Patent Application
20210071138
The present invention relates to methods of inducing gametogenesis in vitro. Reagents and kits for use in the methods of the invention are also provided. The present invention finds use in the field of medicine, particularly in the study and treatment of infertility.
Gametogenesis is the process by which gametes are generated. In animals, gametogenesis proceeds via the division and differentiation of meiotically competent gonocytes in the gonads (testes in males; ovaries in females). In males, spermatogenesis occurs in the testes to produce spermatozoa from spermatogonial stem cells (SSCs) in a multi-step process involving both meiosis and mitosis. The SSCs arise from gonocytes in the postnatal testes and these gonocytes, in turn, arise from primordial germ cells (PGCs; Phillips et al, 2010), which migrate to the genital ridge during embryogenesis.
Specification of PGCs (embryonic precursors of gonocytes) begins around embryonic day 6.25 (E 6.25) in mice1. Following specification, the nascent PGCs undergo pronounced global epigenetic changes2-9, including global reduction of genomic 5-methylcytosine (5mC)3,6,7,10. Following their migration through the developing embryo, further epigenetic reprogramming, which includes global DNA demethylation, proceeds once the PGCs arrive to the developing embryonic gonad. Molecular mechanism(s) implicated in this DNA demethylation of gonadal PGCs have been the focus of intense studies3,4,6,12-19,21 and recently published observations suggest that the 5mC oxygenase Tet1 is a critical factor involved in the correct progression of DNA demethylation in gonadal PGCs12,14,16,17. However, the precise nature of this epigenetic reprogramming has remained elusive. Recent work has shown (Hill et al, 2018) that gonadal epigenetic reprogramming is critically involved in the PGC-to-gonocyte transition, which is required to produce meiotically competent gonocytes (and thus allow gametogenesis to be initiated). Importantly, the Gonadal reprogramming process represents a barrier that until recently has only been overcome in the context of the gonadal somatic environment5,24,25,27.
Recent studies have reported the conversion of somatic precursor cells to meiotically competent cells via induced expression of several germ line-related genes (Medrano et al, 2016). Other studies have identified Tet1 as the critical factor in regulating certain germ line-related genes during the activation of female gametogenesis16. However, manipulation of Tet1 expression has not been shown to be sufficient to convert somatic precursor cells to meiotically competent cells.
In humans, infertility is a major health problem. For instance, male infertility affects 7% of the population, with around 10% of infertile men being azoospermic (Galdon et al, 2016). The provision of meiotically competent cells represents an important step in the in vitro recapitulation of gametogenesis, which will find utility in research and medicine, particularly in the context of infertility.
The inventors have found that two distinct biochemical conditions are required for effective activation of a set of genes required for the progression from PGC to gonocyte stage of germline development (the genes are termed the “germline reprogramming-responsive genes (GRR genes)” herein, and in Hill et al 2018). These genes, required also for the conversion of somatic precursor cells, pluripotent cells or early germ cells into meiotically competent cells, can be activated through firstly a reduction of DNA methylation, and secondly the removal of polycomb driven repression. Once these biochemical conditions are in place, transcriptional factors and activators including the epigenetic activator Tet1 are able to drive GRR gene expression. The recruitment of transcriptional activators such as Tet1 and/or the expression of GRR genes are indicative of the conversion of the precursor (somatic) cell into a meiotically competent cell.
Accordingly, in a first aspect, the invention provides an in vitro method of producing a meiotically competent cell, the method comprising:
In some embodiments, the precursor cell is derived from a sample that has been obtained from a subject. The precursor cell may be a stem cell, a primordial germ cell-like cell (PGCLC) or, an early germ cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPS cell) or a spermatogonial stem cell In some embodiments, the inhibiting step (ii) and the treating step (iii) result in the induction of expression of germline reprogramming responsive (GRR) genes by the precursor cell during propagating step (iv). The expression of the GRR genes may be associated with or induced by recruitment of a transcriptional activator, for instance Tet1. Tet1 may be expressed by the precursor cell, and/or Tet1 may be exogenously provided (e.g. by delivering a nucleic acid that exogenously expresses Tet1, by enhancing or stimulating the endogenous expression of Tet1 and/or by providing Tet1 in the form of an exogenous protein).
Exogenously provided Tet1 may be in the form of a fusion construct that is targeted to one or more specific genomic regions. For instance, a Tet1 fusion construct may be targeted to promoter or enhancer sequences involved in expressing one or more of the GRR genes disclosed herein. Providing an effective level of Tet1 as a transcriptional activator enhances the expression of GRR genes. The methods of this invention enable GRR gene expression to be enhanced, and these methods may include increasing or inducing Tet1 expression and/or targeting Tet1 to one or more GRR genes.
The method of the invention may also include the detection and/or quantification of the expression level of one or more GRR genes in the cell. The GRR genes are listed in Table 1. Methods for detecting and/or quantifying expression levels are well known in the art. For instance, mRNA levels of the gene can be measured e.g. by RT-PCR. Protein expression levels can be measured e.g. by assays such as ELISA. Expression of the one or more GRR genes can be measured before, during or after the conversion of the precursor cell to the meiotically competent cell. Preferably, the expression of one or more GRR genes is measured in the meiotically competent cell following step (iv). The GRR gene to be measured may be one or more of Dazl, Hormad1, Sycp2, Sycp3, Mae1, Fkbp6 (see Table 1). In some embodiments of this invention, the inhibitor of polycomb repressive complex is a PRC1 inhibitor (meaning that the PRC1 complex is selectively inhibited. In other embodiments of this invention, the inhibitor of polycomb repressive complex is a PRC2 inhibitor (meaning that the PRC2 complex is selectively inhibited). In yet further embodiments, the inhibitor of polycomb repressive complex inhibits both PRC1 and PRC2.
In some embodiments, the inhibitor is of polycomb repressive complex is PRT4165. In other embodiments, the inhibitor of polycomb repressive complex is an RNAi molecule, which selectively knocks-down the expression of a component of a polycomb repressive complex, e.g. a component of PRC1 or PRC2.
In some embodiments of the invention, the inhibition of DNA methylation (step (ii) of the method) is performed by treating the precursor cell with an agent that reduces genomic DNA methylation. In the context of this disclosure, ‘treating’ the cell is understood to mean ‘contacting’ the cell, i.e. exposing the cell to an agent. Furthermore, ‘inhibiting’ includes both ‘reducing’ and ‘completely preventing’. For instance, the precursor cell may be treated (contacted) with a DNA methyltransferase inhibitor, with an agent that prevents the deposition of DNA methylation, or with an agent that inhibits the maintenance of DNA methylation. 5-aza-2-deoxycytidine (5-Aza-dc) is an agent that inhibits DNA methylation and also inhibits the maintenance of DNA methylation.
In embodiments where the agent that reduces genomic DNA methylation is a DNA methyltransferase inhibitor, the DNA methyltransferase inhibitor may be a DNMT1 inhibitor. For instance, the DNA methyltransferase inhibitor may be SGI 1027 or 5-azacytidine. Alternatively, the DNA methyltransferase inhibitor may be an RNAi molecule, which knocks-down expression of a component of the DNA methylation machinery. The RNAi molecule may be an siRNA molecule or an miRNA molecule (or a precursor of either).
In alternative embodiments, the inhibition of DNA methylation (step (ii) of the method) may be performed by using a technique such as gene-editing to inactivate a DNA methyltransferase gene. Accordingly, various means of inhibiting DNA methylation can be used in producing the meiotically competent cell. For instance, genetic knock-out of the methylation machinery or chemical blockade of the methylation machinery can be used.
In a second aspect, this invention provides a meiotically competent cell produced by the methods described herein. The meiotically competent cell may be treated with retinoic acid. Retinoic acid is known to induce gametogenesis in meiotically competent cells.
Accordingly, in a third aspect, this invention provides a method of inducing gametogenesis in a meiotically competent cell of the invention, by treating it with retinoic acid. In some embodiments, the gametogenesis is spermatogenesis. In other embodiments, the gametogenesis is oogenesis.
In a further aspect, this invention provides a kit for the in vitro production of meiotically competent cells. The kit of the invention comprises a methylation inhibitor and an inhibitor of a polycomb repressive complex. In some embodiments, the kit also comprises retinoic acid. The kit may also comprise appropriate hardware to be used in the methods of the invention, e.g. test tubes, culture plates, etc.
In a yet further aspect, this invention provides a method of assessing the fertility of a mammal. In this aspect of the invention, the nucleic acid sequence and/or epigenetic status and/or gene expression level of one or more germline reprogramming responsive (GRR) genes is determined in a cell that has been obtained from the mammal.
In a related aspect, this invention provides a method of determining the meiotic competency of a cell, the method comprising determining the nucleic acid sequence, epigenetic status, and/or expression of one or more germline reprogramming responsive (GRR) genes in the genomic DNA of the cell. This invention also provides kits and/or assay plates having a group of probes, which group consists of, or consists essentially of, probes that detect expression or epigenetic status or expression of one or more GRR genes as set forth in Table 1.
As described herein, some embodiments of this invention involve detecting GRR genes (e.g. sequence, epigenetic status or expression level) or inducing GRR expression. These embodiments may involve the detection or induction of a group of genes comprising, consisting of, or consisting essentially of, one or more GRR genes selected from Table 1; for instance any 2, any 3, any 4, any 5, any 6, any 7, any 8, any 9, any 10, any 11, any 12, any 13, any 14, any 15, any 16, any 17, any 18, any 19, or any 20 genes selected from Table 1. In some embodiments, the genes selected from Table 1 may include one or more of Dazl, Hormad1, Sycp2, Sycp3, Mae1, Fkbp6. In other embodiments, the genes selected from Table 1 may exclude any or all of Dazl, Hormad1, Sycp2, Sycp3, Mae1, Fkbp6.
Therapeutic Applications
The methods and products of this invention have therapeutic application, particularly in the treatment of infertility. For instance, as described herein, the meiotically competent cell produced by the methods of the invention can be induced to undergo gametogenesis, e.g. by treatment with retinoic acid (RA). Gametocytes produced in this way (i.e. spermatocytes; oocytes) constitute further aspects of this invention. The gametocyte of the invention has therapeutic applications, for instance in the adoptive transfer to an infertile individual: It is envisaged that the spermatocyte of the invention may be adoptively transferred to the testes of a male infertility patient. It is envisaged that the oocyte of the invention may be adoptively transferred to the ovary of a female infertility patient. These gametocytes may be derived from the patient's own cells, e.g. by performing the method of the invention on an iPS cell, a spermatogonial stem cell (SSC), or a PGCLC derived from a patient cell. This approach allows autologous adoptive transfer of the gametocyte to the patient.
In further aspects of the invention, gametes are derived from the abovementioned gametocyte of the invention in vitro. In this way, the invention provides male gametes, spermatozoa (sperm), and female gametes, ova (eggs), which may be used therapeutically. For instance, the gametes of the invention may be used in in vitro fertilisation (IVF) applications.
Precursor Cells
As explained herein, the methods of the invention are capable of converting a somatic precursor cell into a meiotically competent cell. This subsection discusses various types of cell that may be used as the precursor cell.
In nature, the precursor to meiotically competent gonocytes are primordial germ cells. Current in vitro systems aimed at generating PGC-like cells (PGCLCs)5,24-26 can successfully recapitulate only the early stages of PGC development, with gonadal reprogramming still presenting a barrier that can be overcome and executed only in the context of the gonadal somatic environment5,24,25,27. In some embodiments of this invention, the precursor cell is a PCGLC obtained by the aforementioned prior art methods.
In other embodiments of this invention, the precursor cell is a stem cell, for instance an embryonic stem cell. Human embryonic stem cells represent one type of precursor cell. It is known in the art that human embryonic stem cells can be obtained without destroying the human embryo (Chung et al., 2008). Mouse embryonic stem cells also represent a type of precursor cell that usefully demonstrate the efficacy of this invention. The inventors have found that the epigenetic regulation of GRR genes in PGCs is very similar to that in serum-grown mouse embryonic stem cells.
Pluripotent stem cells that are not of embryonic origin may also be used as the precursor cells in the methods of this invention. Pluripotent stem cells can be obtained by methods including:
Reprogramming by nuclear transfer. This technique involves the transfer of a nucleus from a somatic cell into an oocyte or zygote. In some situations, this may lead to the creation of an animal-human hybrid cell. For example, cells may be created by the fusion of a human somatic cell with an animal oocyte or zygote or fusion of a human oocyte or zygote with an animal somatic cell.
Reprogramming by fusion with embryonic stem cells. This technique involves the fusion of a somatic cell with an embryonic stem cell. This technique may also lead to the creation of animal-human hybrid cells, as in 1 above.
Spontaneous re-programming by culture. This technique involves the generation of pluripotent cells from non-pluripotent cells after long term culture. For example, pluripotent embryonic germ (EG) cells have been generated by long-term culture of primordial germ cells (PGC) (Matsui et al., 1992). The development of pluripotent stem cells after prolonged culture of bone marrow-derived cells has also been reported (Jiang et al., 2002). They designated these cells multipotent adult progenitor cells (MAPCs). Shinohara et al also demonstrated that pluripotent stem cells can be generated during the course of culture of germline stem (GS) cells from neonate mouse testes, which they designated multipotent germline stem (mGS) cells (Kanatsu-Shinohara et al., 2004).
Reprogramming by definedfactors. For example the generation of iPS cells by the retrovirus-mediated introduction of transcription factors (such as Oct-3/4, Sox2, c-Myc, and KLF4) into mouse embryonic or adult fibroblasts, e.g. as described by Kaji et al., 2002 also describe the non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, that can reprogram both mouse and human fibroblasts. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. They succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. Induced pluripotent stem cells have the advantage that they can be obtained by a method that does not cause the destruction of an embryo, more particularly by a method that does not cause the destruction of a human or mammalian embryo.
Pluripotent stem cells may also be obtained from arrested embryos which stopped cleavage and failed to develop to morula and blastocysts in vitro, obtained by parthenogenesis, or derived from hESC lines from single blastomeres or biopsied blastomeres.
As such, aspects of the invention may be performed or put into practice by using cells that have not been prepared exclusively by a method which necessarily involves the destruction of human or animal embryos from which those cells may be derived. This optional limitation is specifically intended to take account of Decision G0002/06 of 25 Nov. 2008 of the Enlarged Board of Appeal of the European Patent Office.
In other embodiments, gametogonia (gamete stem cells) may be used as the precursor cell. For instance spermatogonial stem cells (SSCs) are one preferred precursor cell type for use in the methods of this invention. SSCs may be extracted from the testes, e.g. from a testes biopsy. Testes aspirate is one source of a cell preparation (extract) that contains SSCs. It is envisaged that the methods of the invention can be performed directly on such testes extracts, or could be performed upon SSCs that have been enriched, selected and/or purified.
The precursor cell may be obtained from a subject. The subject may be a mammalian subject, for instance a human subject. In some embodiments of the invention, the subject is an infertility patient.
RNA Interference (RNAi)
The present invention also includes the use of techniques known in the art for the therapeutic down regulation of a component of the polycomb repressive complex or of a component of DNA methylation machinery. These include the use RNA interference (RNAi).
Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.
A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double-stranded RNA (dsRNA)-dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.
In the art, these RNA sequences are termed “short or small interfering RNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNAs are derived by processing of long double stranded RNAs. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.
Accordingly, the present invention provides the use of these sequences for down-regulating the expression of components of a polycomb repressive complex, e.g. PRC1 and/or PRC2.
The siRNA is typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.
miRNA are typically single stranded and have regions that are partially complementary enabling the miRNA to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is known in the art.
Embodiments of the invention will now be described by way of example with reference to the accompanying drawings:
The timely and efficient activation of germline reprogramming responsive (GRR) genes, involved in the PGC-to-gonocyte transition and successful gametogenesis, requires the interplay between the initiation of global DNA demethylation, Tet1 recruitment, and removal of PRC1-mediated repression. Both DNA demethylation-dependent (safeguarding against aberrant residual/de novo promoter DNA methylation) and -independent (such as the potential recruitment of OGT to gene promoters36, thus facilitating deposition of H3K4me3 via SET1/COMPASS38) functions of Tet1 are important for GRR gene activation.
Mouse embryonic stem cells (mESCs) were treated with retinoic acid (RA). The J1 cell line was used in comparison to J1 “TKO” cells lack DNA methylation machinery by virtue of being Dnmt1/Dnmt3a/Dnmt3b triple knock-out. The black bars respectively show the fold-change in Dazl-Hormad1- and Mae1-expression in TKO cells compared with J1 controls (neither treated with RA). The grey bars respectively show the fold-change in Dazl-Hormad1- and Mae1-expression in J1 cells treated with RA compared with J1 cells not treated with RA. The white bars respectively show the fold-change in Dazl-Hormad1- and Mae1-expression in TKO cells treated with RA compared with J1 cells not treated with RA.
While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.
Epigenetic Reprogramming Enables the Primordial Germ Cell-to-Gonocyte Transition
Gametes are highly specialised cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mouse, germ cells are first specified in the developing embryo as primordial germ cells (PGCs) starting around embryonic day (E) 6.251 (
In order to address the potential role and underlying molecular mechanisms of gonadal germline reprogramming, the inventors first set out to investigate the dynamics of and relationship between 5mC and 5-hydroxymethylcytosine (5hmC), which has previously been implicated in DNA demethylation in PGCs3,6,9-11. The inventors did this quantitatively and at single base resolution using liquid chromatography/mass spectrometry (LC/MS) coupled with whole genome bisulphite sequencing (WGBS,
By LC/MS the inventors observed that global levels of genomic 5mC remain stable between migratory (E9.5) and early gonadal (E10.5) PGCs, followed by a significant reduction between E10.5 and E11.5 and much more limited DNA demethylation between E11.5 and E13.5 (
Consistent with our LC/MS measurements, WGBS analysis revealed near complete loss of combined 5mC/5hmC between E10.5 and E11.5 at features within uniquely mapped regions of the genome, with limited further DNA demethylation observed between E11.5 and E12.5 (
Detailed analysis of 5hmC patterns across examined developmental stages uncovered that the majority of 5hmC is lost from uniquely mapped regions of the genome and re-localised to repetitive elements (
We next explored the relationship between 5hmC deposition and DNA demethylation in gonadal PGCs between E10.5 and E12.5 for all initially methylated 2 kb windows (i.e. min. 20% methylation at E10.5). DNA demethylation involving a 5hmC intermediate predicts a direct correlation between 5hmC appearance and 5mC loss (
To expand on this observation, the inventors used a previously published Tet1-KO mouse model18 (
In support of our LC/MS measurements, only a limited number of differentially methylated regions were detected in E14.5 Tet1-KO PGCs by reduced representation bisulphite sequencing (RRBS) (
We next analysed the observed 5mC and 5hmC dynamics in combination with RNA-Seq datasets derived from E10.5-E14.5 PGCs (
As the influence of 5mC on transcriptional activity of a gene has been shown in mammals to be highly dependent on promoter CpG content19, the inventors performed clustering analysis specifically at genes with either high-CpG (HCPs), intermediate-CpG (ICPs) or low-CpG (LCPs) promoters19 (
Considering that GRR genes (n=45) constituted less than 25% of the entire subset of HCP genes that undergo DNA demethylation (n=226;
The observed binding of Tet1 is functionally relevant, as the extent of GRR gene upregulation is considerably lower in Tet1-KO PGCs (
To further mechanistically probe the causal relationship between epigenetic reprogramming and GRR gene activation, the inventors turned to an in vitro model. Serum-grown mESCs represented an ideal system, as these cells are not germ line-restricted yet have highly similar epigenetic modifications at GRR gene promoters to what is observed in vivo in pre-reprogramming gonadal PGCs (
Although these in vitro observations clearly supported our in vivo data with respect to the roles of 5mC and Tet1, the extent to which GRR genes were up-regulated in Dnmt-TKO mESCs (
Our study has identified a set of germline reprogramming responsive (GRR) genes crucial for the correct progression of gametogenesis. These genes have unique promoter sequence characteristics, with high levels of both 5mC and 5hmC, and are targets of Tet1 and PRC1. This disclosure shows that combined loss of DNA methylation and PRC1 repression is uniquely required for GRR gene activation, with this epigenetically poised state further requiring Tet1 to potentiate both full and efficient activation. Tet1 appears to be particularly important in female PGCs9, which initiate meiotic prophase soon after completion of epigenetic reprogramming, thus posing a requirement on the timely high expression of these genes. Importantly, although the inventors observed slight hypermethylation at GRR gene promoters in E14.5 Tet1-KO PGCs, our study clearly documents that Tet1 stimulates transcription of GRR genes also via a DNA demethylation-independent mechanism21,22. In this context, previous studies have shown that Tet1 recruits OGT to gene promoters22, thus facilitating deposition of H3K4me3 via SET1/COMPASS28 leading to transcriptional activation. In further support, GRR gene promoters in mESCs are marked by low but detectable H3K4me3, the levels of which are significantly decreased in the absence of Tet1 without changes in DNA methylation (
All statistical tests are clearly described in the figure legends and/or in the Methods section, and exact p-values or adjusted p-values are given where possible. For WGBS data (
Mice
All animal experiments were carried out under and in accordance with a UK Home Office Project Licence in a Home-Office designated facility. Except for direct comparison with Tet1-KO PGCs, wild type PGCs were isolated from embryos produced by crossing outbred MF1 females with mixed background GOF18ΔPE-EGFP5 transgenic males. The sex of embryos from E12.5 onwards was determined by visual inspection of the gonads. For study of Tet1-KO PGCs, the Tet1 knockout mouse strain (B6;29S4-Tet1tml.IJae/J)18 was purchased from Jackson Laboratory and bred onto the GOF18ΔPE-EGFP5 transgenic mouse line. Wild type and Tet1-KO PGCs were isolated from embryos produced from crosses between Tet1-heterozygous GOF18ΔPE-EGFP-homozygous females and males. For genotyping of embryos produced by crossing Tet1-heterozygous GOF18ΔPE-EGFP-homozygous males and females, PCR was always carried out twice using two different sets of primers (see below) to confirm exon 4 deletion. The sex of the embryos from E12.5 onwards was determined by visual inspection of gonads and additionally confirmed by PCR for Sry. In all cases, the mating is timed in the way that appearance of a vaginal plug at noon is defined as E0.5.
Molecular Biology
The following genotyping primers were used in this study:
PGC Isolation by Flow Cytometry
PGC isolation was carried out as previously described4. Briefly, the embryonic trunk (E10.5) or genital ridge (E11.5-E14.5) was digested at 37° C. for 3 min using 0.05% Trypsin-EDTA (lx) (Gibco) or TrypLE Express (Thermo). Enzymatic digestion was followed by neutralization with DMEM/F-12 (Gibco) containing 15% foetal bovine serum (Gibco) and manual dissociation by pipetting. Following centrifugation, cells were re-suspended in DMEM/F-12 supplemented with hyaluronidase (300 μg/ml; Sigma), and a single cell suspension was generated by manual pipetting. Following centrifugation, cells were re-suspended in ice-cold PBS supplemented with poly-vinyl alcohol (10 μg/ml) and EGTA (0.4 mg/ml, Sigma). GFP positive cells were isolated using an Aria IIu (BD Bioscience) or Aria III (BD Bioscience) flow cytometer and sorted into ice cold PBS supplemented with poly-vinyl alcohol (10 μg/ml) and EGTA (0.4 mg/ml, Sigma).
Generation of Tet1-KO Dnmt-TKO mESCs
Tet1-KO Dnmt-TKO mESC line was generated by CRISPR/Cas9-mediated genome editing. pX330 (Addgene, #42230) with the sgRNA targeting Tet131 (GGCTGCTGTCAGGGAGCTCA) was co-transfected with a reporter GFP plasmid in 5×106 Dnmt-TKO mESCs23 using Lipofectamine 3000. The day after, GFP positive cells were sorted by FACS (BD FACS Aria III) in a 96-well plate. Cells were cultured for a week before being frozen down and extracting gDNA. Colonies were screened for mutations using surveyor assay (Surveyor Mutation Detection Kit from IDT, and Taq DNA polymerase from Qiagen). Tet1-KO Dnmt-TKO mESC selected clone was further analysed by genotype sequencing, which confirmed the presence of a frameshift mutation. Loss of Tet1 was verified by RNA-Seq and western blot. The following primers were used for genotype sequencing and surveyor assay: 5′ TTGTTCTCTCCTCTGACTGC 3′ and 5′ TGATTGATCAAATAGGCCTGC 3′.
mESC Cell Culture
J1 (wild type), Dnmt-TKO23 and Tet1-KO Dnmt-TKO mESCs were cultured in FCS/LIF medium without feeders on 0.1% gelatin. FCS/LIF medium consists of GMEM (Gibco) supplemented with 10% FCS, 0.1 mM MEM nonessential amino acids, 2 mM 1-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol and mouse LIF (ESGRO, Millipore). For inhibitor experiments, mESCs were plated at a density of 1.5×104/cm2 and left overnight. The next morning medium was exchanged for FCS/LIF medium containing either 50 μM PRC1 inhibitor PRT4165 (Ismail et al., 2013) or DMSO control and cells pelleted at the indicated time for analysis.
AbaSeq Library Preparation
Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen). AbaSeq libraries for 5hmC profiling were constructed as previously described15. In brief, genomic DNA was glucosylated, then digested by AbaSI enzyme (NEB). Biotinylated P1 adapters were ligated onto the AbaSI digested DNA then fragmented using a Covaris S2 sonicator (Covaris), following the manufacturer's instructions. Sheared P1-ligated DNA was then captured by mixing with Dynabeads MyOne Streptavidin C1 beads (Life Technologies) according to the manufacturer's specifications. End repair and dA-tailing were carried out on the beads by using the NEBNext End Repair Module (NEB) and the NEBNext dA-tailing Module (NEB) at 20° C. and 37° C. respectively for 30 min. P2 adapters were ligated to the random sheared ends of the dA-tailed DNA. Finally, the entire DNA was amplified using the Phusion DNA polymerase (NEB) with the addition of 300 nM forward primer (PCR_I) and 300 nM reverse primers (PCR_IIpe) for 16 cycles. The libraries were purified using AMPure XP beads (Beckman-Coulter) and sequenced on the Illumina HiSeq 2000 instrument.
Whole Genome Bisulphite Sequencing (WGBS) Library Preparation
Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen). In some cases, unmethylated k phage DNA (Promega) was spiked in following DNA isolation to assess bisulphite conversion rate. DNA was fragmented using a Covaris S2 sonicator (Covaris), as per manufacturer's instructions. Libraries were made following the NEBNext Library Prep protocol, with methylated adaptors and the following modifications: following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma); and PCR enrichment was carried out for 16 cycles using the NEXTflex Bisulphite-Seq Kit for Illumina Sequencing (Bioo Scientific) master mix and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Libraries were sequenced on the Illumina HiSeq 2000 or 2500 instrument.
Reduced Representation Bisulphite Sequencing (RRBS) Library Preparation
Total DNA from FACS-sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and DNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 26 μL final volume using the Savant SpeedVac Concentrator (Thermo) and following the manufacturer's instructions. Genomic DNA was digested by 20 units of MspI enzyme (NEB) in NEB buffer 2 at 37° C. for 3 hrs, and digested DNA was purified using AMPure XP beads (Beckman-Coulter). Libraries were made following the NEBNext Ultra DNA Library Prep protocol with methylated adaptors and the following modifications: following adaptor ligation, bisulphite conversion was carried out using the Imprint Modification Kit (Sigma); and PCR enrichment was carried out for 18 cycles using the KAPA Uracil+DNA polymerase master mix (KAPA Biosystems) and the NEBNext Library Prep universal and index primers (NEB). The libraries were purified by AMPure XP beads (Beckman-Coulter). Pooled libraries were sequenced on the Illumina HiSeq 2500 instrument, using the ‘dark sequencing’ protocol, as previously described32.
RNA-Seq Library Preparation
For study of Tet1-KO PGCs, total RNA from sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and RNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 6 μL final volume using the RNA Clean and Concentrator 5 kit (Zymo). For study of wild type PGCs isolated from embryos produced by crossing MF1 females with GOF18ΔPE-EGFP males, total RNA from 600-1,000 sorted E10.5 PGCs was isolated using the Nucleospin RNA XS kit (Macherey-Nagel). cDNA synthesis and amplification (15 cycles) was performed with the SMARTer Ultra Low Input RNA kit (Clontech) using between 100 pg and 3 ng total RNA and following the manufacturer's instructions. The amplified cDNA was fragmented by a Covaris S2 sonicator (Covaris) and following the manufacturer's instructions. Sheared cDNA was converted to sequencing libraries using the NEBNext DNA Library Prep kit (NEB), following the manufacturer's instructions and using 15 cycles of amplification. For study of mESCs, total RNA was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo). cDNA synthesis and library prep was performed starting with 500 ng total RNA following manufacturer's instructions using the NEBNext Ultra Library Prep Kit (NEB) and the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). All libraries were purified by AMPure XP beads (Beckman-Coulter) and sequenced on the Illumina HiSeq 2500 instrument.
Bioinformatics
Whole Genome Bisulphite Sequencing (WGBS) and Tet-Assisted Bisulphite Sequencing (TAB-Seq) Alignment and Downstream Analysis
Raw reads were first trimmed using Trim Galore (version 0.3.1) with the --paired --trim1 options. Alignments were carried out to the mouse genome (mm9, NCBI build 37) with Bismark (version 0.13.0) with the -n 1 parameter; where appropriate, the λ phage genome was added as an extra chromosome. Aligned reads were deduplicated with deduplicate_bismark. Where appropriate, the bisulphite conversion rate was computed using reads aligned to the λ phage genome and using the to-mr script (parameters: -m bismark) and bsrate script (paramters: -N) of Methpipe (version 3.3.1). CpG methylation calls were extracted from the deduplicated mapping output using the Bismark methylation extractor.
The number of methylated and unmethylated cytosines in a CpG context was extracted using bismark2bedGraph and coverage2cytosine. Symmetric CpGs were merged with custom R script. For all downstream analysis, only symmetric CpGs with minimum 8× coverage were used. All WGBS analysis was carried out on data from merged biological replicates. For assessing DNA modification levels at specific repetitive elements, Bismark (version 0.14.4) was used to map all reads from each data set against consensus sequences constructed from Repbase with the −n 1 parameter set. CpG methylation calls were extracted from the mapping output using the Bismark methylation extractor (version 0.14.4).
The mapBed function of BEDtools (version 2.24.0) was used to compute the combined 5mC/5hmC level for the following genomic features: 1) all 2 kb windows (containing a minimum 4 symmetric CpGs); 2) gene promoters (defined as Ensembl 67 gene start sites −1 kB/+500 bp); 3) gene bodies (defined as the region contained within Ensembl 67 gene start and gene end sites); 4) putative active enhancers in day 6 PGCLCs33; 5) imprint control regions; 6) CpG islands (UCSC); 7) intergenic regions. For metagene plots, a genomic feature was divided into equally sized bins using BEDtools (version 2.24.0), including: 1) gene bodies (defined as the region contained within Ensembl 67 gene start and gene end sites)+/−0.5*gene body length (100 bins); 2) putative active enhancers in day 6 PGCLCs 33 +/−1*putative active enhancer length (90 bins); and 3) CpG islands (UCSC)+/−1*CpG island length (90 bins). In all cases, the combined 5mC/5hmC level was expressed as the mean of individual CpG sites.
For k-means clustering of the combined mean 5mC/5hmC levels, high CpG (HCP), intermediate CpG (ICP) and low CpG (LCP) promoters, as defined using the same parameters as previously published1934. Briefly, LCPs contain no 500-bp window with a CpG ratio >0.45; HCPs contain at least one 500-bp window with a CpG ratio >0.65 and GC content >55%; ICPs do not meet the previous criteria.
For determining locus-specific methylation levels in wild type mESCs grown in serum-containing media, raw WGBS reads were downloaded from GSE4851930 and processed as above. TAB-Seq reads for E14 mESCs were downloaded from GSE3617335 and processed as above, with the exception that only symmetric CpGs with minimum 12× coverage were used.
AbaSeq Alignment and Downstream Analysis
For the uniquely mappable part of the genome, AbaSeq reads were processed as previously described15. In brief, raw sequencing reads were trimmed for adaptor sequences and low quality bases using Trim Galore. The trimmed reads were mapped to the mouse genome (mm9, NCBI build 37) using Bowtie (version 0.12.8) with parameters −n 1 -l25 --best --strata -m 1. Calling of 5hmC was based on the recognition sequence and cleavage pattern of the AbaSI enzyme (5′-C11-13↓N9-10G-3′/3′-GN9-10↓N11-13C-5′) using custom Perl scripts. For assessing relative enrichment of 5hmC at repetitive elements and non-repetitive elements, AbaSeq alignments were divided into two groups: unique (single best alignment) and ambiguous (map to multiple locations with equal alignment score). Both groups were then mapped to the repetitive elements defined by the RepeatMasker track of mm9 (UCSC Genome Browser) separately. For comparison with 5hmC levels in mESCs, AbaSeq reads were downloaded from GSE4289815 and aligned in the same way.
For quantification of relative 5hmC levels at symmetric CpGs in the uniquely mapped part of the genome, the number of counts per symmetric CpG for a given sample were normalised to the combined number of uniquely mapped and ambiguously mapped reads for a given library, and then further multiplied by a stage-specific normalisation factor based on the mean 5hmC level for each stage as computed by LC/MS (E14 ESC=1.64; E10.5=1.0; E11.5=1.13; E12.5F=0.76; E12.5M=1.0). All symmetric CpGs falling within genomic intervals blacklisted by the mouse (mm9) ENCODE project were excluded from all further downstream analysis. Unless stated otherwise, all AbaSeq analysis was carried out on data from merged biological replicates.
The mapBed function of BEDtools (version 2.24.0) was used to compute the 5hmC level for the same genomic features as was carried out with WGBS datasets (see above). In all cases, the 5hmC level was expressed as the mean of individual CpG sites.
To identify 5hmC enriched or depleted regions in E10.5 and E11.5 PGCs, the mm9 genome was first divided into 2 kb windows (minimum 4 symmetric CpGs) and the mean 5hmC level for each window was computed using BEDtools (version 2.24.0). To determine the significance of 5hmC enrichment in each 2 kB window, upper-tail (to determine 5hmC enriched regions) or lower-tail (to determine 5hmC depleted regions) Poisson probability p-values were computed using ppois(x, λ), where x is the observed 5hmC mean value for each 2 kb window and λ is the mean of 5hmC mean values for all 2 kb windows at E10.5. Benjamini-Hochberg correction was then applied to correct for multiple testing, giving a final adjusted upper-tail and lower-tail p-value for each 2 kb window. Windows with adjusted upper-tail p-value<0.05 were considered relatively enriched for 5hmC while windows with adjusted lower-tail p-value<0.05 were considered relatively depleted for 5hmC.
For assessing relative enrichment of 5hmC at specific repetitive elements, Bowtie was used to map all reads from each data set against consensus sequences constructed from Repbase with parameters −n 1−M 1 --strata -best. The number of reads mapped to each sequence within a given sample was first normalised to the library size of that particular sample, and then normalised to both a stage-specific normalisation factor based on the mean 5hmC level for each stage as computed by LC/MS (E10.5=1.0; E11.5=1.13; E12.5F=0.76; E12.5M=1.0) and the mean proportion of reads mapped to a given sequence in E10.5 PGCs.
Reduced Representation Bisulphite Sequencing (RRBS) Alignment and Downstream Analysis
Raw RRBS reads were first trimmed using Trim Galore (version 0.3.1) with --rrbs parameter. Alignments were carried out to the mouse genome (mm9, NCBI build 37) with Bismark (version 0.13.0) with the -n 1 parameter. CpG methylation calls were extracted from the mapping output using the Bismark methylation extractor (version 0.13.0). The number of methylated and unmethylated cytosines in a CpG context was extracted using bismark2bedGraph.
RnBeads (version 1.0.0) and RnBeads.mm9 (version 0.99.0) were used to identify differentially methylated regions between two test groups for the following genomic features, with filtering.missing.value.quantile set to 0.95 and filtering.missing.coverage.threshold set to 8: 1) all 2 kb windows (containing a minimum 4 symmetric CpGs); 2) gene promoters (defined as Ensembl 67 gene start sites −1 kB/+500 bp); and 3) imprint control regions (mm9 genome). The following was extracted from the output of RnBeads: 1) the mean methylation level for each group (i.e. stage, sex and/or genotype) for each commonly covered test region; 2) the difference in methylation means between two groups for each commonly covered test region; and 3) the p-value representing the significance of the difference in methylation means between two groups for each commonly covered test region. Differentially methylated regions were identified as regions with a p-value<0.05 and a difference in methylation means between two groups greater than 10%.
For assessing DNA modification levels at specific repetitive elements, Bismark (version 0.14.4) was used to map all reads from each data set against consensus sequences constructed from Repbase with the -n 1 parameter set. CpG methylation calls were extracted from the mapping output using the Bismark methylation extractor (version 0.14.4). The number of methylated and unmethylated cytosines in a CpG context were extract using bismark2bedGraph and coverage2cytosine. Differentially methylated consensus repeats were identified as regions with a p-value<0.05 (as computed by two-sided Student's t-test) and a difference in methylation means between two groups greater than 10%.
hMeDIP Alignment and Downstream Analysis
Raw hMeDIP-Seq and input reads for E14 mESCs were downloaded from GSE2850036 and aligned to the mouse genome (mm9, NCBI build 37) with Bowtie (version 0.12.8) with parameters -n 2 -l25 -m 1. BEDtools multicov was used to identify the number of hMeDIP and input reads overlapping each 2 kB window (containing a minimum 4 symmetric CpGs). Final 5hmC levels for each 2 kB window were determined by first normalising the number of overlapping hMeDIP reads (normalised to library size) by the number of overlapping input reads (normalised to library size) and then by dividing this value by the number of symmetric CpGs contained within the 2 kB window.
ChIP-Seq Alignment and Downstream Analysis
For putative active enhancer calling, raw ChIP-Seq reads for H3K4me3, H3K27me3 and H3K27Ac in day 6 PGC-like cells (PGCLCs) were downloaded from GSE6020433 and raw ChIP-Seq reads for H3K4me3, H3K27me3, H3K4mel and H3K27Ac in wild type mESCs were downloaded from GSE4851930. Reads were aligned to the mouse genome (mm9, NCBI build 37) with Bowtie (version 0.12.8 or version 1.0.0) with parameters -n 2 -l 25 -m l and -C where appropriate. Subsequent ChIP-Seq analysis was carried out on data from merged biological replicates. To identify putative active enhancers, the inventors first generated an 8-state chromatin model using ChromHMM. Putative active enhancers were defined as all regions not overlapping any potential promoter regions (Ensembl 67 gene start sites −1 kB/+500 bp) and contained within the (H3K27Ac+/H3K4me3−/H3K27me3−) chromatin state in day 6 PGCLCs or (H3K4me+/H3K27Ac+/H3K4me3−/H3K27me3−) in wild type mESCs.
For analysis of epigenetic modifications and modifiers around transcription start sites (Ensembl 67): raw ChIP-Seq reads for: Tet1 binding in wild type serum-grown mESCs was downloaded from GSE2484321; H2AK19Ub1 levels in wild type serum-grown mESCs were downloaded from GSE3452037; Ring1b binding in wild type serum-grown mESCs were downloaded from ERP00557538; and for H3K4me3 in wild type and Tet1-KO serum-grown mESCs were downloaded from GSE4851930. Reads were aligned to the mouse genome (mm9, NCBI build 37) with Bowtie (version 0.12.8 or version 1.0.0) with parameters -n 2 -l 25 -m 1. Subsequent ChIP-Seq analysis was carried out on data from merged biological replicates. For computing ChIP-Seq signal around transcription start sites (TSS), the genomic interval around the Ensembl 67 gene start sites+/−5 kB (or 2 kB) was divided into 100 (or 40) equally sized bins using BED tools make windows. BED tools multicov was then used to compute the number of test and control reads overlapping each bin. The total number of test and control reads per bin for each sample were normalised to the appropriate library size, and fold enrichment for each bin was determined by dividing the number of normalised ChIP-Seq test sample reads by the number of normalised ChIP-Seq control sample reads. For computing ChIP-Seq signal at gene promoters, the genomic interval around the Ensembl 67 gene start sites +500 bp/−1 kB was
RNA-Seq Alignment and Downstream Analysis
For study of Tet1-KO and Tet1-WT PGCs, Illumina and Smart-seq adapters from the sequencing reads were first trimmed using Trimmomatic. For other RNA-Seq libraries, fastq files generated from output of next generation sequencing were used directly for alignment. RNA-Seq reads were aligned to the mouse genome (mm9, NCBI build 37) with Bowtie (version 0.12.8) and Tophat (version 2.0.2) with options -N 2 --b2-very-sensitive --b2-L 25. Annotations from Ensembl Gene version 67 were used as gene model with Tophat. Read counts per annotated gene were computed using HTSeq (version 0.5.3p9) and expression level of each gene was quantified by computing the number of fragments detected per kilobase per million of reads (FPKM) using custom R script. Genes were assigned to an expression level bin based on the mean FPKM values of the two biological replicates. Differential expression analysis was performed using DESeq2 (version 1.6.3), and genes with an adjusted p-value<0.05 were considered differentially expressed. For determining gene expression levels in wild type and Dnmt1-conditional knockout and matched wild type E10.5 PGCs, raw RNA-Seq reads were downloaded from GSE7493824 and processed as above.
HCPs methylated and demethylating in PGCs during epigenetic reprogramming (cluster 3,
For classification of GRR genes (
Tet1 and 5mC/5hmC Detection by Immunofluorescence
The embryonic trunk (E10.5) or genital ridge (E12.5/E13.5) was first fixed in 2% PFA (in PBS) for 30 min at 4° C. Following fixation, tissue was washed in PBS three times for 10 min and then incubated in 15% sucrose in PBS overnight. After rinsing with 1% BSA in PBS the following day, the tissue was embedded in OCT Embedding Matrix (Thermo Scientific Raymond Lamb) and frozen using liquid nitrogen. Samples were subsequently stored at −80° C. A Leica CM 1950 cryostat was used to cut 10 μm sections from the frozen embedded tissue. Sections were settled on poly-lysine slides (Thermo Scientific) and post-fixed with 2% PFA in PBS for 3 minutes.
For detection of Tet1, sections were washed three times for 5 min with PBS. After incubating for 30 min at room temperature in 1% BSA/PBS containing 0.1% Triton X-100, the sections were incubated with primary antibodies listed at 4° C. overnight in the same buffer. Sections were subsequently washed three times in 1% BSA/PBS containing 0.1% Triton X-100 for 5 min and incubated with secondary antibodies in the same buffer for 1 hour in the dark at room temperature. Secondary antibody incubation was followed by three 5 min washes with PBS. DNA was then stained with DAPI (100 ng/ml). After a final wash in PBS for 10 min, the sections were mounted with Vectashield (Vector Laboratories).
For detection of 5hmC/5mC, sections were washed three times for 5 min with PBS. Post-fixed sections were first permeabilized for 30 min with 0.5% Triton X-100 (in 1% BSA/PBS), and subsequently treated with RNase A (10 mg/ml; Roche) in 1% BSA/PBS for 1 hour at 37° C. Following three 5 min washes with PBS, sections were incubated with 4N HCl for 10-20 min at 37° C. to denature genomic DNA, followed by three 10 min washes with PBS. After incubating for 30 min at room temperature in 1% BSA/PBS containing 0.1% Triton X-100, the sections were incubated with primary antibodies listed at 4° C. overnight in the same buffer. Sections were subsequently washed three times in 1% BSA/PBS containing 0.1% Triton X-100 for 5 min and incubated secondary antibodies in the same buffer for 1 hour in the dark at room temperature. Secondary antibody incubation was followed by three 5 min washes with PBS. DNA was then stained with propidium iodide (PI) (0.25 mg/ml). After a final wash in PBS for 10 min, the sections were mounted with Vectashield (Vector Laboratories).
The following primary antibodies were used in this study: anti-SSEA1 (gifted by Dr P. Beverly via Dr G. Durcova Hills); anti-MVH (Abcam 27591 or Abcam 13840); anti-5hmC (Active motif 39791), anti-5mC (Diagenode C15200081-100); anti-Tet1 (GeneTex GTX125888); anti-GFP (Abcam 5450). The following secondary antibodies were used in this study: Alexa Fluor 647 Goat anti-Mouse IgM (Invitrogen A21238); Alexa Fluor 488 Goat anti-Rabbit IgG (Invitrogen A11008); Alexa Fluor 405 Goat anti-Mouse IgG 1:300 (Invitrogen A31553); Alexa Fluor 488 Goat anti-Mouse IgG 1:300 (Invitrogen A11001); Alexa Fluor 405 Goat anti-Rabbit IgG 1:300 (Invitrogen A31556); Alexa Fluor 568 Donkey anti-Rabbit IgG (Invitrogen A10042); Alexa Fluor 488 Donkey anti-Goat IgG (Invitrogen A11055).
Locus-Specific Bisulphite Sequencing
Bisulphite treatment of genomic DNA was carried out using the Imprint DNA modification kit (Sigma). The following primers were used for the semi-nested amplification of the Dazl promoter: F1: GATTTTTGTTATTTTTTAGTTTTTTTAGGAT; F2: TTTATTTAAGTTATTATTTTAAAAATGGTATT; R: AGAAACAAGCTAGGCCAGCTGAGAGAATTCT. The following primers were used for the semi-nested amplification of the IG-DMR ICR: F1: GTGTTAAGGTATATTATGTTAGTGTTAGG; F2: ATATTATGTTAGTGTTAGGAAGGATTGTG; R: TACAACCCTTCCCTCACTCCAAAAATT. The following primers were used for the nested amplification of the Peg3 ICR: F1: TTTTTAGATTTTGTTTGGGGGTTTTTAATA; F2: TTGATAATAGTAGTTTGATTGGTAGGGTGT; R1: AATCCCTATCACCTAAATAACATCCCTACA; R2: ATCTACAACCTTATCAATTACCCTTAAAAA. Methylation levels were assessed by QUMA, using default settings with duplicate bisulphite sequences excluded.
Mass Spectrometry
Genomic DNA from between 100 and 2,000 FACS-sorted PGCs was extracted using ZR-Duet DNA/RNA Miniprep kit (Zymo Reasearch) following manufacturer instructions and eluted in LC/MS grade water. DNA was digested to nucleosides using a digestion enzyme mix provided by NEB. A dilution-series made with known amounts of synthetic nucleosides and the digested DNA were spiked with a similar amount of isotope-labelled nucleosides (provided by Dr T. Carell (LMU, Germany)) and separated on an Agilent RRHD Eclipse Plus C18 2.1×100 mm 1.8u column by using the UHPLC 1290 system (Agilent) and an Agilent 6490 triple quadrupole mass spectrometer. To calculate the quantity of individual nucleosides, standard curves representing the ratio of unlabelled over isotope-labelled nucleosides were generated and used to convert the peak-area values to corresponding quantity. Threshold for quantification is a signal-to-noise (calculated with a peak-to-peak method) above 10.
Western Blot
mESCs were lysed by sonication in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxychlorate, 0.1% sodium dodecylsulfate, 50 mM Tris pH 8.0) and protease-inhibitor cocktail (Roche, 11 697 498 001). Cell debris were removed by centrifugation at 14000 g 5 min 4° C. Protein was quantified using the BCA protein assay (Thermo, 23227). 2 μg (for H2A and H2Aub) or 20 μg (for Tet1) of each protein extract was loaded onto an 15% or 8% SDS polyacrylamide gel and transferred to a PVDF membrane after electrophoresis. Membranes were blocked with 5% BSA for 1 hour and then incubated overnight at 4° C. with primary antibodies at the following dilutions: anti-H2A antibody (Abcam, 18255) 1:2000; anti-ubiquityl H2A antibody (Cell Signalling 8240) 1:2000; anti-Tet1 antibody [N1] (GeneTex GTX125888) 1:1000; anti-Lamin B antibody (C20) (Santa Cruz Biotechnologies, sc-6216) 1:10000. Donkey anti-rabbit IgG-HRP (Santa Cruz Biotechnologies, sc-2077) or donkey anti-goat IgG-HRP (Santa Cruz Biotechnologies, sc-2056) secondary antibody were incubated for 1h at room temperature. Blots were developed by using Luminata Crescendo Western HRP substrate (EMD Milipore).
Alphabetised References:
All references referred to above are hereby incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1721724.1 | Dec 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/086642 | 12/21/2018 | WO | 00 |
