Epigenomics

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

BackgroundUnderstanding the causes of the exceptional rate of evolution of the mammalian placenta is likely to aid the understanding of placental development and the aetiology of the human-specific pregnancy disorder pre-eclampsia (PE). As retroelements (REs) are often lineage-specific and known to be co-opted for placental functioning, here we consider the RE binding of GATA3 and DLX5, these transcription factors being dysregulated in PE, and their downstream consequences. MethodsMultiomics analyses identified the retroviral regulatory sequence LTR8B in the PSG gene array, as a contributor to expression diversification in the placenta. To characterize this genomic domain, we performed copy number variation analysis and whole-genome sequencing. Multiomics data was employed to identify loci that might act as an active chromatin loop boundary around the PSG region. CRISPR-Cas9 knockouts with aligned RNAseq and epigenetic mark data tested for trophoblast-specific cis-regulatory elements (CREs-enhancer and/or promoter sequences) of resulting loci. Functional assays were employed to characterize the phenotypic effects of a candidate locus. Structural analysis of PSG family members also identified an additional RE, MER65-int. RNA-seq and antibody staining was employed to consider polyadenylation and functional diversification. ResultsThe LTR8B CRE facilitates the binding of transcription factors (e.g., GATA3, DLX5, TFAP2A/C), resulting in a diversified PSG gene expression pattern within a primate-specific genomic region that exhibits high intraspecies variability. The LTR8B/PSG9 regulatory element influences other PSG family members. PSG9, unique among PSGs, produces both secreted and membrane-anchored isoforms, MER65-int providing alternative polyA signals, enabling the evolution of secreted PSG variants through the truncation of the ancestral CEACAM proteins transmembrane domain. The LTR8B/PSG9 locus regulates the differentiation of multinucleated trophoblasts (syncytialization) and, like chorionic gonadotropin and syncytin1, determines the identity of syncytiotrophoblasts. Notably, PSG9 is the most upregulated PSG in PE, with levels correlated with GATA3 and DLX5 levels. ConclusionsREs contribute to the structural and expression evolution of PSG genes, facilitating lineage-specific placental evolution. The LTR8B/PSG9 regulatory network plays a central role in syncytiotrophoblast differentiation. Given the association between DLX5/GATA3 dysregulation and elevated PSG9 levels, along with PSG9s expression in the first trimester, PSG9 shows potential as a predictive biomarker for PE.

Pregnancy results in profound physiological changes driven by dynamic and precisely programmed molecular processes. Maternal peripheral blood is generally the specimen of choice for studying these processes, as it is easily accessible and essential for many aspects of maintaining a healthy pregnancy. Here, we present a high-resolution atlas of the dynamic temporal changes in the transcriptome of maternal peripheral blood in healthy human pregnancy. We generated comprehensive RNA sequencing data in 802 weekly samples from 31 healthy pregnant women from the first trimester until after delivery. Using a strict discovery and replication setup, our longitudinal analysis of gene expression identified 720 genes with robust pregnancy-specific expression patterns. Using weighted graph correlation network analysis, we identified nine pregnancy-associated transcriptional modules that reveal a strong, coordinated enrichment of innate/neutrophil and antiviral immune programs, alongside changes in adaptive immunity (T cell differentiation and signaling), erythropoiesis and hemoglobin metabolism. Cell-type deconvolution revealed that these transcriptomic shifts were accompanied by increased relative neutrophil proportions and reduced naive CD4 and CD8 T cells in pregnancy. We provide a comprehensive characterization of dynamic changes across pregnancy, highlighting maternal blood as a key systemic regulator in healthy gestation. Together, our findings establish a reference atlas of healthy pregnancy, which can be used to identify dysregulated processes and mechanisms in women with pregnancy complications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/715300v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@2a4b28org.highwire.dtl.DTLVardef@ac49d9org.highwire.dtl.DTLVardef@12468c8org.highwire.dtl.DTLVardef@15b282f_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LI720 genes showed robust pregnancy specific expression patterns. C_LIO_LICo-expression analysis clustered the genes into nine modules with distinct dynamics. C_LIO_LIEnrichment in pathways involved in innate and neutrophil-mediated immunity, antiviral responses, T cell differentiation and signaling, erythropoiesis and hemoglobin metabolism. C_LIO_LICell-type deconvolution showed increases in neutrophils and decreases in naive CD4 and CD8 T cells. C_LIO_LIThe atlas of detailed longitudinal transcriptional changes provides a baseline reference for healthy pregnancy. C_LIO_LIResults for all genes and protein-protein interaction networks are made available for interactive exploration. C_LI

Introduction: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation with complex etiology involving both genetic and environmental factors. Epigenetic mechanisms may mediate environmental contributions, but separating genetic from environmental effects remains challenging. Methods: We present an epigenome-wide association study with 32 monozygotic and 22 dizygotic twin pairs discordant for NSCL/P on blood and saliva samples. Differential methylation analysis was conducted using linear models to identify CpG sites showing significant methylation differences between affected and unaffected twins followed by functional annotation and pathway enrichment analysis. Results: The top-ranked finding is a differentially methylated region comprising two CpG sites at the CYP26A1 locus, cg12110262 (P = 3.21x10-7) and cg15055355 (P = 1.39x10-3). CYP26A1 is essential for retinoic acid catabolism and craniofacial patterning. The chromatin regulator ANKRD11, which causes KBG syndrome featuring cleft palate was the second best hit. Differentially methylated CpG sites showed significant enrichment in craniofacial enhancers and overlap with multiple GWAS-validated cleft genes including VAX1, PVRL1, SMAD3, and PRDM16. Conclusions: Our findings implicate retinoic acid signaling and chromatin regulation in NSCL/P etiology and demonstrate the value of discordant twin designs for distinguishing environmental from genetic epigenetic contributions to complex malformations.

BackgroundAcute appendicitis (AA) is a common surgical emergency. Observational studies have reported associations between obesity-related anthropometric traits and AA, but these associations may be affected by confounding and reverse causation. We used Mendelian randomization (MR) to investigate the potential causal effects of body mass index (BMI) and waist-to-hip ratio (WHR) on AA risk. Methods and FindingsWe obtained genome-wide association study (GWAS) summary statistics for BMI (ukb-b-19953), WHR (ieu-a-73), and AA (finn-b-K11_APPENDACUT) from the IEU Open GWAS database. We conducted single-variable MR (SVMR) and multivariable MR (MVMR) analyses. The primary estimator was inverse-variance weighted (IVW) MR, complemented by MR-Egger, weighted median, weighted mode, and simple mode methods. Instrument strength was assessed using the variance explained and F-statistics. Sensitivity analyses included Cochrans Q for heterogeneity, MR-Egger intercept and MR-PRESSO global test for horizontal pleiotropy, leave-one-out analysis, and Steiger directionality testing. We mapped instrumental variants to cis-eQTL genes (eQTLGen) and performed GO and KEGG enrichment analyses. In SVMR, genetically predicted BMI (OR 1.145, P = 0.0006) and WHR (OR 1.336, P = 0.0040) were associated with higher AA risk. Instruments were strong (BMI: [Formula]; mean/min F-statistic= 64.15/29.76; WHR: [Formula]; mean/min F-statistic= 48.41/29.75). Sensitivity analyses did not show strong evidence of heterogeneity or directional pleiotropy, and Steiger tests supported the hypothesized direction (exposure -> outcome). In MVMR including both traits, WHR remained associated with AA risk (OR 1.374, P = 0.0110), whereas BMI was not (P = 0.8000). Enrichment analyses suggested WHR-mapped genes were enriched in pathways related to adipocyte differentiation, while BMI-mapped genes were enriched in terms including nuclear envelope and endocytosis-related pathways. ConclusionsThese MR analyses are consistent with a potential causal relationship between obesity-related traits and AA risk, with WHR showing an association independent of BMI in multivariable models. Further work in diverse populations and with additional sensitivity analyses is warranted to assess robustness to pleiotropy and generalizability.

BackgroundWnt/{beta}-catenin signalling is dysregulated in acute myeloid leukaemia (AML), where it lacks effective targeting strategies. Previously, we discovered that {beta}-catenin interacts with several RNA-binding proteins (RBP), indicating post-transcriptional influence which is yet to be therapeutically interrogated in AML. MethodsCo-immunoprecipitation confirmed protein interactions, and TCF/LEF reporters were used to assess Wnt signalling output in leukaemia cells. Regulatory crosstalk was assessed using immunoblotting and RT-qPCR approaches following lentiviral transduction of myeloid cell lines. Targeting of {beta}-catenin and LIN28B was tested through combinations of genetic and pharmacological inhibition in AML cells. ResultsThe most frequent RBP-binding motif amongst {beta}-catenin-bound mRNAs was the GGAG motif targeted by oncofetal miRNA-regulating RBP; LIN28B. {beta}-Catenin:LIN28B interactions were detected in lymphoid and myeloid cell lines, plus primary human CD34 fetal-liver HSCs. LIN28B positively regulated Wnt signalling output through LEF1 regulation involving a post-transcriptional let7 miRNA mechanism. Further miRNA sequencing of {beta}-catenin- and LIN28B-depleted myeloid cells revealed potential cooperative and antagonistic function in miRNA regulation. Finally, dual-targeting both {beta}-catenin and LIN28B through either genetic and/or pharmacological means preferentially reduced AML cell viability. ConclusionThe {beta}-catenin:LIN28B axis could represent a novel synthetically lethal relationship in AML which could be exploited in rare subtypes where LIN28B expression becomes reactivated.

Early achievement of deep remission improves patients outcome in chronic myeloid leukemia (CML) treatment, highlighting the need for predictive indicators before therapy initiation. This study aimed to develop a tool to predict CML treatment responses to guide optimal therapy selection. Using hierarchical clustering of complete blood count (CBC) data at diagnosis, patients were stratified into two clusters. Patients in Cluster 1 had higher BCR::ABL1IS mRNA levels at 3 and 6 months post-treatment and lower rates of major molecular response compared to cluster 2. Cluster 1 also showed increased granulocyte and immature white blood cell counts and decreased erythroid parameters. Flow cytometric analysis of bone marrow mononuclear cells revealed that cluster 1 had a significant increase in hematopoietic stem cell fractions and a higher ratio of granulocyte-macrophage progenitors to megakaryocyte-erythroid progenitors compared to cluster 2. These findings suggest that differences in bone marrow progenitor cell differentiation affect peripheral blood profiles. Artificial intelligence-driven ghost cytometry (GC) was evaluated for its ability to comprehensively capture these changes and successfully distinguished patients with poorer treatment responses, with GC scores at diagnosis strongly correlating with BCR::ABL1IS mRNA levels at 3 and 6 months post-treatment initiation. The study indicates that multivariate analysis of CBC or GC analysis may enable simple, early prediction of CML treatment efficacy, potentially contributing to effective and individualized CML therapy.

BackgroundAberrant DNA methylation is a hallmark of colorectal cancer (CRC). Yet, how DNA methylation is linked to transcriptional states, immune programs, and tissue resident microbiome within the same tumors has not been systematically analyzed. MethodsWe profiled genome-wide DNA methylation (Illumina MethylationEPIC) in 182 colon tumors and 76 adjacent normals from AC-ICAM, and integrated with matched transcriptomes, whole exome, microbiome, and clinical data. Tumor-specific methylation, promoter methylation-expression links, microbiome associations, and survival were analyzed and validated in TCGA-COAD. ResultsTumor and normal tissues exhibited distinct DNA methylation patterns, reflecting widespread epigenetic alterations in cancer. Pathway analysis identified two major tumor pathways regulated by DNA methylation. The first involved extracellular signaling and adhesion genes, with higher methylation linked to increased proliferation and lower immune infiltration. Similarly, higher tumor methylation in nitric oxide signaling was associated with reduced adaptive immune activity and interestingly, influenced immune-related survival. These findings were also validated in the TCGA-COAD cohort. An inverse methylation-expression pattern implicated modifications of TCR signaling in naive CD8, and interferon-/{beta} signaling which were hypermethylated and hypomethylated in tumors compared to normal, respectively. Combining methylation and microbiome revealed connections between Akkermansia muciniphila and TGF-{beta} and Prevotella nigrescens with MAPK signaling pathways. Finally, a methylation-based model using 43 promoters CpGs successfully identified patients with different survival outcomes, underscoring the clinical relevance of these epigenetic alterations in colon cancer. ConclusionDNA methylation shapes the molecular and immune landscape of colon cancer, altering signaling pathways and immune programs, interacting with the microbiome, and impacting patients survival.

Background and AimMASLD affects 30-38% of Indian adults, yet the contribution of genetic risk variants to disease susceptibility and fibrosis progression remains poorly characterised. We investigated the association of 12 candidate SNPs with MASLD susceptibility and fibrosis severity in North Indian patients, benchmarking allele frequencies against IndiGenomes and global populations. MethodsSixty-nine MASLD patients (75.4% male; median BMI 29.8 kg/m{superscript 2}) from a tertiary care liver clinic in New Delhi were genotyped for 12 SNPs using Illumina custom BeadChip array and Sanger sequencing. Patients were stratified by liver stiffness measurement (LSM): significant fibrosis ([&ge;]8 kPa, n=38) versus no significant fibrosis (<8 kPa, n=31). Allele frequencies were compared with IndiGenomes ([~]1,020 Indian individuals) and 1000 Genomes populations. ResultsPNPLA3 rs738409 G allele was the strongest within-cohort predictor of significant fibrosis (allelic OR 2.89, 95% CI 1.35-6.19, P=0.006; dominant model OR 3.94, P=0.008), with carriers demonstrating higher LSM (median 15.6 vs. 7.5 kPa, P=0.005). SAMM50 rs3761472 (OR 2.12, P=0.065) and FTO rs9939609 (OR 2.08, P=0.089) showed non-significant trends. In the population-level comparison, APOC3 rs2854116 T allele was the only variant significantly enriched after Bonferroni correction (64.0% vs. 47.9%; OR 1.93, 95% CI 1.35-2.77, P<0.001), followed by PNPLA3 (33.3% vs. 24.1%, OR 1.57, P=0.019) and SAMM50 (31.2% vs. 22.6%, OR 1.55, P=0.028). Notably, APOC3 showed no association with fibrosis (OR 0.96, P=1.000), suggesting a role in susceptibility rather than progression. All SNPs were in Hardy-Weinberg equilibrium. ConclusionsThis study reveals a dissociation between genetic determinants of MASLD susceptibility and fibrosis progression in North Indian patients. APOC3 rs2854116 predisposes to MASLD at the population level, while PNPLA3 rs738409 drives fibrosis severity within established disease, underscoring the need for ancestry-specific genetic risk stratification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/26347059v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@a07808org.highwire.dtl.DTLVardef@12882adorg.highwire.dtl.DTLVardef@9b33a1org.highwire.dtl.DTLVardef@15aa5e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe global landscape of induced pluripotent stem cell (iPSC) resources remains heavily skewed toward European, North American, and East Asian populations, leaving the Middle East and North Africa (MENA) region critically underrepresented. This disparity hinders the application of precision medicine in populations with unique genetic backgrounds, particularly those with high rates of consanguinity and distinctive rare disease profiles. To address this gap, we established the Saudi Bank of Induced Pluripotent Stem Cells (SBiPSCs), at King Abdullah International Medical Research Centre (KAIMRC). The bank comprises two major complementary arms: one dedicated to the derivation and biobanking of iPSCs from individuals with rare and common genetic disorders, and a second focused on Human Leukocyte Antigen (HLA)-based iPSC banking to support the development of immunocompatible cell therapies. MethodsSBiPSCs operates within King Abdulaziz Medical City in Jeddah under the Ministry of National Guard for Health Affairs (MNGHA)s ethical and clinical framework. To establish the repository, we implemented a clinic-guided enrolment strategy in which treating physicians, briefed on the banks objectives, recruited patients with confirmed genetic diagnoses. Peripheral blood samples were collected, processed, and cells were reprogrammed using non-integrating episomal plasmids. All derived lines underwent rigorous quality control in accordance with International Society for Stem Cell Research (ISSCR) standards, including assessment of pluripotency markers, genomic integrity, and trilineage differentiation potential. To demonstrate our iPS characterization workflow and translational utility, iPSCs from a Saudi patient with familial Long QT Syndrome (LQTS) and a healthy sibling were differentiated into functional cardiac organoids. Simultaneously, for the HLA-based banking arm, the Saudi Stem Cell Donor Registry (SSCDR) database was leveraged to identify donors predicted to provide maximal coverage for the Saudi population. ResultsTo date, SBiPSCs has successfully generated 37 iPSC lines derived from 19 Saudi patients and healthy donors. All lines exhibit robust expression of pluripotency markers, maintain normal karyotypes, and demonstrate differentiation capacity. To demonstrate our characterization pipeline and translational utility, iPSCs from an LQTS patient and a healthy sibling were generated, validated, and differentiated into beating cardiac organoids that recapitulated the disease phenotype, with microelectrode array analysis confirming prolonged field potential durations mirroring the clinical QT prolongation. Furthermore, the HLA-based banking arm has expanded to include two homozygous iPSC lines, which together provide immunological compatibility for approximately 9% of the Saudi population. ConclusionsSBiPSCs represents the first centralized iPSC repository in the MENA region. The SBiPSCs is well-positioned to accelerate the translation of stem cell research into scalable, immunocompatible cell therapies and precision medicine applications aligned with national and regional healthcare priorities.

During early embryonic development, cells transition from naive to primed pluripotent state. Various culture conditions have been established to revert primed cells back to naive state, to increase differentiation potential and to reset epigenetic abnormalities. In this study, we modified culture conditions to allow primed-to-naive conversion under feeder-independent and normoxic conditions (FINO medium), which exemplified the need for a quantitative measure of pluripotent states. DNA methylation (DNAm) profiling revealed extensive hypomethylation at naive state, but also significant gains of methylation at specific sites in the genome. We demonstrate that DNAm patterns can be used to benchmark culture protocols. Furthermore, we developed a naive-score based on DNAm at two genomic sites, which can be analyzed by digital PCR to monitor transition between pluripotent states. Our study describes a simplified culture protocol for primed-to-naive conversion, offers insights into the specific DNAm changes, and introduces a robust DNAm-based biomarker to track this process effectively.

The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, Human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that are wildtype or contain a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thus providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants. HighlightsO_LIUsed CRISPR-Cas9 to delete TOP2B from hiPSC C_LIO_LIProduced beating cardiomyocytes from both WT and TOP2B null hiPSC C_LIO_LITranscriptome analysis of WT and TOP2B null hiPSC and derived cardiomyocytes C_LIO_LIRNA seq showed he specific TOP2B inhibitor topobexin largely phenocopies TOP2B gene inactivation in iPSC derived cardiomyocytes. C_LIO_LITopobexin inhibition could be used as an alternative to a TOP2B gene knockout in many different cell types, speeding up the analysis of the function of TOP2B. C_LI

The microRNAs miR-15a and miR-16 are key regulators of the anti-apoptotic oncogene BCL2, playing a significant role in tumorigenesis. These miRNAs function as tumor suppressors by directly targeting BCL2, whose overexpression contributes to cell survival and resistance to therapy in multiple malignancies, including chronic lymphocytic leukemia (CLL). The downregulation or deletion miR-15a/miR-16-1 cluster located on chromosome 13q occurs in about 50% of CLL patients and leads to the overexpression of the oncogenic BCL2, contributing to the survival and proliferation of cancer cells. In this confirmatory study, we provide additional evidence supporting the mechanism by which these miRNAs mediate the inhibition of BCL2 translation, leading to reduced levels of BCL2 protein with no significant effect on BCL2 mRNA degradation. This mechanism has been previously established as a critical pathway in the regulation of apoptosis, particularly in cancer cells where BCL2 overexpression is often associated with resistance to cell death. Our findings reinforce the notion that miRNAs, such as miR-15 and miR-16, bind to the 3-UTR of BCL2 messenger RNA (mRNA), specifically repressing its translation without inducing mRNA degradation. The results from our study align with previous research, confirming that the miRNA-mediated inhibition of BCL2 translation serves as a precise regulatory mechanism that targets protein synthesis rather than mRNA stability. These findings highlight the role of miRNAs in fine-tuning post-transcriptional gene regulation, offering a targeted approach to downregulate oncogenic proteins like BCL2 without disrupting the underlying mRNA, which could be leveraged for more refined therapeutic strategies.

Ehmt2 is a key H3K9 methyltransferase that regulates genome silencing and structural integrity during animal development. In addition to this canonical function, Ehmt2 has also been implicated in neural tissues mediating both direct and indirect transcriptional activation, and exon splicing, to facilitate proper neural cell differentiation and survival. Several germline loss-of-function animal models have been developed showing both conserved and divergent phenotypes that range from embryonic lethality to behavioural deficits in adult, fertile animals. Here, we generated the first maternal-zygotic ehmt2 loss of function mutant in zebrafish using CRISPR-Cas9 mutagenesis. An assessment of the pattern of H3K9 methylation in mutant embryos by ChIP-seq indicates that there are aberrant levels of this repressive mark, including reduction in discrete 5 non-coding regions of genes, but with no significant change in the overall pattern distribution of these marks across the genome. Global transcriptome and morphological analyses demonstrated that mutant embryos displayed greater variation in the timing of developmental progression that is, on average, slower compared to controls. Despite this, mutant embryos ultimately survive and are fertile. Through examination of progenitor cell dynamics and gene expression profiles, we found that the delay in embryonic development was associated with longer rates of S-M phases of the progenitor cell cycle in mutants leading to deficits in tissue growth. Finally, our data suggest a robust network of epigenetic regulators can potentially compensate for Ehmt2 loss of function and permit embryonic development and survival in ehmt2 mutant zebrafish. Our work establishes a zebrafish ehmt2 loss of function model that will facilitate examination of the complex and varied roles of Ehmt2 in vertebrate development.

BackgroundGestational exposures may contribute to the newborns lifetime risk of inflammatory bowel disease (IBD). While gestational influences are associated with IBD onset, the causality and confounding of such exposures are difficult to ascertain. The neonatal metabolome provides a metabolic snapshot of gestational influences. ObjectiveWe tested the neonatal metabolomes ability to predict future IBD, to assess whether gestational exposures are reflected in early molecular precursors of the disease. MethodsWe profiled dried blood spots from 520 newborns who later developed IBD and matched controls using high-resolution untargeted mass spectrometry metabolomics (1,350 QC-passing metabolites). Genotyping was available for 1,009 of these individuals. PERMANOVA confirmed assay sensitivity to gestational exposures, gradient boosting was used for prediction. ResultsThe neonatal metabolome significantly captured maternal smoking, birth weight, and gestational age (p < 0.001), but explained minimal variance in IBD status (R2 = 0.09%, p = 0.390) and showed no predictive power for IBD (AUC = 0.51, 95% CI 0.50-0.52, p = 0.585). Stratifying by disease subtype and age of onset did not improve performance. In contrast, genetic risk scores were modestly predictive (CD: AUC = 0.64, p < 5.11x10-14; UC: AUC = 0.63, p < 7.65x10-{superscript 1}{superscript 2}), but uncorrelated with neonatal metabolomic profiles (CD: p = 0.650; UC: p = 0.970), suggesting a later-age effect. ConclusionsUsing a large, comprehensively profiled cohort, we demonstrate that neonatal metabolomic profiles sensitively capture gestational signatures, but not the overall future IBD risk. Our findings suggest that most IBD risk accumulates later in life, beyond gestational molecular imprints.

Infertility is a complex condition affecting both the male and female population. Influenced by multiple factors, it remains a constant challenge due to limited understanding of endometrial abnormalities. With this study we aim to investigate the molecular basis of infertility using transcriptomic analysis of endometrial tissue from the NCBI GEO dataset GSE92324. We performed exploratory data analysis using Principal Component Analysis (PCA) to find samples variance followed by differential gene expression (DGE) analysis using DESeq2 package where we identified 168 significant genes with adjusted p-value < 0.05 and |log2FC| > 2. Upregulated genes included GPX3, CXCL14, and PPARGC1A and downregulated genes included WNK4, GJB2, and TRPM6. Functional enrichment using KEGG and GO showed that differentially expressed genes (DEGs) are involved in immune-inflammatory pathways, lipid metabolism and steroid biosynthesis pathways. Through Ingenuity Pathway Analysis (IPA) we identified affected canonical pathways such as increased innate immune responses, altered lipid metabolism and inhibition of mitochondrial dysfunction. Upstream regulator analysis highlighted PTEN, PRKAA1, HDAC4, IL10RA, and RAD51, which were impacting metabolic pathways and anti-inflammatory signalling. Further, through Weighted Gene Co-expression Network Analysis (WGCNA) we found a Turquoise module that had very strong and highly significant negative correlation (cor = - 0.84, respectively and P < 0.0001) with traits of interest. This led to the discovery of C7orf50 as a novel insight involved in cholesterol metabolism linked to infertility. This integrative approach reveals crucial genes, co-expression modules, and underlying pathways involved in female infertility. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/701467v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@4418a6org.highwire.dtl.DTLVardef@ae7900org.highwire.dtl.DTLVardef@89f581org.highwire.dtl.DTLVardef@154f1a9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIFrom the dataset GSE92324 total of 168 significant DEGs associated with unexplained infertility were identified using adjusted p-value < 0.05 and |log2FC| > and < 2. C_LIO_LIIn comparison with the CTD list we identified five genes C1orf106, C15orf59, LINC00461, C15orf48, and C10orf99 previously unknown as having direct evidence of involvement in infertility. C_LIO_LIWGCNA analysis highlighted the turquoise module as highly associated and gave the novel gene C7orf50 associated with cholesterol metabolism. C_LIO_LIIPA revealed PTEN, PRKAA1, IL10RA, and RAD51 as potential upstream regulators and inflammatory pathways, mitochondrial dysfunction as canonical pathways. C_LIO_LIThe study highlights a novel link between GI inflammation and endometrial receptivity. C_LI

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and a leading cause of female infertility, with complex genetic, metabolic, and hormonal etiologies. Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes, yet their roles in PCOS remain underexplored. Here, we identified and characterized PCOS differentially expressed gene-associated lncRNAs (PDEGAL) with an integrative approach combining expression data, genetic association, and evolutionary analysis. MethodsThirty-three PCOS-associated protein-coding genes were obtained from our prior study, and all their nearby and overlapping lncRNAs were annotated. These candidates were analyzed using UCSC Genome Browser-mapped annotations and datasets, including NCBI RefSeq, GENCODE, GTEx, GWAS SNPs, and conservation, as well as the FANTOM5 cap analysis of gene expression (CAGE) promoter data, to assess their expression, regulatory potential, genetic variant overlaps, and evolutionary conservation. ResultsTwenty-three PDEGALs (18 antisense to, and 5 sharing bidirectional promoters with, known PCOS-associated protein-coding genes) were identified. 17 PDEGALs contained GWAS SNPs with statistically significant disease associations, 9 of which were associated with PCOS-related traits. 5 PDEGALs demonstrated expression in the KGN granulosa cell model of PCOS. Key gene structure element (KGSE) analysis revealed that most PDEGALs are primate-specific. Integrating four criteria--GTEx expression, GWAS SNPs, FANTOM promoterome, and KGSE conservation--highlighted HELLPAR as the only lncRNA fulfilling all four, while five others--PGR-AS1, MTOR-AS1, ENSG00000265179, ENSG00000256218, and LOC105377276--fulfilled three of the four criteria. ConclusionsWe have systematically identified candidate PCOS regulatory lncRNAs with convergent genetic, expression, and evolutionary evidence. These results provide a framework for functional validation and highlight lncRNAs as potential biomarkers and therapeutic targets in PCOS that function by regulating their nearby and overlapping protein-coding genes.

Very preterm infants (<30 weeks gestation) are at elevated risk for neurodevelopmental and social-behavioral challenges. DNA methylation (DNAm) may provide a biological link between preterm birth and later behavioral outcomes. We examined associations between DNAm profiles at neonatal intensive care unit (NICU) discharge and at age 5 with Social Responsiveness Scale (SRS) scores which measure social communication, social interaction, and repetitive behaviors at age 5, including sex-specific effects, in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study. Epigenome-wide buccal DNAm was profiled at NICU discharge (n=218) and at 5 years (n=188). We identified 38 neonatal and 6 age-5 CpG sites associated with SRS scores (all q<0.05) using epigenome-wide association studies (EWAS) at each time point. Several CpGs mapped to genes involved in neurodevelopment including TCF4, KLC4, CAP2, PTDSS1, ADAM12, SENP1, CHN2, SH3D19, and ITGA1, with sex-specific effects observed for CpGs in CAMTA1 and GABBR1. Enriched pathways included neurodevelopment, cytoskeletal regulation, stress-response, and metabolic processes. DNAm patterns during early life, particularly the neonatal period, were associated with social-behavioral development in very preterm children. Findings in key genes such as TCF4 and CAMTA1 highlight potential epigenetic mechanisms linking early-life biology to later behavioral challenges.

Long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) are candidate preventive agents for breast cancer. With emerging evidence of epigenetic regulation of the tumor microenvironment, tissue-level epigenetic effects may represent an important target for cancer prevention. In a randomized Phase II sub-study (high-dose 5 g/day vs low-dose 1 g/day for 12 months; n = 17; Clinicaltrials.gov: NCT02295059), DNA methylation (DNAm) of the breast environment was profiled by reduced-representation bisulfite sequencing (RRBS). DNAm was assessed genome-wide, at individual gene promoters, and for locus-level heterogeneity which has been linked to epigenetic dysregulation that can precede breast cancer. Both doses induced promoter DNAm changes, but their responses diverged: low-dose samples showed increased CpG variance and more differentially methylated promoters without pathway enrichment, whereas high-dose samples had reduced DNAm heterogeneity and promoter enrichment in inflammation signaling pathways. Many overlapping differentially methylated promoters changed in opposite directions between doses. The finding that high-dose n-3 PUFA affects DNAm fidelity in the breast adipose suggests a new potential mechanism for n-3 PUFA-mediated prevention of breast cancer development. Together with the dose-specific, directionally discordant DNAm responses in breast adipose, this study has important implications for both advancing n-3 PUFA for breast cancer prevention and dose selection in future n-3 PUFA supplementation trials.

Acute promyelocytic leukemia is a distinct subtype of acute myeloid leukemia characterized by the t(15;17) translocation, leading to the PML (Promyelocytic leukemia protein)-RARA (Retinoic Acid Receptor Alpha) fusion protein. Although PML-RARA fusion is common, there are 20 more fusion events also reported in APL. All -trans retinoic acid (ATRA) is a standard drug for APL, leading to significant improvement in patient outcomes; nevertheless, a small fraction of patients still experience relapse, and some patients exhibit resistance to the drug. Long non-coding RNAs (LncRNAs) are recognized as promising biomarkers for cancer diagnosis, prognosis, and treatment response. In this study, we used ATRA-Resistant (AP1060) and ATRA -Sensitive (NB4), both treated and untreated cell line transcriptomic data retrieved from the NCBI Gene Expression Omnibus(GEO) database to perform transcriptomic analysis with bioinformatic tools. We utilized the LncRAnalyzer pipeline to predict the lncRNAs, followed by differential expression analysis using DESeq2. Weighted Gene Co-expression Network Analysis (WGCNA) was employed to construct lncRNA co-expression modules associated with ATRA resistance. BEDTools is used to identify cis-acting target genes of lncRNAs.LncRNA -miRNA sponging identified by miRanda algorithm. The identified miRNAs reveal their significant role in APL and other leukemia subtypes. The results of the study show that the identified lncRNAs from the miRNA-LncRNA network are promising biomarkers for ATRA resistance.