Claudia Baumann

Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis

During meiosis, the arrangement of homologous chromosomes is tightly regulated by the synaptonemal complex (SC). Each SC consists of two axial/lateral elements (AEs/LEs), and numerous transverse filaments. SC protein 2 (SYCP2) and SYCP3 are integral components of AEs/LEs in mammals. We find that SYCP2 forms heterodimers with SYCP3 both in vitro and in vivo. An evolutionarily conserved coiled coil domain in SYCP2 is required for binding to SYCP3. We generated a mutant Sycp2 allele in mice that lacks the coiled coil domain. The fertility of homozygous Sycp2 mutant mice is sexually dimorphic; males are sterile because of a block in meiosis, whereas females are subfertile with sharply reduced litter size. Sycp2 mutant spermatocytes exhibit failure in the formation of AEs and chromosomal synapsis. Strikingly, the mutant SYCP2 protein localizes to axial chromosomal cores in both spermatocytes and fetal oocytes, but SYCP3 does not, demonstrating that SYCP2 is a primary determinant of AEs/LEs and, thus, is required for the incorporation of SYCP3 into SCs.

Lsh is required for meiotic chromosome synapsis and retrotransposon silencing in female germ cells

Lymphoid specific helicase (Lsh) is a major epigenetic regulator that is essential for DNA methylation and transcriptional silencing of parasitic elements in the mammalian genome. However, whether Lsh is involved in the regulation of chromatin-mediated processes during meiosis is not known. Here, we show that Lsh is essential for the completion of meiosis and transcriptional repression of repetitive elements in the female gonad. Oocytes from Lsh knockout mice exhibit demethylation of transposable elements and tandem repeats at pericentric heterochromatin, as well as incomplete chromosome synapsis associated with persistent RAD51 foci and γH2AX phosphorylation. Failure to load crossover-associated foci results in the generation of non-exchange chromosomes. The severe oocyte loss observed and lack of ovarian follicle formation, together with the patterns of Lsh nuclear compartmentalization in the germ line, demonstrate that Lsh has a critical and previously unidentified role in epigenetic gene silencing and maintenance of genomic stability during female meiosis.

Loss of Maternal ATRX Results in Centromere Instability and Aneuploidy in the Mammalian Oocyte and Pre-Implantation Embryo

The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA–containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

NEDD1 is crucial for meiotic spindle stability and accurate chromosome segregation in mammalian oocytes.

Defects in meiotic spindle structure contribute to chromosome segregation errors leading to genomic instability in oocytes and embryos upon fertilization. In this study, we analyzed the mechanisms that control spindle microtubule nucleation and stability in mammalian oocytes, and identified NEDD1/GCP-WD as a key regulator. NEDD1 specifically co-localizes with gamma-tubulin and pericentrin at microtubule-organizing centers (MTOCs) in mouse oocytes arrested at prophase-I. During metaphase-I and metaphase-II, the protein remains associated with MTOCs, in a pericentrin dependent manner. Notably, knockdown of Nedd1 transcripts using specific siRNAs resulted in a high incidence (65-70%) of metaphase-I arrest. The arrested oocytes were characterized by disrupted meiotic spindle structure, reduced microtubule density and significant chromosome misalignment. Detection of MAD2 at kinetochores indicated an absence of stable chromosome-microtubule attachment as well as activation of the spindle assembly checkpoint (SAC). Importantly, the disruption of meiotic spindle stability was associated with decreased gamma-tubulin at MTOCs in NEDD1-depleted oocytes, as well as a high frequency of chromosome non-disjunction errors leading to aneuploidy (50%) in the oocytes that did progress to metaphase-II. This study demonstrates that NEDD1 is an essential component of acentriolar oocyte MTOCs, which functions in the regulation of meiotic spindle stability. Moreover, it underscores that disruption of spindle stability in oocytes can lead to chromosomes segregation errors that are not fully resolved by SAC.

Role of ATRX in chromatin structure and function: implications for chromosome instability and human disease

Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in Poly (ADP-ribose) polymerase (Parp-1) null oocytes

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1((-/-)) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1((-/-)) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia.

BackgroundEstablishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.ResultsOur results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.ConclusionThese results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.

ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. HIGHLIGHTED ARTICLE: The chromatin remodelling protein ATRX is transmitted to the early zygote through the maternal germ line and is required to silence major satellite transcripts and control chromosome stability.

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.

Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.