Epigenetic State Research Articles

Genome-wide DNA methylation status of Mongolians exhibits signs of cellular stress response related to their nomadic lifestyle

BackgroundEpigenetics is crucial for connecting environmental stresses with physiological responses in humans. Mongolia, where nomadic livestock pastoralism has been the primal livelihood, has a higher prevalence of various chronic diseases than the surrounding East Asian regions, which are more suitable for crop farming. The genes related to dietary stress and pathogenesis of related disorders may have varying epigenetic statuses among the human populations with diverse dietary cultures. Hence, to understand such epigenetic differences, we conducted a comparative analysis of genome-wide DNA methylation of Mongolians and crop-farming East Asians.MethodsGenome-wide DNA methylation status of peripheral blood cells (PBCs) from 23 Mongolian adults and 24 Thai adults was determined using the Infinium Human Methylation 450K arrays and analyzed in combination with previously published 450K data of 20 Japanese and 8 Chinese adults. CpG sites/regions differentially methylated between Mongolians and crop-farming East Asians were detected using a linear model adjusted for sex, age, ethnicity, and immune cell heterogeneity on RnBeads software.ResultsOf the quality-controlled 389,454 autosomal CpG sites, 223 CpG sites were significantly differentially methylated among Mongolians and the four crop farming East Asian populations (false discovery rate < 0.05). Analyses focused on gene promoter regions revealed that PM20D1 (peptidase M20 domain containing 1), which is involved in mitochondrial uncoupling and various processes, including cellular protection from reactive oxygen species (ROS) and thermogenesis, was the top differentially methylated gene. Moreover, gene ontology enrichment analysis revealed that biological processes related to ROS metabolism were overrepresented among the top 1% differentially methylated genes. The promoter regions of these genes were generally hypermethylated in Mongolians, suggesting that the metabolic pathway detoxifying ROS might be globally suppressed in Mongolians, resulting in the high susceptibility of this population to various chronic diseases.ConclusionsThis study showed a significantly diverse DNA methylation status among Mongolians and crop-farming East Asians. Further, we found an association between the differentially methylated genes and various metabolic and neurodegenerative diseases. Knowledge of the epigenetic regulators might help in proper understanding, treatment, and control of such disorders, and physiological adaptation in the future.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

Chronic stress-driven glucocorticoid receptor activation programs key cell phenotypes and functional epigenomic patterns in human fibroblasts

SummaryChronic environmental stress can profoundly impact cell and body function. Although the underlying mechanisms are poorly understood, epigenetics has emerged as a key link between environment and health. The genomic effects of stress are thought to be mediated by the action of glucocorticoid stress hormones, primarily cortisol in humans, which act via the glucocorticoid receptor (GR). To dissect how chronic stress-driven GR activation influences epigenetic and cell states, human fibroblasts underwent prolonged exposure to physiological stress levels of cortisol and/or a selective GR antagonist. Cortisol was found to drive robust changes in cell proliferation, migration, and morphology, which were abrogated by concomitant GR blockade. The GR-driven cell phenotypes were accompanied by widespread, yet genomic context-dependent, changes in DNA methylation and mRNA expression, including gene loci with known roles in cell proliferation and migration. These findings provide insights into how chronic stress-driven functional epigenomic patterns become established to shape key cell phenotypes.

Characterizing the variation in chromosome structure ensembles in the context of the nuclear microenvironment.

Inside the nucleus, chromosomes are subjected to direct physical interaction between different components, active forces, and thermal noise, leading to the formation of an ensemble of three-dimensional structures. However, it is still not well understood to what extent and how the structural ensemble varies from one chromosome region or cell-type to another. We designed a statistical analysis technique and applied it to single-cell chromosome imaging data to reveal the heterogeneity of individual chromosome structures. By analyzing the resulting structural landscape, we find that the largest dynamic variation is the overall radius of gyration of the chromatin region, followed by domain reorganization within the region. By comparing different human cell-lines and experimental perturbation data using this statistical analysis technique and a network-based similarity quantification approach, we identify both cell-type and condition-specific features of the structural landscapes. We identify a relationship between epigenetic state and the properties of chromosome structure fluctuation and validate this relationship through polymer simulations. Overall, our study suggests that the types of variation in a chromosome structure ensemble are cell-type as well as region-specific and can be attributed to constraints placed on the structure by factors such as variation in epigenetic state.

Single-cell multiomics sequencing reveals the reprogramming defects in embryos generated by round spermatid injection.

Round spermatid injection (ROSI) technique holds great promise for clinical treatment of a proportion of infertile men. However, the compromised developmental potential of ROSI embryos largely limits the clinical application, and the mechanisms are not fully understood. Here, we describe the transcriptome, chromatin accessibility, and DNA methylation landscapes of mouse ROSI embryos derived from early-stage round spermatids using a single-cell multiomics sequencing approach. By interrogating these data, we identify the reprogramming defects in ROSI embryos at the pronuclear stages, which are mainly associated with the misexpression of a cohort of minor zygotic genome activation genes. We screen a small compound, A366, that can significantly increase the developmental potential of ROSI embryos, in which A366 can partially overcome the reprogramming defects by amending the epigenetic and transcriptomic states. Collectively, our study uncovers the reprogramming defects in ROSI embryos for understanding the mechanisms underlying compromised developmental potential and offers an avenue for ROSI technique optimization.

Polydopamine-Mediated Protein Adsorption Alters the Epigenetic Status and Differentiation of Primary Human Adipose-Derived Stem Cells (hASCs).

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and β1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Transient nuclear deformation primes epigenetic state and promotes cell reprogramming.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering.

The epigenetic state of EED-Gli3-Gli1 regulatory axis controls embryonic cortical neurogenesis.

SummaryMutations in the embryonic ectoderm development (EED) cause Weaver syndrome, but whether and how EED affects embryonic brain development remains elusive. Here, we generated a mouse model in which Eed was deleted in the forebrain to investigate the role of EED. We found that deletion of Eed decreased the number of upper-layer neurons but not deeper-layer neurons starting at E16.5. Transcriptomic and genomic occupancy analyses revealed that the epigenetic states of a group of cortical neurogenesis-related genes were altered in Eed knockout forebrains, followed by a decrease of H3K27me3 and an increase of H3K27ac marks within the promoter regions. The switching of H3K27me3 to H3K27ac modification promoted the recruitment of RNA-Pol2, thereby enhancing its expression level. The small molecule activator SAG or Ptch1 knockout for activating Hedgehog signaling can partially rescue aberrant cortical neurogenesis. Taken together, we proposed a novel EED-Gli3-Gli1 regulatory axis that is critical for embryonic brain development.

Therapeutic Potential of Human Microbiome-Based Short-Chain Fatty Acids and Bile Acids in Liver Disease

Microbiome-derived short chain fatty acids (SCFAs: acetate, propionate, and butyrate) and bile acids (BAs: primary BAs and secondary BAs) widely influence liver metabolic inflammation, immune responses, and carcinogenesis. In recent literature, the role of SCFAs and BAs in various liver diseases has been discussed. SCFAs and BAs are two types of microbiome-derived metabolites and they have been shown to have immunoregulatory ability in autoimmunity, inflammation, and liver-cancer microcellular environments. SCFAs and BAs are dependent on dietary components. The numerous regulatory processes in lymphocytes and non-immune cells that underpin both the positive and harmful effects of microbial metabolites include variations in metabolic signaling and epigenetic states. As a result, histone deacetylase (HDAC) inhibitors, SCFAs, and BAs, which are powerful immunometabolism modulators, have been explored. BAs have also been shown to alter the microbiome as well as adaptive and innate immune systems. We therefore emphasize the important metabolites in liver disease for clinical therapeutic applications. A deep understanding of SCFAs and Bas, as well as their molecular risk, could reveal more about certain liver-disease conditions.

The Variable CTCF Site from Drosophila melanogasterUbx Gene is Redundant and Has no Insulator Activity

CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin. One such variable CTCF site (vCTCF) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx.

Engagement of Foxh1in chromatin regulation revealed by protein interactome analyses.

Early embryonic cell fates are specified through coordinated integration of transcription factor activities and epigenetic states of the genome. Foxh1 is a key maternal transcription factor controlling the mesendodermal gene regulatory program. Proteomic interactome analyses using FOXH1 as a bait in mouse embryonic stem cells revealed that FOXH1 interacts with PRC2 subunits and HDAC1. Foxh1 physically interacts with Hdac1, and confers transcriptional repression of mesendodermal genes in Xenopus ectoderm. Our findings reveal a central role of Foxh1 in coordinating the chromatin states of the Xenopus embryonic genome.

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice.

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

Balanced gene dosage control rather than parental origin underpins genomic imprinting

Mammalian parental imprinting represents an exquisite form of epigenetic control regulating the parent-specific monoallelic expression of genes in clusters. While imprinting perturbations are widely associated with developmental abnormalities, the intricate regional interplay between imprinted genes makes interpreting the contribution of gene dosage effects to phenotypes a challenging task. Using mouse models with distinct deletions in an intergenic region controlling imprinting across the Dlk1-Dio3 domain, we link changes in genetic and epigenetic states to allelic-expression and phenotypic outcome in vivo. This determined how hierarchical interactions between regulatory elements orchestrate robust parent-specific expression, with implications for non-imprinted gene regulation. Strikingly, flipping imprinting on the parental chromosomes by crossing genotypes of complete and partial intergenic element deletions rescues the lethality of each deletion on its own. Our work indicates that parental origin of an epigenetic state is irrelevant as long as appropriate balanced gene expression is established and maintained at imprinted loci.

Regulation of Embryonic Stem Cell Self-Renewal.

Embryonic stem cells (ESCs) are a type of cells capable of self-renewal and multi-directional differentiation. The self-renewal of ESCs is regulated by factors including signaling pathway proteins, transcription factors, epigenetic regulators, cytokines, and small molecular compounds. Similarly, non-coding RNAs, small RNAs, and microRNAs (miRNAs) also play an important role in the process. Functionally, the core transcription factors interact with helper transcription factors to activate the expression of genes that contribute to maintaining pluripotency, while suppressing the expression of differentiation-related genes. Additionally, cytokines such as leukemia suppressor factor (LIF) stimulate downstream signaling pathways and promote self-renewal of ESCs. Particularly, LIF binds to its receptor (LIFR/gp130) to trigger the downstream Jak-Stat3 signaling pathway. BMP4 activates the downstream pathway and acts in combination with Jak-Stat3 to promote pluripotency of ESCs in the absence of serum. In addition, activation of the Wnt-FDZ signaling pathway has been observed to facilitate the self-renewal of ESCs. Small molecule modulator proteins of the pathway mentioned above are widely used in in vitro culture of stem cells. Multiple epigenetic regulators are involved in the maintenance of ESCs self-renewal, making the epigenetic status of ESCs a crucial factor in this process. Similarly, non-coding RNAs and cellular energetics have been described to promote the maintenance of the ESC’s self-renewal. These factors regulate the self-renewal and differentiation of ESCs by forming signaling networks. This review focused on the role of major transcription factors, signaling pathways, small molecular compounds, epigenetic regulators, non-coding RNAs, and cellular energetics in ESC’s self-renewal.

Regulation of CD4+ and CD8+ T Cell Biology by Short-Chain Fatty Acids and Its Relevance for Autoimmune Pathology.

The gut microbiota encodes a broad range of enzymes capable of synthetizing various metabolites, some of which are still uncharacterized. One well-known class of microbiota-derived metabolites are the short-chain fatty acids (SCFAs) such as acetate, propionate, butyrate and valerate. SCFAs have long been considered a mere waste product of bacterial metabolism. Novel results have challenged this long-held dogma, revealing a central role for microbe-derived SCFAs in gut microbiota-host interaction. SCFAs are bacterial signaling molecules that act directly on host T lymphocytes by reprogramming their metabolic activity and epigenetic status. They have an essential biological role in promoting differentiation of (intestinal) regulatory T cells and in production of the anti-inflammatory cytokine interleukin-10 (IL-10). These small molecules can also reach the circulation and modulate immune cell function in remote tissues. In experimental models of autoimmune and inflammatory diseases, such as inflammatory bowel disease, multiple sclerosis or diabetes, a strong therapeutic potential of SCFAs through the modulation of effector T cell function was observed. In this review, we discuss current research activities toward understanding a relevance of microbial SCFA for treating autoimmune and inflammatory pathologies from in vitro to human studies.

Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

Fetal origin of sex-bias brain aging.

DNA methylation plays crucial roles during fetal development as well as aging. Whether the aging of the brain is programmed at the fetal stage remains untested. To test this hypothesis, mouse epigenetic clock (epiclock) was profiled in fetal (gestation day 15), postnatal (day 5), and aging (week 70) brain of male and female C57BL/6J inbred mice. Data analysis showed that on week 70, the female brain was epigenetically younger than the male brain. Predictive modeling by neural network identified specific methylations in the brain at the developing stages that were predictive of epigenetic state of the brain during aging. Transcriptomic analysis showed coordinated changes in the expression of epiclock genes in the fetal brain relative to the placenta. Whole-genome bisulfite sequencing identified sites that were methylated both in the placenta and fetal brain in a sex-specific manner. Epiclock genes and genes associated with specific signaling pathways, primarily the gonadotropin-releasing hormone receptor (GnRHR) pathway, were associated with the sex-bias methylations in the placenta as well as the fetal brain. Transcriptional crosstalk among the epiclock and GnRHR pathway genes was evident in the placenta that was maintained in the brain during development as well as aging. Collectively, these findings suggest that sex differences in the aging of the brain are of fetal origin and epigenetically linked to the placenta.

Structural Mechanism of TAF-Iβ Chaperone Function on Linker Histone H1.10

Linker histone H1, facilitated by its chaperones, plays an essential role in regulating gene expression by maintaining chromatin’s higher-order structure and epigenetic state. However, we know little about the structural mechanism of how the chaperones recognize linker histones and conduct their function. Here, we used biophysical and biochemical methods to investigate the recognition of human linker histone isoform H1.10 by the TAF-Iβ chaperone. Both H1.10 and TAF-Iβ proteins consist of folded cores and disordered tails. We found that H1.10 formed a complex with TAF-Iβ in a 2:2 stoichiometry. Using distance restraints obtained from methyl-TROSY NMR and spin labels, we built a structural model for the core region of the complex. In the model, the TAF-Iβ core interacts with the globular domain of H1.10 mainly through electrostatic interactions. We confirmed the interactions by measuring the effects of mutations on the binding affinity. A comparison of our structural model with the chromatosome structure shows that TAF-Iβ blocks the DNA binding sites of H1.10. Our study provides insights into the structural mechanism whereby TAF-Iβ functions as a chaperone by preventing H1.10 from interacting with DNA directly.

β-hydroxybutyrate reduces blastocyst viability via trophectoderm-mediated metabolic aberrations in mice.

STUDY QUESTIONWhat is the effect of the ketone β-hydroxybutyrate (βOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability?SUMMARY ANSWER In vitro βOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner.WHAT IS KNOWN ALREADYA maternal KD in humans elevates gamete and offspring βOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to βOHB at supraphysiological concentrations (8–80 mM) perturbs preimplantation mouse embryo development.STUDY DESIGN, SIZE, DURATIONA mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with βOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured βOHB-exposed embryos was assessed.PARTICIPANTS/MATERIALS, SETTING, METHODSMouse embryos were cultured in vitro with or without βOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of βOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, βOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 β-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed.MAIN RESULTS AND THE ROLE OF CHANCEAll βOHB concentrations tested slowed embryo development (P < 0.05), and βOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, βOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of βOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25–4 mM) βOHB elevated H3K27bhb levels. Preimplantation βOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of βOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of βOHB was detected, whereby female fetuses from βOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses.LIMITATIONS, REASONS FOR CAUTIONThis study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro βOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of βOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown.WIDER IMPLICATIONS OF THE FINDINGSThese findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to βOHB within a physiological range. Maternal diets which increase βOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of βOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

Heterogeneity of Organization of Subcompartments in DSB Repair Foci.

Cells assemble compartments around DNA double-strand breaks (DSBs). The assembly of this compartment is dependent on the phosphorylation of histone H2AX, the binding of MDC1 to phosphorylated H2AX, and the assembly of downstream signaling and repair components. The decision on whether to use homologous recombination or nonhomologous end-joining repair depends on competition between 53BP1 and BRCA1. A major point of control appears to be DNA replication and associated changes in the epigenetic state. This includes dilution of histone H4 dimethylation and an increase in acetylation of lysine residues on H2A and H4 that impair 53BP1 binding. In this article, we examined more closely the spatial relationship between 53BP1 and BRCA1 within the cell cycle. We find that 53BP1 can associate with early S-phase replicated chromatin and that the relative concentration of BRCA1 in DSB-associated compartments correlates with increased BRCA1 nuclear abundance as cells progress into and through S phase. In most cases during S phase, both BRCA1 and 53BP1 are recruited to these compartments. This occurs for both IR-induced DSBs and breaks targeted to an integrated LacO array through a LacI-Fok1-mCherry fusion protein. Having established that the array system replicates this heterogeneity, we further examined the spatial relationship between DNA repair components. This enabled us to precisely locate the DNA containing the break and map other proteins relative to that DNA. We find evidence for at least three subcompartments. The damaged DNA, single-stranded DNA generated from end resection of the array, and nuclease CtIP all localized to the center of the compartment. BRCA1 and 53BP1 largely occupied discrete regions of the focus. One of BRCA1 or 53BP1 overlaps with the array, while the other is more peripherally located. The array-overlapping protein occupied a larger volume than the array, CtIP, or single-stranded DNA (ssDNA). Rad51 often occupied a much larger volume than the array itself and was sometimes observed to be depleted in the array volume where the ssDNA exclusively localizes. These results highlight the complexity of molecular compartmentalization within DSB repair compartments.