Epigenetic Regulation Of Gene Expression Research Articles

Mitochondrial dysfunction and oxidative stress in selective fetal growth restriction

IntroductionPlacental dysfunction is the primary cause of selective fetal growth restriction (sFGR), and the specific role of mitochondria remains unclear. This study aims to elucidate mitochondrial functional defects in sFGR placentas and explore the roles of mitochondrial genomic and epigenetic alterations in its pathogenesis. MethodsThe placental villi of MCDA twins with sFGR were collected and the morphology and number of mitochondria were observed by transmission electron microscopy. Meanwhile, the levels of reactive oxygen species (ROS), ATP and oxidative damage markers were assessed. Mitochondrial DNA (mtDNA) copy number detection, targeted sequencing and methylation sequencing were performed. The expression of placental cytochrome c oxidase subunit I (COX I) and mitochondrial long non-coding RNAs (lncRNAs) were evaluated by Western blotting and qPCR. ResultsCompared with placentae from normal fetuses, pronounced mitochondrial damage within cytotrophoblast was revealed in sFGR placentae, alongside augmented mitochondrial number in syncytiotrophoblast. Enhanced oxidative stress in these placentae was evidenced by elevated markers of oxidative damage, accompanied by increased ROS production and diminished ATP generation. In sFGR placentae, a notable rise in mitochondrial copy number and one heterozygous mutation in the MT-RNR2 gene were observed, along with decreased COX Ⅰ levels, increased lncND5, lncND6, lncCyt b, and MDL1 synthesis, and decreased RMRP synthesis. DiscussionFindings collectively confirmed an exacerbation of oxidative stress within sFGR placentae, coinciding with mitochondrial dysfunction, compromised energy production, and ultimately the failure of compensatory mechanisms to restore energy balance, which may result from mutations in the mitochondrial genome and abnormal expression of epigenetic regulatory genes.

LMNA-Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-Derived iPSC Differentiation Support Cell Type and Lineage-Specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency.

LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Bio-Pathological Functions of Posttranslational Modifications of Histological Biomarkers in Breast Cancer.

Proteins are the most common types of biomarkers used in breast cancer (BC) theranostics and management. By definition, a biomarker must be a relevant, objective, stable, and quantifiable biomolecule or other parameter, but proteins are known to exhibit the most variate and profound structural and functional variation. Thus, the proteome is highly dynamic and permanently reshaped and readapted, according to changing microenvironments, to maintain the local cell and tissue homeostasis. It is known that protein posttranslational modifications (PTMs) can affect all aspects of protein function. In this review, we focused our analysis on the different types of PTMs of histological biomarkers in BC. Thus, we analyzed the most common PTMs, including phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, palmitoylation, myristoylation, and glycosylation/sialylation/fucosylation of transcription factors, proliferation marker Ki-67, plasma membrane proteins, and histone modifications. Most of these PTMs occur in the presence of cellular stress. We emphasized that these PTMs interfere with these biomarkers maintenance, turnover and lifespan, nuclear or subcellular localization, structure and function, stabilization or inactivation, initiation or silencing of genomic and non-genomic pathways, including transcriptional activities or signaling pathways, mitosis, proteostasis, cell-cell and cell-extracellular matrix (ECM) interactions, membrane trafficking, and PPIs. Moreover, PTMs of these biomarkers orchestrate all hallmark pathways that are dysregulated in BC, playing both pro- and/or antitumoral and context-specific roles in DNA damage, repair and genomic stability, inactivation/activation of tumor-suppressor genes and oncogenes, phenotypic plasticity, epigenetic regulation of gene expression and non-mutational reprogramming, proliferative signaling, endocytosis, cell death, dysregulated TME, invasion and metastasis, including epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET), and resistance to therapy or reversal of multidrug therapy resistance. PTMs occur in the nucleus but also at the plasma membrane and cytoplasmic level and induce biomarker translocation with opposite effects. Analysis of protein PTMs allows for the discovery and validation of new biomarkers in BC, mainly for early diagnosis, like extracellular vesicle glycosylation, which may be considered as a potential source of circulating cancer biomarkers.

Synergistic Cytotoxicity of Histone Deacetylase and Poly-ADP Ribose Polymerase Inhibitors and Decitabine in Breast and Ovarian Cancer Cells: Implications for Novel Therapeutic Combinations.

Breast and ovarian cancers pose significant therapeutic challenges. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACis), poly(ADP-ribose) polymerase inhibitors (PARPis), and decitabine in breast (MDA-MB-231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cancer cell lines that were exposed to HDACi (panobinostat or vorinostat), PARPi (talazoparib or olaparib), decitabine, or their combinations. HDACi, PARPi, and decitabine combinations had synergistic cytotoxicity (assessed by MTT and clonogenic assays) in all cell lines (combination index < 1). Clonogenic assays confirmed the sensitivity of breast and ovarian cancer cell lines to the three-drug combinations (panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine; vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine). Cell proliferation was inhibited by 48-70%, and Annexin V positivity was 42-59% in all cell lines exposed to the three-drug combinations. Western blot analysis showed protein PARylation inhibition, caspase 3 and PARP1 cleavage, and c-MYC down-regulation. The three-drug combinations induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs, impaired the DNA repair pathways, and altered the epigenetic regulation of gene expression. These results indicate that HDACi, PARPi, and decitabine combinations should be further explored in these tumor types. Further clinical validation is warranted to assess their safety and efficacy.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Histone (de)acetylation in epigenetic regulation of Phytophthora pathobiology.

Phytophthora species are oomycetes that have evolved a broad spectrum of biological processes and improved strategies to cope with host and environmental challenges. A growing body of evidence indicates that the high pathogen plasticity is based on epigenetic regulation of gene expression linked to Phytophthora's rapid adjustment to endogenous cues and various stresses. As 5mC DNA methylation has not yet been identified in Phytophthora, the reversible processes of acetylation/deacetylation of histone proteins seem to play a pivotal role in the epigenetic control of gene expression in oomycetes. To explore this issue, we review the structure, diversity, and phylogeny of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in six plant-damaging Phytophthora species: P. capsici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, and P. sojae. To further integrate and improve our understanding of the phylogenetic classification, evolutionary relationship, and functional characteristics, we supplement this review with a comprehensive view of HATs and HDACs using recent genome- and proteome-level databases. Finally, the potential functional role of transcriptional reprogramming mediated by epigenetic changes during Phytophthora species saprophytic and parasitic phases under nitro-oxidative stress is also briefly discussed.

Vascular remodelling in cardiovascular diseases: hypertension, oxidation, and inflammation.

Optimal vascular structure and function are essential for maintaining the physiological functions of the cardiovascular system. Vascular remodelling involves changes in vessel structure, including its size, shape, cellular and molecular composition. These changes result from multiple risk factors and may be compensatory adaptations to sustain blood vessel function. They occur in diverse cardiovascular pathologies, from hypertension to heart failure and atherosclerosis. Dynamic changes in the endothelium, fibroblasts, smooth muscle cells, pericytes or other vascular wall cells underlie remodelling. In addition, immune cells, including macrophages and lymphocytes, may infiltrate vessels and initiate inflammatory signalling. They contribute to a dynamic interplay between cell proliferation, apoptosis, migration, inflammation, and extracellular matrix reorganisation, all critical mechanisms of vascular remodelling. Molecular pathways underlying these processes include growth factors (e.g., vascular endothelial growth factor and platelet-derived growth factor), inflammatory cytokines (e.g., interleukin-1β and tumour necrosis factor-α), reactive oxygen species, and signalling pathways, such as Rho/ROCK, MAPK, and TGF-β/Smad, related to nitric oxide and superoxide biology. MicroRNAs and long noncoding RNAs are crucial epigenetic regulators of gene expression in vascular remodelling. We evaluate these pathways for potential therapeutic targeting from a clinical translational perspective. In summary, vascular remodelling, a coordinated modification of vascular structure and function, is crucial in cardiovascular disease pathology.

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

Increased expression of miR-320b in blood plasma of patients in response to SARS-CoV-2 infection

Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Recent research has demonstrated how epigenetic mechanisms regulate the host–virus interactions in COVID-19. It has also shown that microRNAs (miRNAs) are one of the three fundamental mechanisms of the epigenetic regulation of gene expression and play an important role in viral infections. A pilot study published by our research group identified, through next-generation sequencing (NGS), that miR-4433b-5p, miR-320b, and miR-16–2-3p are differentially expressed between patients with COVID-19 and controls. Thus, the objectives of this study were to validate the expression of these miRNAs using quantitative real-time polymerase chain reaction (qRT-PCR) and to perform in silico analyses. Patients with COVID-19 (n = 90) and healthy volunteers (n = 40) were recruited. MiRNAs were extracted from plasma samples and validated using qRT-PCR. In addition, in silico analyses were performed using mirPath v.3 software. MiR-320b was the only miRNA upregulated in the case group com-pared to the control group. The in silico analyses indicated the role of miR-320b in the regulation of the KITLG gene and consequently in the inflammatory process. This study confirmed that miR-320b can distinguish patients with COVID-19 from control participants; however, further research is needed to determine whether this miRNA can be used as a target or a biomarker.

LMNA -Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-derived iPSC Differentiation Support Cell type and Lineage-specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency.

LMNA -Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C ( LMNA ) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. Molecular mechanisms of disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA -Related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes (CM) and Epicardium-Derived Cells (EPDC). Data integration and comparative analyses of Patient and Control cells found cell type and lineage differentially expressed genes (DEG) with enrichment to support pathway dysregulation. Top DEG and enriched pathways included: 10 ZNF genes and RNA polymerase II transcription in Pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CM; LMNA and epigenetic regulation and DDIT4 and mTORC1 signaling in EPDC. Top DEG also included: XIST and other X-linked genes, six imprinted genes: SNRPN , PWAR6 , NDN , PEG10 , MEG3 , MEG8 , and enriched gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Maternal Hypermethylated Genes Contribute to Intrauterine Growth Retardation of Piglets in Rongchang Pigs.

The placenta is a crucial determinant of fetal survival, growth, and development. Deficiency in placental development directly causes intrauterine growth retardation (IUGR). IUGR can lead to fetal growth restriction and an increase in the mortality rate. The genetic mechanisms underlying IUGR development, however, remain unclear. In the present study, we integrated whole-genome DNA methylation and transcriptomic analyses to determine distinct gene expression patterns in various placental tissues to identify pivotal genes that are implicated with IUGR development. By performing RNA-sequencing analysis, 1487 differentially expressed genes (DEGs), with 737 upregulated and 750 downregulated genes, were identified in IUGR pigs (H_IUGR) compared with that in normal birth weight pigs (N_IUGR) (p < 0.05); furthermore, 77 miRNAs, 1331 lncRNAs, and 61 circRNAs were differentially expressed. The protein-protein interaction network analysis revealed that among these DEGs, the genes GNGT1, ANXA1, and CDC20 related to cellular developmental processes and blood vessel development were the key genes associated with the development of IUGR. A total of 495,870 differentially methylated regions were identified between the N_IUGR and H_IUGR groups, which included 25,053 differentially methylated genes (DMEs); moreover, the overall methylation level was higher in the H_IUGR group than in the N_IUGR group. Combined analysis showed an inverse correlation between methylation levels and gene expression. A total of 1375 genes involved in developmental processes, tissue development, and immune system regulation exhibited methylation differences in gene expression levels in the promoter regions and gene ontology regions. Five genes, namely, ANXA1, ADM, NRP2, SHH, and SMAD1, with high methylation levels were identified as potential contributors to IUGR development. These findings provide valuable insights that DNA methylation plays a crucial role in the epigenetic regulation of gene expression and mammalian development and that DNA-hypermethylated genes contribute to IUGR development in Rongchang pigs.

Hdac3-deficiency increases senescence-associated distention of satellite DNA and telomere-associated foci in osteoprogenitor cells.

Histone deacetylase 3 (Hdac3) is an epigenetic regulator of gene expression and interacts with skeletal transcription factors such as Runx2. We previously reported that conditional deletion of Hdac3 in Osterix-Cre recombinase-expressing osteoprogenitor cells (Hdac3 CKOOsx) caused osteopenia and increased marrow adiposity, both hallmarks of skeletal aging. We also showed that Runx2+ cells within osteogenic cultures of Hdac3-depleted bone marrow stromal cells (BMSCs) contain lipid droplets (LDs). Cellular senescence, a nonproliferative metabolically active state, is associated with increased marrow adiposity, bone loss, and aging. In this study, we sought to determine if Hdac3 depleted Runx2+ pre-osteoblasts from young mice exhibit chromatin changes associated with early cellular senescence and how these events correlate with the appearance of LDs. We first confirmed that BMSCs from Hdac3 CKOOsx mice have more Runx2 + LD+ cells compared with controls under osteogenic conditions. We then measured senescence-associated distention of satellite (SADS) DNA and telomere-associated foci (TAFs) in Hdac3 CKOOsx and control BMSCs. In situ, Runx2+ cells contained more SADS per nuclei in Hdac3 CKOOsx femora than in controls. Runx2+ BMSCs from Hdac3 CKOOsx mice also contained more SADS and TAFs per nuclei than Runx2+ cells from age-matched control mice in vitro. SADs and TAFs were present at similar levels in Runx2 + LD+ cells and Runx2 + LD- cells from Hdac3 CKOOsx mice. Hdac inhibitors also increased the number of SADS in Runx2 + LD+ and Runx2 + LD- WT BMSCs. Senolytics reduced viable cell numbers in Hdac3 CKOOsx BMSC cultures. These data demonstrate that the depletion of Hdac3 in osteochondral progenitor cells triggers LD formation and early events in cellular senescence in Runx2+ BMSCs through mutually exclusive mechanisms.

Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: Implications for novel therapy.

Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes.

Heat stress induced cytosine methylation in the coding region of Rubisco activase (Rca) reveals its genotype-specific expression in contrasting wheat genotypes

Rising environmental temperature has become a major concern in global wheat production since it has critical effects on plant growth,development and yield. Elevated temperatures have become. Further understanding of the processes behind the development of heattolerance is necessary for significant agricultural crops. Cytosine methylation in DNA plays an essential function in epigenetic regulation of gene expression at various developmental stages and environmental stress in plants. In this study, we report the comparative analysis of cytosine methylation in the coding region of the Rubisco activase (Rca) gene in heat tolerant (RAJ3765) and heat susceptible (HUW510)genotypes of wheat in four growth stages under control and heat-stressed conditions in CG, CHG and CGG contexts. We found that the overall 5-mC increased due to heat stress in HUW510 during tillering (25%), boot stage (25%), heading (48%) and anthesis (50%) as compared to RAJ3765 during tillering (21%), boot stage (4%), heading (4%) and anthesis (11%). Additionally, gene expression profiling by using qPCR at the different plant growth stages also showed the decline in the gene expression in the leaf samples in both the genotypes due to heat stress, with a minimum in the susceptible genotype at the anthesis stage. The study will increase the knowledge on the molecular regulation of photosynthetic pathways by cytosine methylation which would assist in selection and manipulation of heat tolerance in wheat.

Temporal dynamics and metagenomics of phosphorothioate epigenomes in the human gut microbiome.

Epigenetic regulation of gene expression and host defense is well established in microbial communities, with dozens of DNA modifications comprising the epigenomes of prokaryotes and bacteriophage. Phosphorothioation (PT) of DNA, in which a chemically-reactive sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is catalyzed by dnd and ssp gene families widespread in bacteria and archaea. However, little is known about the role of PTs or other microbial epigenetic modifications in the human microbiome. Here we optimized and applied fecal DNA extraction, mass spectrometric, and metagenomics technologies to characterize the landscape and temporal dynamics of gut microbes possessing PT modifications. Exploiting the nuclease-resistance of PTs, mass spectrometric analysis of limit digests of PT-containing DNA reveals PT dinucleotides as part of genomic consensus sequences, with 16 possible dinucleotide combinations. Analysis of mouse fecal DNA revealed a highly uniform spectrum of 11 PT dinucleotides in all littermates, with PTs estimated to occur in 5-10% of gut microbes. Though at similar levels, PT dinucleotides in fecal DNA from 11 healthy humans possessed signature combinations and levels of individual PTs. Comparison with a widely distributed microbial epigenetic mark, m6dA, suggested temporal dynamics consistent with expectations for gut microbial communities based on Taylor's Power Law. Application of PT-seq for site-specific metagenomic analysis of PT-containing bacteria in one fecal donor revealed the larger consensus sequences for the PT dinucleotides in Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria, which differed from unbiased metagenomics and suggested that the abundance of PT-containing bacteria did not simply mirror the spectrum of gut bacteria. PT-seq further revealed low abundance PT sites not detected as dinucleotides by mass spectrometry, attesting to the complementarity of the technologies. The results of our studies provide a benchmark for understanding the behavior of an abundant and chemically-reactive epigenetic mark in the human gut microbiome, with implications for inflammatory conditions of the gut.

MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

The microbiota-gut-brain-immune interface in the pathogenesis of neuroinflammatory diseases: a narrative review of the emerging literature.

Research is beginning to elucidate the sophisticated mechanisms underlying the microbiota-gut-brain-immune interface, moving from primarily animal models to human studies. Findings support the dynamic relationships between the gut microbiota as an ecosystem (microbiome) within an ecosystem (host) and its intersection with the host immune and nervous systems. Adding this to the effects on epigenetic regulation of gene expression further complicates and strengthens the response. At the heart is inflammation, which manifests in a variety of pathologies including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Multiple Sclerosis (MS). Generally, the research to date is limited and has focused on bacteria, likely due to the simplicity and cost-effectiveness of 16s rRNA sequencing, despite its lower resolution and inability to determine functional ability/alterations. However, this omits all other microbiota including fungi, viruses, and phages, which are emerging as key members of the human microbiome. Much of the research has been done in pre-clinical models and/or in small human studies in more developed parts of the world. The relationships observed are promising but cannot be considered reliable or generalizable at this time. Specifically, causal relationships cannot be determined currently. More research has been done in Alzheimer's disease, followed by Parkinson's disease, and then little in MS. The data for MS is encouraging despite this. While the research is still nascent, the microbiota-gut-brain-immune interface may be a missing link, which has hampered our progress on understanding, let alone preventing, managing, or putting into remission neurodegenerative diseases. Relationships must first be established in humans, as animal models have been shown to poorly translate to complex human physiology and environments, especially when investigating the human gut microbiome and its relationships where animal models are often overly simplistic. Only then can robust research be conducted in humans and using mechanistic model systems.

Reprogramming tendon healing: a guide to novel molecular tools.

Tendons are a frequent site of injury, which greatly impairs the movement and locomotion of patients. Regrettably, injuries at the tendon frequently require surgical intervention, which leads to a long path to recovery. Moreover, the healing of tendons often involves the formation of scar tissue at the site of injury with poor mechanical properties and prone to re-injury. Tissue engineering carries the promise of better and more effective solutions to the improper healing of tendons. Lately, the field of regenerative medicine has seen a significant increase in the focus on the potential use of non-coding RNAs (e.g., siRNAs, miRNAs, and lncRNAs) as molecular tools for tendon tissue engineering. This class of molecules is being investigated due to their ability to act as epigenetic regulators of gene expression and protein production. Thus, providing a molecular instrument to fine-tune, reprogram, and modulate the processes of tendon differentiation, healing, and regeneration. This review focuses particularly on the latest advances involving the use of siRNAs, miRNAs, and lncRNAs in tendon tissue engineering applications.

High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

Role of sirtuins in epigenetic regulation and aging control.

Advances in modern healthcare in developed countries make it possible to extend the human lifespan, which is why maintaining active longevity is becoming increasingly important. After the sirtuin (SIRT) protein family was discovered, it started to be considered as a significant regulator of the physiological processes associated with aging. SIRT has deacetylase, deacylase, and ADP-ribosyltransferase activity and modifies a variety of protein substrates, including chromatin components and regulatory proteins. This multifactorial regulatory system affects many processes: cellular metabolism, mitochondrial functions, epigenetic regulation, DNA repair and more. As is expected, the activity of sirtuin proteins affects the manifestation of classic signs of aging in the body, such as cellular senescence, metabolic disorders, mitochondrial dysfunction, genomic instability, and the disruption of epigenetic regulation. Changes in the SIRT activity in human cells can also be considered a marker of aging and are involved in the genesis of various age-dependent disorders. Additionally, experimental data obtained in animal models, as well as data from population genomic studies, suggest a SIRT effect on life expectancy. At the same time, the diversity of sirtuin functions and biochemical substrates makes it extremely complicated to identify cause-and-effect relationships and the direct role of SIRT in controlling the functional state of the body. However, the SIRT influence on the epigenetic regulation of gene expression during the aging process and the development of disorders is one of the most important aspects of maintaining the homeostasis of organs and tissues. The presented review centers on the diversity of SIRT in humans and model animals. In addition to a brief description of the main SIRT enzymatic and biological activity, the review discusses its role in the epigenetic regulation of chromatin structure, including the context of the development of genome instability associated with aging. Studies on the functional connection between SIRT and longevity, as well as its effect on pathological processes associated with aging, such as chronic inflammation, fibrosis, and neuroinflammation, have been critically analyzed.