Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance

The distribution of 5-methylcytosine (5-mC) in DNA within the eukaryotic genome is known to greatly affect gene regulation and is currently a major topic of research. Studies on DNA methylation have been aided by advancements in bisulfite conversion and Next-Gen sequencing technologies which can provide single-base resolution of 5-mC across the entire genome. Many whole-genome bisulfite sequencing (WGBS) library preparation protocols designed for analysis of 5-mC distribution employ bisulfite to chemically convert unmethylated cytosine bases into uracil following the library preparation step. While these protocols produce reliable results, degradation of DNA is always an inherent issue when dealing with bisulfite conversion, and a large proportion of the adapterized library is often fragmented and can no longer be amplified. This requires high levels of DNA to serve as the input material. However, by rearranging the order of library preparation and bisulfite conversion, we developed a streamlined protocol that necessitates less input material for accurate whole-genome methylation analysis at single-base resolution. This unique workflow accommodates picogram quantities of starting material, making it ideal for analysis of precious samples that are of limited availability (e.g., FFPE). Comparison of sequencing data generated using this new library preparation method and data generated with established Reduced Representation Bisulfite Sequencing demonstrated a correlation of 0.95 in CpG sites with > 10X coverage in human DNA. Also, with only slight modification, this protocol is versatile enough to be used in the preparation of libraries for ChIP-Seq and RNA-Seq analyses. Citation Format: Karolyn Giang, TzuHung Chung, Xueguang Sun, Marc E. Van Eden, Xi Yu Jia. A fast and simple method for whole-genome bisulfite library preparation from ultra-low DNA inputs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2014-2293

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

Effects of ERα-specific antagonist on mouse preimplantation embryo development and zygotic genome activation

Transcription and development of intranuclear bodies and particles during the 2-cell stage in cattle

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor β (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Spermatozoa have been shown to carry key RNAs which, according to animal evidence, seem to play a role in early embryo development. In this context, a potential key growth regulator is insulin-like growth factor 2 (IGF2), a highly conserved paternally expressed imprinted gene involved in cell growth and proliferation which, recent observations indicate, is expressed in human spermatozoa. We herein hypothesized that sperm IGF2 gene expression and transmission at fertilization is required to support early embryo development. To test this hypothesis, we analyzed sperm IGF2 mRNA levels in the same semen aliquot used for homologous assisted reproductive technique (ART) in infertile couples and correlated these levels with embryo morphokinetics. To find a mechanistic explanation for the observed results, the transcriptomes of blastocysts obtained after injection of Igf2 mRNA in mouse parthenotes were analyzed. Sperm IGF2 mRNA negatively correlated with time of 2-cell stage (t2), t3, t4, t5, and time of expanded blastocyst (tEB), independently of maternal age, body mass index, anti-Müllerian hormone levels, and oocyte quality. An IGF2 mRNA index >4.9 predicted the ability of the embryos to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; P < 0.001). In the animal study, transcriptome analysis demonstrated that 65 and 36 genes were, respectively, up- and down-regulated in the experimental group compared to the control group. These genes belong to pathways that regulate early embryo development, thus supporting the findings found in humans. This study has the potential to challenge the longstanding tenet that spermatozoa are simply vehicles carrying paternal DNA. Instead, it suggests that IGF2 mRNA in healthy spermatozoa provides critical support for early embryo development. Pre-ART sperm-carried IGF2 mRNA levels may be used as a marker to predict the chances of obtaining blastocysts to be transferred for infertile couples undergoing ART.

Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10-5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases. Trial registration number not applicable

Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10–5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings: Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases Trial registration number Not applicable

Bisulfite modification of cytosine residues is a widely used method for analyzing genomic DNA methylation. Despite its robustness, degradation of DNA during modification has hampered the application to the analysis of small amounts of DNA. We show that the addition of large volume bisulfite-treated heterogeneous DNA promotes the amplification of the targeted region in bisulfite-treated DNA by PCR. The addition of untreated DNA did not promote the amplification. The addition of large volume bisulfite-treated heterogeneous DNA neither promoted the amplification when untreated DNA was used as template. PCR products were detected when a ten thousandth of aliquot of bisulfite-treated 1 μg of human genomic DNA (0.1 ng) was used as template. This figure is equivalent to that of human genomic DNA derived from as little as 15 cells. This procedure permits the analysis of genomic DNA methylation when only limited numbers of cells are available.

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

BackgroundTransposable Elements (TE) are mobile sequences that make up large portions of eukaryote genomes. The functions they play within the complex cellular architecture are still not clearly understood, but it is becoming evident that TE have a role in several physiological and pathological processes. In particular, it has been shown that TE transcription is necessary for the correct development of mice embryos and that their expression is able to finely modulate transcription of coding and non-coding genes. Moreover, their activity in the central nervous system (CNS) and other tissues has been correlated with the creation of somatic mosaicisms and with pathologies such as neurodevelopmental and neurodegenerative diseases as well as cancers.ResultsWe analyzed TE expression among different cell types of the Caenorhabditis elegans (C. elegans) early embryo asking if, where and when TE are expressed and whether their expression is correlated with genes playing a role in early embryo development. To answer these questions, we took advantage of a public C. elegans embryonic single-cell RNA-seq (sc-RNAseq) dataset and developed a bioinformatics pipeline able to quantify reads mapping specifically against TE, avoiding counting reads mapping on TE fragments embedded in coding/non-coding transcripts. Our results suggest that i) canonical TE expression analysis tools, which do not discard reads mapping on TE fragments embedded in annotated transcripts, may over-estimate TE expression levels, ii) Long Terminal Repeats (LTR) elements are mostly expressed in undifferentiated cells and might play a role in pluripotency maintenance and activation of the innate immune response, iii) non-LTR are expressed in differentiated cells, in particular in neurons and nervous system-associated tissues, and iv) DNA TE are homogenously expressed throughout the C. elegans early embryo development.ConclusionsTE expression appears finely modulated in the C. elegans early embryo and different TE classes are expressed in different cell types and stages, suggesting that TE might play diverse functions during early embryo development.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.