Stella Regulates the Development of Female Germline Stem Cells by Modulating Chromatin Structure and DNA Methylation

Female germline stem cells (FGSCs) have the ability to self-renew and differentiate into oocytes. Stella, encoded by a maternal effect gene, plays an important role in oogenesis and early embryonic development. However, its function in FGSCs remains unclear. In this study, we showed that CRISPR/Cas9-mediated knockout of Stella promoted FGSC proliferation and reduced the level of genome-wide DNA methylation of FGSCs. Conversely, Stella overexpression led to the opposite results, and enhanced FGSC differentiation. We also performed an integrative analysis of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), high-throughput genome-wide chromosome conformation capture (Hi-C), and use of our published epigenetic data. Results indicated that the binding sites of STELLA and active histones H3K4me3 and H3K27ac were enriched near the TAD boundaries. Hi-C analysis showed that Stella overexpression attenuated the interaction within TADs, and interestingly enhanced the TAD boundary strength in STELLA-associated regions. Taking these findings together, our study not only reveals the role of Stella in regulating DNA methylation and chromatin structure, but also provides a better understanding of FGSC development.     

Degradation of Ccnb3 is essential for maintenance of MII arrest in oocyte

Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MII), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MII arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MII arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MII arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Wee1B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MII arrest and exit in mouse oocytes.     

Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome

Background

Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively.

Principal Findings

We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%.

Conclusions

Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.     

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.     

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.     

Identification of an Epi-metabolic dependency on EHMT2/G9a in T-cell acute lymphoblastic leukemia

Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.Subject terms: Translational research, Cancer metabolism, Acute lymphocytic leukaemia