The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation

The sex locus is tightly linked to factors conferring sex-specific lethal effects in the mosquito Aedes aegypti

In many taxa, sex chromosomes are heteromorphic and largely non-recombining. Evolutionary models predict that spread of recombination suppression on the Y chromosome is fueled by the accumulation of sexually antagonistic alleles in close linkage to the sex determination region. However, empirical evidence for the existence of sexually antagonistic alleles is scarce. In the mosquito Aedes aegypti, the sex-determining chromosomes are homomorphic. The region of suppressed recombination, which surrounds the male-specific sex-determining gene, remains very small, despite ancient origin of the sex chromosomes in the Aedes lineage. We conducted a genetic analysis of the A. aegypti chromosome region tightly linked to the sex locus. We used a strain with an enhanced green fluorescent protein (EGFP)-tagged transgene inserted near the male-determining gene to monitor crossing-over events close to the boundary of the sex-determining region (SDR), and to trace the inheritance pattern of the transgene in relation to sex. In a series of crossing experiments involving individuals with a recombinant sex chromosome we found developmental abnormalities leading to 1:2 sex biases, caused by lethality of half of the male or female progeny. Our results suggest that various factors causing sex-specific lethal effects are clustered within the neighborhood of the SDR, which in the affected sex are likely lost or gained through recombination, leading to death. These may include genes that are recessive lethal, vital for development and/or sexually antagonistic. The sex chromosome fragment in question represents a fascinating test case for the analysis of processes that shape stable boundaries of a non-recombining region

Genetic Analysis of Genome-Scale Recombination Rate Evolution in House Mice

The rate of meiotic recombination varies markedly between species and among individuals. Classical genetic experiments demonstrated a heritable component to population variation in recombination rate, and specific sequence variants that contribute to recombination rate differences between individuals have recently been identified. Despite these advances, the genetic basis of species divergence in recombination rate remains unexplored. Using a cytological assay that allows direct in situ imaging of recombination events in spermatocytes, we report a large (∼30%) difference in global recombination rate between males of two closely related house mouse subspecies (Mus musculus musculus and M. m. castaneus). To characterize the genetic basis of this recombination rate divergence, we generated an F2 panel of inter-subspecific hybrid males (n = 276) from an intercross between wild-derived inbred strains CAST/EiJ (M. m. castaneus) and PWD/PhJ (M. m. musculus). We uncover considerable heritable variation for recombination rate among males from this mapping population. Much of the F2 variance for recombination rate and a substantial portion of the difference in recombination rate between the parental strains is explained by eight moderate- to large-effect quantitative trait loci, including two transgressive loci on the X chromosome. In contrast to the rapid evolution observed in males, female CAST/EiJ and PWD/PhJ animals show minimal divergence in recombination rate (∼5%). The existence of loci on the X chromosome suggests a genetic mechanism to explain this male-biased evolution. Our results provide an initial map of the genetic changes underlying subspecies differences in genome-scale recombination rate and underscore the power of the house mouse system for understanding the evolution of this trait

Recombination study by MLH1 immunostaining of mouse spermatocytes after dietary treatments

The dataset is an Excel file with five sheets that contain the following information: Sheet 1 ("1st experiment, 3 strains"): MLH1 foci count per spermatocyte per mice, strain and diet Sheet 2 ("2nd experiment, B6 males"): MLH1 foci count per spermatocyte per C57BL/6 mice treated with two diets (2nd experiment). Columns indicate the mouse ID and number of spermatocytes analyzed in parenthesis. Sheet 3 ("intercrossover distances"): Interfocus distances in control mice (maintenance diet) of 3 strains. Values are shown as percentage of synaptonemal complex length. Sheet 4 ("synaptonemal c. length, 1st"): Total autosomal length of synaptonemal complexes per strain, control groups (maintenance diets) Sheet 5 ("synaptonemal c. length, 2nd"): Total autosomal length of synaptonemal complexes per diet in C57BL/6 mice (2nd experiment)We performed two studies: in the initial one, adult males from the three strains were analyzed for the effect of two diets on recombination (undernourishment (reduction to 50% daily intake) and breeding diets (Teklad Global 18% Protein Rodent Diet)) provided during 24 days relative to a control group kept ad libitum with maintenance diet (Teklad Global 14% Protein Rodent Maintenance Diet). After the 24-day diet period, adult male mice were euthanized by cervical dislocation and weighed. After removing and weighing the testes, chromosome spreads for immunostaining as previously described (Anderson et al. 1999; de Boer et al. 2009; Milano et al. 2019). MLH1 immunostaining allows for identification of about 90% of mammalian crossover sites (Anderson et al. 1999; Cole et al. 2012). All slides were imaged on a Zeiss LSM 710 confocal microscope and analyzed using Zeiss Zen lite software. Only mid and mid-late pachytene stage spermatocytes were scored. For each spermatocyte, we counted the number of foci localizing to the SC of the 19 autosomes (Anderson et al. 1999); total SC length and interfocus distances were also measured in autosomes only.Meiotic recombination is a critical process for sexually reproducing organisms. This exchange of genetic information between homologous chromosomes during meiosis is important not only because it generates genetic diversity, but also because it is often required for proper chromosome segregation. Consequently, the frequency and distribution of crossovers are tightly controlled to ensure fertility and offspring viability. However, in many systems it has been shown that environmental factors can alter the frequency of crossover events. We have explored for the first time the effect of dietary changes on crossover frequency per nucleus. Our study was performed in spermatocytes of 3 mouse inbred strains by analyzing the number and position of crossovers along the synaptonemal complexes, as well as the length of such synaptonemal complexes, by immunostaining with antibodies against MLH1 (which allows the identification of the crossover sites) and SYCP3 (a component of the synaptonemal complex). Our results show that male recombination rate is sensitive to dietary changes, and this sensitivity depends on the genetic background in mice. This is first to report a nutrition effect on genome-wide levels of recombination.Peer reviewe

Diet effects on mouse meiotic recombination: a warning for recombination studies

Meiotic recombination is a critical process for sexually reproducing organisms. This exchange of genetic information between homologous chromosomes during meiosis is important not only because it generates genetic diversity, but also because it is often required for proper chromosome segregation. Consequently, the frequency and distribution of crossovers are tightly controlled to ensure fertility and offspring viability. However, in many systems, it has been shown that environmental factors can alter the frequency of crossover events. Two studies in flies and yeast point to nutritional status affecting the frequency of crossing over. However, this question remains unexplored in mammals. Here, we test how crossover frequency varies in response to diet in Mus musculus males. We use immunohistochemistry to estimate crossover frequency in multiple genotypes under two diet treatments. Our results indicate that while crossover frequency was unaffected by diet in some strains, other strains were sensitive even to small composition changes between two common laboratory chows. Therefore, recombination is both resistant and sensitive to certain dietary changes in a strain-dependent manner and, hence, this response is genetically determined. Our study is the first to report a nutrition effect on genome-wide levels of recombination. Moreover, our work highlights the importance of controlling diet in recombination studies and may point to diet as a potential source of variability among studies, which is relevant for reproducibility.E.d.l.C.-E. received financial support through the program “Plan Propio de Investigacion” of the University of Castilla-La Mancha (2018/11744), cofunded by the European Regional Development Fund (FEDER, UE).Peer reviewe