Serge McGraw

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

Generations affected by histone changes Parent and even grandparent environmental exposure can transmit adverse health effects to offspring. The mechanism of transmission is unclear, but some studies have implicated variations in DNA methylation. In a mouse model, Siklenka et al. found that alterations in histone methylation during sperm formation in one generation leads to reduced survival and developmental abnormalities in three subsequent generations (see the Perspective by McCarrey). Although changes in DNA methylation were not observed, altered sperm RNA content and abnormal gene expression in offspring were measured. Thus, chromatin may act as a mediator of molecular memory in transgenerational inheritance. Science, this issue p. 10.1126/science.aab2006; see also p. 634 Overexpression of a histone demethylase in the mouse germ line reveals a mode of transgenerational epigenetic inheritance. [Also see Perspective by McCarrey] INTRODUCTION Despite the father transmitting half of the heritable information to the embryo, the focus of preconception health has been the mother. Paternal effects have been linked to complex diseases such as cancer, diabetes, and obesity. These diseases are increasing in prevalence at rates that cannot be explained by genetics alone and highlight the potential for disease transmission via nongenetic inheritance, through epigenetic mechanisms. Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, and noncoding RNA. Studies in humans and animals suggest that epigenetic mechanisms may serve in the transmission of environmentally induced phenotypic traits from the father to the offspring. Such traits have been associated with altered gene expression and tissue function in first and second offspring generations, a phenomenon known as intergenerational or transgenerational inheritance, respectively. The mechanisms underlying such paternal epigenetic transmission are unclear. RATIONALE Sperm formation involves rapid cell division and distinctive transcription programs, resulting in a motile cell with highly condensed chromatin. Within the highly compacted sperm nucleus, few histones are retained in a manner that suggests an influential role in development. Despite being the major focus of studies in epigenetic inheritance, the role of DNA methylation in paternal epigenetic inheritance is unresolved, as only minor changes in DNA methylation in sperm at CpG-enriched regions have been associated with transmission of environmentally induced traits. Instead, there may be a combination of molecular mechanisms underlying paternal transgenerational epigenetic inheritance involving changes in histone states and/or RNA in sperm. The function of sperm histones and their modifications in embryonic development, offspring health, and epigenetic inheritance is unknown. By overexpressing the human KDM1A histone lysine 4 demethylase during mouse spermatogenesis, we generated a mouse model producing spermatozoa with reduced H3K4me2 within the CpG islands of genes implicated in development, and we studied the development and fitness of the offspring. RESULTS Male transgenic offspring were bred with C57BL/6 females, generating the experimental heterozygous transgenic (TG) and nontransgenic (nonTG) brothers. Each generation from TG and nonTG animals (F1 to F3 in our transgenerational studies) was bred with C57BL/6 females, and the offspring (pups from generations F1 to F4) were analyzed for intergenerational and transgenerational effects. We found that KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of KDM1A germline expression. We characterized histone and DNA methylation states in the sperm of TG and nonTG sires. Overexpression of KDM1A was associated with a specific loss of H3K4me2 at more than 2300 genes, including many developmental regulatory genes. Unlike in other examples of paternal transgenerational inheritance, we observed no changes in sperm DNA methylation associated with primarily CpG-enriched regions. Instead, we measured robust and analogous changes in sperm RNA content of TG and nonTG descendants, as well as in their offspring, at the two-cell stage. These changes in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at genes in sperm. This study demonstrates that KDM1A activity during sperm development has major developmental consequences for offspring and implicates histone methylation and sperm RNA as potential mediators of transgenerational inheritance. Our data emphasize the complexity of transgenerational epigenetic inheritance likely involving multiple molecular factors, including the establishment of chromatin states in spermatogenesis and sperm-borne RNA. CONCLUSION Correct histone methylation during spermatogenesis is critical for offspring development and survival over multiple generations. These findings demonstrate the potential of histone methylation as a molecular mechanism underlying paternal epigenetic inheritance. Its modification by environmental influences may alter embryo development and complex disease transmission across generations. An essential next step is to establish functional links between environmental exposures, the composition of the sperm epigenome, and consequent altered gene expression and metabolic processes in offspring. Considering the mounting evidence, it may soon be reasonable to suggest that future fathers protect their sperm epigenome. Disruption of histone methylation in developing sperm by exposure to the KDM1A transgene in one generation severely impaired development and survivability of offspring. These defects were transgenerational and occurred in nonTG descendants in the absence of KDM1A germline expression. Developmental defects in offspring and embryos were associated with altered RNA expression in sperm and embryos. A father’s lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

Transcription Factor Expression Patterns in Bovine In Vitro-Derived Embryos Prior to Maternal-Zygotic Transition

Abstract Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

Unveiling the bovine embryo transcriptome during the maternal-to-embryonic transition.

Bovine early embryos are transcriptionally inactive and subsist through the initial developmental stages by the consumption of the maternal supplies provided by the oocyte until its own genome activation. In bovine, the activation of transcription occurs during the 8- to 16-cell stages and is associated with a phase called the maternal-to-embryonic transition (MET) where maternal mRNA are replaced by embryonic ones. Although the importance of the MET is well accepted, since its inhibition blocks embryonic development, very little is known about the transcripts expressed at this crucial step in embryogenesis. In this study, we generated and characterized a cDNA library enriched in embryonic transcripts expressed at the MET in bovine. Suppression subtractive hybridization followed by microarray hybridization was used to isolate more than 300 different transcripts overexpressed in untreated late eight-cell embryos compared with those treated with the transcriptional inhibitor, alpha-amanitin. Validation by quantitative RT-PCR of 15 genes from this library revealed that they had remarkable consistency with the microarray data. The transcripts isolated in this cDNA library have an interesting composition in terms of molecular functions; the majority is involved in gene transcription, RNA processing, or protein biosynthesis, and some are potentially involved in the maintenance of pluripotency observed in embryos. This collection of genes associated with the MET is a novel and potent tool that will be helpful in the understanding of particular events such as the reprogramming of somatic cells by nuclear transfer or for the improvement of embryonic culture conditions.

Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations

Significance Our study shows that the clotting protein tissue factor (TF) controls the state of tumor dormancy and does so in conjunction with recruitment of inflammatory cells and blood vessels. We show that indolent glioma cells remain harmless in mice unless rendered TF positive. Our work also demonstrates the ability of TF to indirectly influence the DNA of cancer cells by facilitating gene mutations and silencing. This ability is important because injury, cardiovascular disease, or other conditions may activate the clotting system and contribute to the awakening of occult cancer cells. This understanding also may suggest a prophylactic use of blood thinners in cases where dormant cancer cells and clotting are suspected to coexist (e.g., after surgery). The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105+) and myeloid (CD11b+ and F4/80+) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat−/− mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.

Modulation of imprinted gene expression following superovulation.

Although assisted reproductive technologies increase the risk of low birth weight and genomic imprinting disorders, the precise underlying causes remain unclear. Using a mouse model, we previously showed that superovulation alters the expression of imprinted genes in the placenta at 9.5days (E9.5) of gestation. Here, we investigate whether effects of superovulation on genomic imprinting persisted at later stages of development and assess the surviving fetuses for growth and morphological abnormalities. Superovulation, followed by embryo transfer at E3.5, as compared to spontaneous ovulation (controls), resulted in embryos of normal size and weight at 14.5 and 18.5days of gestation. The normal monoallelic expression of the imprinted genes H19, Snrpn and Kcnq1ot1 was unaffected in either the placentae or the embryos from the superovulated females at E14.5 or E18.5. However, for the paternally expressed imprinted gene Igf2, superovulation generated placentae with reduced production of the mature protein at E9.5 and significantly more variable mRNA levels at E14.5. We propose that superovulation results in the ovulation of abnormal oocytes with altered expression of imprinted genes, but that the coregulated genes of the imprinted gene network result in modulated expression.

Transient DNMT1 suppression reveals hidden heritable marks in the genome

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Spatiotemporal expression of transcriptional regulators in concert with the maternal-to-embryonic transition during bovine in vitro embryogenesis

Cleavage-stage bovine embryos are transcriptionally quiescent until they reach the 8- to 16-cell stage, and thus rely on the reserves provided by the stored maternal mRNAs and proteins found in the oocytes to achieve their first cell divisions. The objective of this study was to characterize the expression and localization of the transcriptional and translational regulators, Y box binding protein 2 (YBX2), TATA box-binding protein (TBP), and activating transcription factor 2 (ATF2), during bovine early embryo development. Germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, as well as 2-, 4-, 8-, 16-cell-stage embryos, morula, and blastocysts, produced in vitro were analyzed for temporal and spatial protein expression. Using Q-PCR, ATF2 mRNA expression was shown to remain constant from the GV-stage oocyte to the four-cell embryo, and then decreased through to the blastocyst stage. By contrast, the protein levels of ATF2 remained constant throughout embryo development and were found in both the cytoplasm and the nucleus. Both TBP and YBX2 showed opposite protein expression patterns, as YBX2 protein levels decreased throughout development, while TBP levels increased through to the blastocyst stage. Immunolocalization studies revealed that TBP protein was localized in the nucleus of 8- to 16-cell-stage embryos, whereas the translational regulator YBX2 was exclusively cytoplasmic and disappeared from the 16-cell stage onward. This study shows that YBX2, TBP, and ATF2 are differentially regulated through embryo development, and provides insight into the molecular events occurring during the activation of the bovine genome during embryo development in vitro.

The Dnmt1 Intrinsically Disordered Domain Regulates Genomic Methylation During Development

The DNMT1 cytosine methyltransferase enzyme contains a large ∼300-aa intrinsically disordered domain (IDD) that we previously showed regulated DNA methylation patterns in mouse ES cells. Here we generated seven mouse lines with different mutations in the IDD. Homozygous mutant mice of five lines developed normally, with normal levels of methylation on both imprinted and nonimprinted DNA sequences. The other two lines, however, had alterations in imprinted and/or nonimprinted (global) DNA methylation appearing during embryonic development. Embryos of one line expressing a DNMT1 variant containing a 6-aa rat orthologous sequence in the IDD maintained imprinted methylation, showed very reduced levels of global methylation and occasionally completed fetal development. These in vivo studies demonstrate that at least two DNMT1-dependent methylation processes can be distinguished during fetal development. One process maintains the bulk of genomic methylation on nonimprinted sequences. The other process maintains methylation on a much smaller class of sequences including but not limited to gametic differentially methylated domains (gDMDs) that transmit essential imprinted parent-specific methylation for embryonic development.

Stability of DNA Methylation Patterns in Mouse Spermatogonia Under Conditions of MTHFR Deficiency and Methionine Supplementation

ABSTRACT Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr+/−) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr+/− as compared to wild-type, Mthfr+/+ mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr+/+ and Mthfr+/− spermatogonia. Using spermatogonia from Mthfr+/+ and Mthfr+/− mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr+/− cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr+/+ cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.