Mouse primordial germ cells (PGC) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5-E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is a significant component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.Specification of primordial germ cells (PGCs) from epiblast cells at ~E6.25 is linked with extensive epigenetic reprogramming, including global DNA demethylation, chromatin reorganisation and imprint erasure, that is vital for generating totipotency (1, 2). The erasure of CpG methylation (5mC) is a key component of this program, but the dynamics and underlying mechanisms of the process remain unclear (3). Here we report a comprehensive analysis of PGCs by combining immunofluorescence, genome-wide (h)meDIP-seq, single cell RNA-seq, bisulfite-seq and functional analyses to address the mechanistic basis of epigenetic reprogramming in PGCs.We investigated Tet expression using single cell RNA-seq, which revealed that Tet1 and Tet2 are expressed in PGCs and peak between E10.5-E11.5, but that Tet3 is undetectable (Fig. 1A). Immunofluorescence (IF) showed that TET1 and TET2 are nuclear and expressed at significantly higher levels in PGCs than neighbouring somatic cells between E9.5-E11.5 (Fig. 1B & S1-S2). This suggests that erasure of 5mC in PGCs could occur through conversion to 5-hydroxymethylcytosine (5hmC) by TET1/TET2 (4, 5).We pursued this possibility by IF and found a progressive reduction of 5mC in PGCs between E9.5-E10.5, until it became undetectable by E11.5 (Fig. 1C). The loss of 5mC occurs concurrently with a global enrichment of 5hmC in PGCs between E9.5-E10.5, † To whom correspondence should be addressed. a.surani@gurdon.cam.ac.uk.

MicroRNAs (miRNAs) have important roles in diverse cellular processes, but little is known about their identity and functions during early mammalian development. Here, we show the effects of the loss of maternal inheritance of miRNAs following specific deletion of Dicer from growing oocytes. The mutant mature oocytes were almost entirely depleted of all miRNAs, and they failed to progress through the first cell division, probably because of disorganized spindle formation. By comparing single-cell cDNA microarray profiles of control and mutant oocytes, our data are compatible with the notion that a large proportion of the maternal genes are directly or indirectly under the control of miRNAs, which demonstrates that the maternal miRNAs are essential for the earliest stages of mouse embryonic development.Supplemental material is available at http://www.genesdev.org.Received November 20, 2006; revised version accepted January 29, 2007. MicroRNAs (miRNAs) are a large family of short noncoding RNAs (17-25 nucleotides) (He and Hannon 2004). A key function of miRNAs is to repress expression of their target genes through sequence complementation, which reduces the abundance of the target mRNAs and/ or inhibits their translation (Bartel 2004;Bagga et al. 2005). MiRNA genes are first transcribed into miRNA primary transcripts by RNA polymerase II (Kim 2005). These primary transcripts are then processed into miRNA precursors by the Drosha/DGCR8 complex and transported from the nucleus to the cytoplasm. Finally, Dicer processes the miRNA precursors into mature miRNAs. From previous studies, Dicer seems to be critical for early mouse development since its loss of function is embryonic lethal at embryonic day 7.5 (E7.5) (Bernstein et al. 2003).In this study, we have examined the role of miRNAs in the mouse oocyte. The mature oocyte contains a number of molecules that are manufactured during oocyte maturation and utilized during early stages of development before activation of the embryonic genome (Dean 2002). It is likely that miRNAs would also be present in the oocyte, but no information is yet available in the mouse. The purpose of this study was to determine if there is significant inheritance of maternal miRNAs in mammalian zygotes, and to investigate if they play a critical role in early mammalian development. We have investigated how the loss of Dicer affects synthesis of miRNA during oocyte maturation and their impact on mRNA and early development. Results and DiscussionFirst, we decided to investigate if there is significant biogenesis of miRNAs in developing oocytes, and their inheritance in the zygote. We therefore examined expression of miRNAs in single cells during oogenesis by a real-time PCR-based miRNA expression profiling method that we recently developed (C. Tang et al. 2006a,b). We compared the miRNA expression profiles of growing oocytes obtained from females 15-16 d after birth (postnatal days 15-16 [P15-P16]) and at P20-P21, and of mature oocytes from adult females. This analysis reveled dynamic chan...

The germ cell lineage exhibits unique characteristics, which are essential towards generating totipotency. Among the distinctive events in this lineage is DNA demethylation and the erasure of parental imprints, which occur on embryonic day 11.5 (E11.5) after the primordial germ cells (PGCs) have entered into the developing gonads 12 . Little is yet known about the mechanism involved, except that this appears to be an active process. Here we have examined the associated changes in the chromatin to gain further insights into this reprogramming event. We show that chromatin changes during this process occur in two-steps. The first changes observed in nascent PGCs at E8.5 establish a distinctive chromatin signature with some characteristics associated with pluripotency. Subsequently, at E11.5 when these PGCs are residing in the gonads, major changes occur in nuclear architecture with an extensive erasure of several histone modification marks along with exchange of histone variants. Furthermore, at this time, the histone chaperones, HIRA and NAP1, which are implicated in histone exchange, show accumulation in PGC nuclei undergoing reprogramming. We thus suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The striking chromatin changes we show here are intimately linked with the process of genome-wide DNA demethylation. Based on the timing of the observed events, we propose, that if DNA demethylation entails DNA repair based mechanism, the evident histone replacement would rather than being a prerequisite, represent a repair-induced response event.The specification of about 40 primordial germ cells (PGCs) from Blimp1 expressing PGCs precursors is accompanied by expression of stella on embryonic day 7.25 (E7.25) 34 . PGCs then migrate into the developing gonads by E10.5. Between E11.5-E12.5 when there are approximately 2000 PGCs per gonad, PGCs exhibit a striking genome-wide DNA demethylation, including erasure of genomic imprints, which is supposedly an active process 12 (Supplementary Figure 1). The mechanism of this DNA demethylation process is unknown but we reasoned that it might be linked with changes in chromatin and histone modifications. However, investigations of this fundamental event are technically difficult since only limited numbers of PGCs are available for analysis, and they are temporally asynchronous and difficult to culture in vitro.

SummaryDuring the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.

The mouse germ-line presents a unique opportunity to study epigenetic reprogramming in vivo1. Recently we showed that genome-wide active DNA demethylation in primordial germ cells (PGCs) is linked to changes in nuclear architecture and extensive loss of histone modifications brought about by widespread histone replacement2. Notably, this chromatin remodelling follows the onset of genome-wide DNA demethylation, which raises a possibility that DNA demethylation may be linked to a DNA repair process2. Here we show the activation of components of the base excision repair (BER) pathway and the presence of single-strand DNA (ssDNA) breaks at the time of genome-wide DNA demethylation in PGCs. We found high levels of expression of critical BER components as well as chromatin-bound XRCC1 together with nuclear poly-ADP-ribosylation (PAR), specifically at the time when PGCs are undergoing DNA demethylation. A similar wave of genome-wide DNA demethylation occurs in the zygote affecting only the paternal genome3-5 where we observed a strikingly similar activation of BER components. Notably, maternally inherited Stella promotes this epigenetic asymmetry, since in zygotes lacking this protein, DNA demethylation is detected in both pronuclei. Crucially, zygotes lacking Stella exhibit aberrant targeting of active BER to both pronuclei. Finally we demonstrate that small molecule inhibitors of diverse BER components administered during in vitro fertilisation interfere with the progress of DNA demethylation. Our combined observations demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation and provides a vital mechanistic link to the extensive chromatin remodelling in developing PGCs2.

Plasma exposure to rosuvastatin and its metabolites was significantly higher in Chinese, Malay, and Asian-Indian subjects compared with white subjects living in the same environment.

SummaryBackgroundWhole brain radiotherapy (WBRT) and dexamethasone are widely used to treat brain metastases from non-small cell lung cancer (NSCLC), although there have been no randomised clinical trials showing that WBRT improves either quality of life or overall survival. Even after treatment with WBRT, the prognosis of this patient group is poor. We aimed to establish whether WBRT could be omitted without a significant effect on survival or quality of life.MethodsThe Quality of Life after Treatment for Brain Metastases (QUARTZ) study is a non-inferiority, phase 3 randomised trial done at 69 UK and three Australian centres. NSCLC patients with brain metastases unsuitable for surgical resection or stereotactic radiotherapy were randomly assigned (1:1) to optimal supportive care (OSC) including dexamethasone plus WBRT (20 Gy in five daily fractions) or OSC alone (including dexamethasone). The dose of dexamethasone was determined by the patients' symptoms and titrated downwards if symptoms improved. Allocation to treatment group was done by a phone call from the hospital to the Medical Research Council Clinical Trials Unit at University College London using a minimisation programme with a random element and stratification by centre, Karnofsky Performance Status (KPS), gender, status of brain metastases, and the status of primary lung cancer. The primary outcome measure was quality-adjusted life-years (QALYs). QALYs were generated from overall survival and patients' weekly completion of the EQ-5D questionnaire. Treatment with OSC alone was considered non-inferior if it was no more than 7 QALY days worse than treatment with WBRT plus OSC, which required 534 patients (80% power, 5% [one-sided] significance level). Analysis was done by intention to treat for all randomly assigned patients. The trial is registered with ISRCTN, number ISRCTN3826061.FindingsBetween March 2, 2007, and Aug 29, 2014, 538 patients were recruited from 69 UK and three Australian centres, and were randomly assigned to receive either OSC plus WBRT (269) or OSC alone (269). Baseline characteristics were balanced between groups, and the median age of participants was 66 years (range 38–85). Significantly more episodes of drowsiness, hair loss, nausea, and dry or itchy scalp were reported while patients were receiving WBRT, although there was no evidence of a difference in the rate of serious adverse events between the two groups. There was no evidence of a difference in overall survival (hazard ratio 1·06, 95% CI 0·90–1·26), overall quality of life, or dexamethasone use between the two groups. The difference between the mean QALYs was 4·7 days (46·4 QALY days for the OSC plus WBRT group vs 41·7 QALY days for the OSC group), with two-sided 90% CI of −12·7 to 3·3.InterpretationAlthough the primary outcome measure result includes the prespecified non-inferiority margin, the combination of the small difference in QALYs and the absence of a difference in survival and quality of life between the two groups suggests that WBRT provides little additional ...

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