can be considered as the biggest DNA repair “focus” in cell, which may reveal the dynamic recruitment of DNA damage response proteins as well as the repair mechanism in future.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was funded by project 81471494 supported by National Natural Science Foundation of China (to LY) and grants from National Institutes of Health (CA132755, CA130899, and CA187209 to XY). XY is a recipient of Era of Hope Scholar Award from the Department of Defense.