Sex-based disparities in DNA methylation and gene expression in late-gestation mouse placentas.

BACKGROUND: The placenta is vital for fetal development and its contributions to various developmental issues, such as pregnancy complications, fetal growth restriction, and maternal exposure, have been extensively studied in mice. The placenta forms mainly from fetal tissue and therefore has the same biological sex as the fetus it supports. Extensive research has delved into the placenta's involvement in pregnancy complications and future offspring development, with a notable emphasis on exploring sex-specific disparities. However, despite these investigations, sex-based disparities in epigenetic (e.g., DNA methylation) and transcriptomic features of the late-gestation mouse placenta remain largely unknown. METHODS: We collected male and female mouse placentas at late gestation (E18.5, n = 3/sex) and performed next-generation sequencing to identify genome-wide sex differences in transcription and DNA methylation. RESULTS: Our comparison between male and female revealed 358 differentially expressed genes (DEGs) on autosomes, which were associated with signaling pathways involved in transmembrane transport and the responses to viruses and external stimuli. X chromosome DEGs (n = 39) were associated with different pathways, including those regulating chromatin modification and small GTPase-mediated signal transduction. Differentially methylated regions (DMRs) were more common on the X chromosomes (n = 3756) than on autosomes (n = 1705). Interestingly, while most X chromosome DMRs had higher DNA methylation levels in female placentas and tended to be included in CpG dinucleotide-rich regions, 73% of autosomal DMRs had higher methylation levels in male placentas and were distant from CpG-rich regions. Several DEGs were correlated with DMRs. A subset of the DMRs present in late-stage placentas were already established in mid-gestation (E10.5) placentas (n = 348 DMRs on X chromosome and 19 DMRs on autosomes), while others were acquired later in placental development. CONCLUSION: Our study provides comprehensive lists of DEGs and DMRs between male and female that collectively cause profound differences in the DNA methylation and gene expression profiles of late-gestation mouse placentas. Our results demonstrate the importance of incorporating sex-specific analyses into epigenetic and transcription studies to enhance the accuracy and comprehensiveness of their conclusions and help address the significant knowledge gap regarding how sex differences influence placental function.

The placenta is a crucial organ for a healthy pregnancy and proper fetal development, and its functions are often studied in mice. The placenta stems from the developing embryo, and therefore shares its sex. Male fetuses have higher risks of pregnancy complications and neurodevelopmental disorders, and these risks are linked to placenta functions. However, how the placenta's sex influences the proteins it contains­and therefore, how it helps the fetus develop­remains largely unknown. We used cutting-edge techniques to systematically examine late-pregnancy mouse placentas, cataloging the genes being expressed (i.e., sections of DNA used to make proteins) and the patterns of a specific DNA mark (called methylation) that controls gene expression. We identified several genes with important placental functions, such as protecting the fetus from viruses and responding to environmental changes, whose expression levels were sex-specific. We also observed differences in DNA methylation between male and female placentas. Most DNA methylation differences were on the X chromosomes, and the majority had higher methylation levels in female placentas. Conversely, on other chromosomes, most differences present an increased level of DNA methylation in male placentas. As methylation affects gene expression, we found links between the changes. Additionally, we found that some sex differences in the placenta were already present earlier in pregnancy. Our findings provide important insights into the molecular differences between male and female mouse placentas during late pregnancy. Including sex-specific analyses in placenta studies will improve our understanding of how the placenta ensures the healthy development of male and female fetuses.

Mitigating the detrimental developmental impact of early fetal alcohol exposure using a maternal methyl donor-enriched diet.

Fetal alcohol exposure at any stage of pregnancy can lead to fetal alcohol spectrum disorder (FASD), a group of life-long conditions characterized by congenital malformations, as well as cognitive, behavioral, and emotional impairments. The teratogenic effects of alcohol have long been publicized; yet fetal alcohol exposure is one of the most common preventable causes of birth defects. Currently, alcohol abstinence during pregnancy is the best and only way to prevent FASD. However, alcohol consumption remains astoundingly prevalent among pregnant women; therefore, additional measures need to be made available to help protect the developing embryo before irreparable damage is done. Maternal nutritional interventions using methyl donors have been investigated as potential preventative measures to mitigate the adverse effects of fetal alcohol exposure. Here, we show that a single acute preimplantation (E2.5; 8-cell stage) fetal alcohol exposure (2 × 2.5 g/kg ethanol with a 2h interval) in mice leads to long-term FASD-like morphological phenotypes (e.g. growth restriction, brain malformations, skeletal delays) in late-gestation embryos (E18.5) and demonstrate that supplementing the maternal diet with a combination of four methyl donor nutrients, folic acid, choline, betaine, and vitamin B12, prior to conception and throughout gestation effectively reduces the incidence and severity of alcohol-induced morphological defects without altering DNA methylation status of imprinting control regions and regulation of associated imprinted genes. This study clearly supports that preimplantation embryos are vulnerable to the teratogenic effects of alcohol, emphasizes the dangers of maternal alcohol consumption during early gestation, and provides a potential proactive maternal nutritional intervention to minimize FASD progression, reinforcing the importance of adequate preconception and prenatal nutrition.

Alteration of the brain methylation landscape following postnatal inflammatory injury in rat pups.

Preterm infants are vulnerable to inflammation-induced white matter injury (WMI), which is associated with neurocognitive impairment and increased risk of neuropsychiatric diseases in adulthood. Epigenetic mechanisms, particularly DNA methylation, play a role in normal development and modulate the response to pathological challenges. Our aims were to determine how WMI triggered DNA methylation alterations in brains of neonatal rats and if such changes persisted over time. We used a robust model of WMI by injecting lipopolysaccharide (LPS) or sterile saline in the corpus callosum of 3-day-old (P3) rat pups. Brains were collected 24 hours (P4) and 21 days post-injection (P24). We extracted genomic DNA from the brain to establish genome-wide quantitative DNA methylation profiles using reduced representation bisulfite sequencing. Neonatal LPS exposure induced a persistent increased methylation of genes related to nervous system development and a reduced methylation of genes associated with inflammatory pathways. These findings suggest that early-life neuroinflammatory exposure impacts the cerebral methylation landscape with determining widespread epigenetic modifications especially in genes related to neurodevelopment.

ATAT1 regulates forebrain development and stress-induced tubulin hyperacetylation.

α-Tubulin acetyltransferase 1 (ATAT1) catalyzes acetylation of α-tubulin at lysine 40 in various organisms ranging from Tetrahymena to humans. Despite the importance in mammals suggested by studies of cultured cells, the mouse Atat1 gene is non-essential for survival, raising an intriguing question about its real functions in vivo. To address this question, we systematically analyzed a mouse strain lacking the gene. The analyses revealed that starting at postnatal day 5, the mutant mice display enlarged lateral ventricles in the forebrain, resembling ventricular dilation in human patients with ventriculomegaly. In the mice, ventricular dilation is due to hypoplasia in the septum and striatum. Behavioral tests of the mice uncovered deficits in motor coordination. Birth-dating experiments revealed that neuronal migration to the mutant septum and striatum is impaired during brain development. In the mutant embryonic fibroblasts, we found mild defects in cell proliferation and primary cilium formation. Notably, in these cells, ATAT1 is indispensable for tubulin hyperacetylation in response to high salt, high glucose, and hydrogen peroxide-induced oxidative stress. We investigated the role of ATAT1 in the hematopoietic system using multicolor flow cytometry and found that this system remains normal in the mutant mice. Although tubulin acetylation was undetectable in a majority of mutant tissues, residual levels were detected in the heart, skeletal muscle, trachea, oviduct, thymus and spleen. This study thus not only establishes the importance of ATAT1 in regulating mouse forebrain development and governing tubulin hyperacetylation during stress responses, but also suggests the existence of an additional α-tubulin acetyltransferase.

Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-ß Signaling and Epigenomics.

BACKGROUND & AIMS: A generalized human pacemaking syndrome, chronic atrial and intestinal dysrhythmia (CAID) (OMIM 616201), is caused by a homozygous SGO1 mutation (K23E), leading to chronic intestinal pseudo-obstruction and arrhythmias. Because CAID patients do not show phenotypes consistent with perturbation of known roles of SGO1, we hypothesized that noncanonical roles of SGO1 drive the clinical manifestations observed. METHODS: To identify a molecular signature for CAID syndrome, we achieved unbiased screens in cell lines and gut tissues from CAID patients vs wild-type controls. We performed RNA sequencing along with stable isotope labeling with amino acids in cell culture. In addition, we determined the genome-wide DNA methylation and chromatin accessibility signatures using reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing. Functional studies included patch-clamp, quantitation of transforming growth factor-ß (TGF-ß) signaling, and immunohistochemistry in CAID patient gut biopsy specimens. RESULTS: Proteome and transcriptome studies converge on cell-cycle regulation, cardiac conduction, and smooth muscle regulation as drivers of CAID syndrome. Specifically, the inward rectifier current, an important regulator of cellular function, was disrupted. Immunohistochemistry confirmed overexpression of Budding Uninhibited By Benzimidazoles 1 (BUB1) in patients, implicating the TGF-ß pathway in CAID pathogenesis. Canonical TGF-ß signaling was up-regulated and uncoupled from noncanonical signaling in CAID patients. Reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing experiments showed significant changes of chromatin states in CAID, pointing to epigenetic regulation as a possible pathologic mechanism. CONCLUSIONS: Our findings point to impaired inward rectifier potassium current, dysregulation of canonical TGF-ß signaling, and epigenetic regulation as potential drivers of intestinal and cardiac manifestations of CAID syndrome. Transcript profiling and genomics data are as follows: repository URL: https://www.ncbi.nlm.nih.gov/geo; SuperSeries GSE110612 was composed of the following subseries: GSE110309, GSE110576, and GSE110601.

Rapid Multiplexed Reduced Representation Bisulfite Sequencing Library Prep (rRRBS).

DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.

Pre-implantation alcohol exposure and developmental programming of FASD: an epigenetic perspective.

Exposure to alcohol during in-utero development can permanently change the developmental programming of physiological responses, thereby increasing the risk of neurological illnesses during childhood and later adverse health outcomes associated with fetal alcohol spectrum disorder (FASD). There is an increasing body of evidence indicating that exposure to alcohol during gestation triggers lasting epigenetic alterations in offspring, long after the initial insult; together, these studies support the role of epigenetics in FASD etiology. However, we still have little information about how ethanol interferes with the fundamental epigenetic reprogramming wave (e.g., erasure and re-establishment of DNA methylation marks) that characterizes pre-implantation embryo development. This review examines key epigenetic processes that occur during pre-implantation development and especially focus on the current knowledge regarding how prenatal exposure to alcohol during this period could affect the developmental programming of the early stage pre-implantation embryo. We will also outline the current limitations of studies examining the in-vivo and in-vitro effects of alcohol exposure on embryos and underline the next critical steps to be taken if we want to better understand the implicated mechanisms to strengthen the translational potential for epigenetic markers for non-invasive early detection, and the treatment of newborns that have higher risk of developing FASD.