Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

AbstractThe imprinted genes of primate embryonic stem cells (ESCs) often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs), SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates

Molecular and functional features demarcate embryonic and induced pluripotent stem cells in rabbits

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Progressive establishment of differential methylation on POU5F1 upstream sequence over blastocyst development

International audienceThe POU5F1 gene encodes one of the "core" transcription factors necessary to maintain pluripotency but very few data are available concerning the precise epigenetic regulation of its expression in early embryo. We analysed the progressive modifications of DNA methylation of POU5F1 upstream region in the different compartments of the developing blastocyst. Therefore we used the rabbit embryo as a model for most mammalian blastocysts, where contrarily to the mouse, epiblast differentiates in a plane embryonic disk at the surface of the conceptus and in direct contact with maternal environment. Embryos were micro dissected at Day4,5, and6. POU5F1 expression was quantified by RTqPCR. Methylation profile of four regions encompassing conserved cis-elements (Kobolak et al. 2009 BMC Mol. Biol.10 88.) was determined by bisulfite treatment, cloning and sequencing. POU5F1 is highly expressed in Day4 trophectoderm but significantly enriched in the inner cell mass. At Day5, the POU5F1 relative expression decreased but the difference between embryonic and trophectoderm compartments tends to increase. At Day6, POU5F1 transcripts were restricted to the epiblast, with a very reduced expression in the hypoblast. Noticeably POU5F1 expression progressively decreases in the trophectoderm/trophoblast compartment over the Day4-Day6 period. The four regions were hypomethylated in pluripotent Inner Cell Mass and epiblast. Differences in DNA methylation between pluripotent and differentiated layers occurred as early as Day4 and progressively increased. A principal component analysis segregated pluripotent and highly differentiated samples and identified the CpGs mostly involved in this separation. Interestingly while differentiation-related changes in methylation occurred in the four analyzed regions, theyare more pronounced in the region encompassing the proximal enhancer 1A

Methylation profile of KCNQ1OT1 gene in in vitro maturated human oocytes and non growing embryos

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Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the VL_L domain in estradiol recognition

International audienceThe high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K$_a$ = 1.2 Â 10$^9$ M$^{-1}$) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6-and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the Aring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17b-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with aor b-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V$_H$ and V$_L$) and scFv hybrids have been constructed. The binding activity of V$_L$ 9D3 as well as the specificity of the V$_L$ 9D3/V$_H$ 15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V$_L$ in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates

Investigation of DNA methylation role periconceptional programming in early stage embryos from rabbit diabetic pregnancy model

International audienceDNA methylation is a conservative epigenetic marker. A correct DNA methylation pattern is essential for embryonic development. Mammalian embryos gain a complete de novo DNA methylation design around implantation. DNA methylation is a potential mechanism of periconceptional programming, suggesting that the DNA methylation pattern of developing embryo might be affected in distinct ways, depending on nutritional and hormonal signals. To expand this idea we have investigated the DNA methylation pattern of the promoter region of the POU5F1 (Oct4) pluripotency gene in preimplantation rabbit embryos at day 6 p.c (early gastrulation stage). The blastocysts were collected from healthy and diabetic rabbits. The POU5F1 promoter contains four conservative regions, which are embracing important elements for its transcription: proximal and distal enhancers (PE-1A, PE-1B, DE-1A), a SOX2/Oct4 binding site and hormone responding element (HRE). We have characterized the CpG islands methylation at all mentioned regions in two embryonic tissues: embryoblast and trophoblast, using bisulfite treatment, cloning and sequencing. The analysis has been successfully performed for the trophoblast tissue yet. A higher methylation of the CpG islands for HRE and at the beginning of first exon has been noticed in the trophoblast of diabetic embryos. A hypomethylation in trophoblast from diabetic embryos comparing to trophoblasts from healthy pregnancies was visible for proximal enhancer 1A (PE-1A) in frame of conservative region 3 (CR3). In this context, DNA methylation can be considered as a form of embryo developmental plasticity, which can promote metabolic disorders in adult life