Khursheed Iqbal

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine.

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

BackgroundExposure to environmental endocrine-disrupting chemicals during pregnancy reportedly causes transgenerationally inherited reproductive defects. We hypothesized that to affect the grandchild, endocrine-disrupting chemicals must alter the epigenome of the germ cells of the in utero-exposed G1 male fetus. Additionally, to affect the great-grandchild, the aberration must persist in the germ cells of the unexposed G2 grandchild.ResultsHere, we treat gestating female mice with vinclozolin, bisphenol A, or di-(2-ethylhexyl)phthalate during the time when global de novo DNA methylation and imprint establishment occurs in the germ cells of the G1 male fetus. We map genome-wide features in purified G1 and G2 prospermatogonia, in order to detect immediate and persistent epigenetic aberrations, respectively. We detect changes in transcription and methylation in the G1 germline immediately after endocrine-disrupting chemicals exposure, but changes do not persist into the G2 germline. Additional analysis of genomic imprints shows no persistent aberrations in DNA methylation at the differentially methylated regions of imprinted genes between the G1 and G2 prospermatogonia, or in the allele-specific transcription of imprinted genes between the G2 and G3 soma.ConclusionsOur results suggest that endocrine-disrupting chemicals exert direct epigenetic effects in exposed fetal germ cells, which are corrected by reprogramming events in the next generation. Avoiding transgenerational inheritance of environmentally-caused epigenetic aberrations may have played an evolutionary role in the development of dual waves of global epigenome reprogramming in mammals.

Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.

Effects of endocrine disruptors on imprinted gene expression in the mouse embryo

Environmental endocrine disruptors (EDs) are synthetic chemicals that resemble natural hormones and are known to cause epigenetic perturbations. EDs have profound effects on development and fertility. Imprinted genes had been identified as susceptible loci to environmental insults by EDs because they are functionally haploid, and because the imprints undergo epigenetic resetting between generations. To screen for possible epigenetic perturbations caused by EDs at imprinted loci, we treated pregnant mice daily between 8.5 and 12.5 days post coitum (dpc) with di-(2-ethylhexyl)-phthalate (DEHP), bisphenol A (BPA), vinclozolin (VZ), or control oil vehicle. After isolating RNA from the placenta, yolk sac, amnion, head, body, heart, liver, lung, stomach, and intestines of 13.5 dpc embryos we measured the allele-specific expression of 38 imprinted transcripts using multiplex single nucleotide primer extension (SNuPE) assays. In this representative data set we identified only a small number of transcripts that exhibited a substantial relaxation of imprinted expression with statistical significance: Slc22a18 with 10% relaxation in the embryo after BPA treatment; Rtl1as with 11 and 16% relaxation in the lung and placenta, respectively after BPA treatment; and Rtl1 with 12% relaxation in the yolk sac after DEHP treatment. Additionally, the standard deviation of allele-specificity increased in various organs after ED treatment for several transcripts including Igf2r, Rasgrf1, Usp29, Slc38a4, and Xist. Our data suggest that the maintenance of strongly biased monoallelic expression of imprinted genes is generally insensitive to EDs in the 13.5 dpc embryo and extra-embryonic organs, but is not immune to those effects.

Species-Specific Telomere Length Differences Between Blastocyst Cell Compartments and Ectopic Telomere Extension in Early Bovine Embryos by Human Telomerase Reverse Transcriptase

The enzyme telomerase is active in germ cells and is critically involved in maintenance of telomere length in successive generations. In preimplantation mammalian embryos, telomerase activity is present from the morula stage onward and is associated with an increase in telomere length in blastocysts. Herein, we show that telomere length regulation in murine and bovine blastocysts differed between trophectodermal and inner cell mass cells in a species-specific manner. Ectopic expression of human telomerase reverse transcriptase (TERT) in bovine embryos increased telomerase activity and in turn increased telomere length. Transient expression of human TERT could be targeted to the 4-cell to morula stages and to the morula to blastocyst stages using unmodified and cytosine-methylated expression plasmids, respectively. Introduction of human TERT constructs in bovine embryos resulted in functional telomerase expression and effective telomere elongation, allowing us to study the effects on embryonic development. Ultimately, these studies may lead to a large-animal model for telomere regulation and aging.

RNA-Seq Transcriptome Profiling of Equine Inner Cell Mass and Trophectoderm

ABSTRACT Formation of the inner cell mass (ICM) and trophectoderm (TE) marks the first differentiation event in mammalian development. These two cell types have completely divergent fates for the remainder of the developmental process. The molecular mechanisms that regulate ICM and TE formation are poorly characterized in horses. The objective of this study was to establish the transcriptome profiles of ICM and TE cells from horse blastocysts using RNA sequencing (RNA-seq). A total of 12 270 genes were found to be expressed in either lineage. Global analysis of the transcriptome profiles by unsupervised clustering indicated that ICM and TE samples presented different gene expression patterns. Statistical analysis indicated that 1662 genes were differentially expressed (adjusted P < 0.05 and fold change > 2) between ICM and TE. Genes known to be specific to the ICM and TE were expressed primarily in their respective tissue. Transcript abundance for genes related to biological processes important for horse blastocyst formation and function is presented and discussed. Collectively, our data and analysis serve as a valuable resource for gene discovery and unraveling the fundamental mechanisms of early horse development.

High type I error and misrepresentations in search for transgenerational epigenetic inheritance: response to Guerrero-Bosagna.

In a recent paper, we described our efforts in search for evidence supporting epigenetic transgenerational inheritance caused by endocrine disrupter chemicals. One aspect of our study was to compare genome-wide DNA methylation changes in the vinclozolin-exposed fetal male germ cells (n = 3) to control samples (n = 3), their counterparts in the next, unexposed, generation (n = 3 + 3) and also in adult spermatozoa (n = 2 + 2) in both generations. We reported finding zero common hits in the intersection of these four comparisons. In our interpretation, this result did not support the notion that DNA methylation provides a mechanism for a vinclozolin-induced transgenerational male infertility phenotype. In response to criticism by Guerrero-Bosagna regarding our statistical power in the above study, here we provide power calculations to clarify the statistical power of our study and to show the validity of our conclusions. We also explain here how our data is misinterpreted in the commentary by Guerrero-Bosagna by leaving out important data points from consideration.Please see related Correspondence article: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0982-4 and related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0619-z

147 GENE KNOCKDOWN OF MATERNAL- AND EMBRYONIC-EXPRESSED GREEN FLUORESCENT PROTEIN IN MURINE EMBRYOS BY THE INJECTION OF A SHORT INTERFERING RNA (siRNA)

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. Table 1.siRNA microinjection into zygotes with maternally and paternally inherited GFP