In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12⁻/⁻ knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.

In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas.

In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols.

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes.

Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI) results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids.

CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear.

The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution.

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions.

Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ-specific ageing, we utilized progeroid repair-deficient Ercc1Δ/- mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1Δ/- intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over-proliferation. ISCs retain their organoid-forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1-KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1Δ/- liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA-damaging agents, and lower lesion removal. Our findings reveal organ-specific anti-ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self-renewal mainly invokes replication-linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA-repair-deficient progerias.

In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses.

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

In meiotic prophase of male placental mammals, the heterologous X and Y chromosomes remain largely unsynapsed, which activates meiotic sex chromosome inactivation (MSCI), leading to formation of the transcriptionally silenced XY body. MSCI is most likely related to meiotic silencing of unsynapsed chromatin (MSUC), a mechanism that can silence autosomal unsynapsed chromatin. However, heterologous synapsis and escape from silencing also occur. In mammalian species, formation of DNA double strand breaks (DSBs) during leptotene precedes meiotic chromosome pairing. These DSBs are essential to achieve full synapsis of homologous chromosomes. We generated 25% extra meiotic DSBs by whole body irradiation of mice. This leads to a significant increase in meiotic recombination frequency. In mice carrying translocation chromosomes with synaptic problems, we observed an approximately 35% increase in asynapsis and MSUC of the nonhomologous region in the smallest chromosome pair following irradiation. However, the same nonhomologous region in the largest chromosome pair, shows complete synapsis and escape from MSUC in almost 100% of the nuclei, irrespective of exposure to irradiation. We propose that prevention of synapsis and associated activation of MSUC is linked to the presence of unrepaired meiotic DSBs in the nonhomologous region. Also, spreading of synaptonemal complex formation from regions of homology may act as an opposing force, and drive heterologous synapsis.

In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify properly targeted ES cell clones. We demonstrate that knockout alleles can be generated either by targeting of an RFLP located in the open reading frame thereby disrupting the RFLP and ablating gene function, or by introduction of a transcription stop cassette that prematurely stops transcription of an RFLP located downstream of the stop cassette. With both methods we have generated Rnf12 heterozygous knockout ES cells, which were identified by allele specific PCR using genomic DNA or cDNA as a template. Our results indicate that this novel strategy is efficient and precise, by combining a high targeting efficiency with a convenient PCR based readout and reliable detection of correct targeting events.

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.

RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.