Epigenomic Changes Research Articles

ASH2L Controls Ureteric Bud Morphogenesis through the Regulation of RET/GFRA1 Signaling Activity in a Mouse Model.

Causes of congenital anomalies of the kidney and urinary tract (CAKUT) remain unclear. The authors investigated whether and how inactivation of Ash2l -which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide histone H3 lysine K4 (H3K4) methylation-might contribute to CAKUT. In a mouse model, inactivation of Ash2l in the ureteric bud (UB) lineage led to CAKUT-like phenotypes. Removal of ASH2L led to deficient H3K4 trimethylation, which slowed cell proliferation at the UB tip, delaying budding and impairing branching morphogenesis. The absence of ASH2L also downregulated the expression of Ret , Gfra1 , and Wnt11 genes involved in RET/GFRA1 signaling. These findings identify ASH2L-mediated H3K4 methylation as an upstream epigenetic regulator of signaling crucial for UB morphogenesis and indicate that deficiency or dysregulation of these processes may lead to CAKUT. Ureteric bud (UB) induction and branching morphogenesis are fundamental to the establishment of the renal architecture and are key determinants of nephron number. Defective UB morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor rearranged during transfection (RET) and coreceptor GFRA1 seems to be particularly important in UB development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT. To investigate whether and how inactivation of Ash2l , which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the UB lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with Cleavage Under Targets and Tagmentation sequencing. UB-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the UB tip, delaying budding and impairing UB branching morphogenesis. These effects were associated with downregulation of Ret , Gfra1 , and Wnt11 , which participate in RET/GFRA1 signaling. These experiments identify ASH2L-dependent H3K4 methylation in the UB lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in UB morphogenesis, which, if deficient, may lead to CAKUT.

Emerging Molecular Targets for Anti-Epileptogenic and Epilepsy Modifying Drugs.

The pharmacological treatment of epilepsy is purely symptomatic. Despite many decades of intensive research, causal treatment of this common neurologic disorder is still unavailable. Nevertheless, it is expected that advances in modern neuroscience and molecular biology tools, as well as improved animal models may accelerate designing antiepileptogenic and epilepsy-modifying drugs. Epileptogenesis triggers a vast array of genomic, epigenomic and transcriptomic changes, which ultimately lead to morphological and functional transformation of specific neuronal circuits resulting in the occurrence of spontaneous convulsive or nonconvulsive seizures. Recent decades unraveled molecular processes and biochemical signaling pathways involved in the proepileptic transformation of brain circuits including oxidative stress, apoptosis, neuroinflammatory and neurotrophic factors. The "omics" data derived from both human and animal epileptic tissues, as well as electrophysiological, imaging and neurochemical analysis identified a plethora of possible molecular targets for drugs, which could interfere with various stages of epileptogenetic cascade, including inflammatory processes and neuroplastic changes. In this narrative review, we briefly present contemporary views on the neurobiological background of epileptogenesis and discuss the advantages and disadvantages of some more promising molecular targets for antiepileptogenic pharmacotherapy.

The influence of exogenic lactoferrin on DNA methylation in postimplantation mouse embryos developed from zygotes exposed to bisphenol A

BACKGROUND: Bisphenol A is a chemical agent ubiquitous in plastic consumer products and a toxin capable of disrupting key epigenetic mechanisms in early embryogenesis. It becomes more and more clear that early development changes in epigenetic pathways caused by exposure to toxic substances are associated with various adult diseases. Therefore the need to identify new agents capable of eliminating epigenetic mechanisms failures caused by the bisphenol A toxin becomes evident. Here we suggest lactoferrin as a normalizer of toxicant-induced epigenomic changes. Currently there is no data on the role of lactoferrin as a normalizer of epigenomic disorders under the influence of toxicants. We assume that in mammalian embryogenesis lactoferrin might function as an epigenetic modulating factor.
 AIM: The aim of the research is to study effects of lactoferrin on the epigenetic status of postimplantation mouse embryos, exposed to bisphenol A in utero.
 MATERIALS AND METHODS: In this study, 3 experimental groups of mice and two control group were used. 1. Mice on the first day of pregnancy, injected with 40 mg/kg of body weight of bisphenol A; 2. Mice on the first day of pregnancy, injected with 50 mg/kg of body weight of lactoferrin; 3. Mice on the first day of pregnancy, successively injected with 50 mg/kg body weight of lactoferrin and 40 mg/kg of body weight of bisphenol A. On the 15th day of embryonic development, the level of genome-wide DNA methylation was evaluated in different body parts of the embryos by methyl-sensitive restriction and ImageJ visualization analysis.
 RESULTS: We demonstrated that in post-implantation mouse embryos, exposure to bisphenol A in the prenatal period caused an increased level of genome-wide DNA methylation. The most prominent effects were observed in brain and abdominal section of the embryos. Together, the present findings confirmed that lactoferrin administration at a dose of 50 mg/kg of body weight resulted in normalization of genome-wide DNA methylation levels after bisphenol A-induced epigenetic alterations.
 CONCLUSIONS: We assume that lactoferrin may partially neutralize the harmful effects of bisphenol A caused aberrant methylation, and thus can potentially be used as a pharmaceutical product. Factual findings of the present study may help by development of new therapeutic approaches. Nevertheless, further research of the bisphenol A, lactoferrin and lactoferrin + bisphenol A effects on reactive oxygen species and/or antioxidant enzymes is needed.

Chromatin Landscape Governing Murine Epidermal Differentiation

Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells invivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.

Activation of Notch signaling in endothelium cause upregulation of N-terminal acetylated histone 1

Highlights. Notch signaling is known to be important regulator of endothelium homeostasis and cardiovascular disease. Particularly, Notch seems to be associated with pathological changes in endothelium epigenome although no such Notch effects have been found. We have discovered that activation of Notch signaling alters histone 1 repertoire in the human endothelial cells and this is the first example of epigenomic Notch targets.Aim. The disturbance of blood flow and alteration of physiological shear stress is one of the main reasons for endothelial dysfunction. Mechanosensitive and dosedependent Notch pathway is assumed to be an important player of endothelial dysfunction progression, but the molecular mechanisms of the influence of Notch dysregulation on endothelium are still not understood. In particular, there is no data about possible targets of Notch in the endothelial epigenome.Methods. Here we focused on the analysis of changes in histone code of human umbilical vein endothelial cells (HUVEC) after activation of Notch. For this purpose, we transduced cells by lentiviruses with construction for Notch 1 intracellular domain (N1ICD) overexpression or by empty vector (control). Then we isolated histone enriched fraction and secretome proteins and performed their shotgun proteomics analysis on timsToF Pro instrument. Proteomics data are available via ProteomeXchange with identifier PXD032978.Results. We found the shift in proteomics profile of HUVEC caused by Notch activation and, particularly, the increase in the levels of N-terminal acetylated forms of histone 1: H1-0, H1-3, H1-4, H1-5, H1-10. We also found changes in the cell secretome profile which are associated with the decrease in proangiogenic effect of HUVEC secretome.Conclusion. Our data identified epigenomic Notch targets and we assume that changes in H1 repertoire might be associated with cardiovascular disease progression in vivo.

Set7 methyltransferase roles in myocardial protection from chronic stressors.

Epigenome changes in chronic states of cardiovascular stress including diabetes, pressure overload and cardiomyopathies frequently involve changes in open chromatin and post-translation modifications of histone lysine residues at specific amino acid positions by acetylation, methylation and phosphorylation. Since the discovery of Set7 as an important regulator of histone H3 lysine 4 methylation state, there has been wide interest in its role in cardiovascular remodeling and cardiac dysfunction. Recent transcriptome and Fourier transform infrared spectroscopy analyses and in vivo assessments of cardiac function by Lunardon and colleagues now reveal a clear role of Set7 in the regulation of the extracellular matrix composition and cardiac hypertrophy in response to chronic isoproterenol induced cardiac stress.

Epigenetic effects of high-fat diet on intestinal tumorigenesis in C57BL/6J-ApcMin/+ mice

Obesity and obesogenic diets might partly accelerate cancer development through epigenetic mechanisms. To determine these early effects, we investigated the impact of three days of a high-fat diet on epigenomic and transcriptomic changes in Apc Min/+ murine intestinal epithelia. ChIP-Seq and RNA-Seq were performed on small intestinal epithelia of WT and Apc Min/+ male mice fed high-fat diet (HFD) or low-fat diet (LFD) for three days to identify genomic regions associated with differential H3K27ac levels as a marker of variant enhancer loci (VELs) as well as differentially expressed genes (DEGs). Regarding epigenetic and transcriptomic changes, diet type (LFD vs. HFD) showed a significant impact, and genotype (WT vs.Apc Min/+) showed a small impact. Compared to LFD, HFD resulted in 1306 gained VELs, 230 lost VELs, 133 upregulated genes, and 127 downregulated genes in WT mice, with 1056 gained VELs, 371 lost VELs, 222 upregulated genes, and 182 downregulated genes in Apc Min/+ mice. Compared to the WT genotype, the Apc Min/+ genotype resulted in zero changed VELs for either diet type group, 21 DEGs for LFD, and 48 DEGs for HFD. Most gained VELs, and upregulated genes were associated with lipid metabolic processes. Gained VELs were also associated with Wnt signaling. Downregulated genes were associated with antigen presentation and processing. Three days of HFD-induced epigenomic and transcriptomic changes involving metabolic and immunologic pathways that may promote tumor growth in the genetically predisposed murine intestine without affecting key cancer signaling pathways.

SEX-DIMORPHIC REGULATION OF INNATE IMMUNITY DURING AGING

Abstract Aging is accompanied by striking changes in chromatin and gene expression across cell types and species. Yet, how chromatin landscapes change with age and regulate transcription, and how epigenomic changes in turn influence aging in response to external or internal cues, is largely unknown. In addition, accumulating evidence indicates that sex hormones play a key role in driving aspects of cellular and molecular sex-dimorphism. In parallel to sex hormones, karyotypic sex (i.e. XX vs. XY) is also likely to have important impact outside of gonadal sex determination. A key compartment whose activity can be actively modulated by sex-dimorphic mechanisms throughout life is the immune system, whose function declines sharply with aging and may be actively modulated by sex. Indeed, aspects of the immune responses differ between sexes, with a more robust immune response in females and an increased susceptibility to infection in males. Thus, our main cell models of study are key components of the innate immune system and the inflammatory response: macrophages, which accomplish key tasks such as phagocytosis, antigen presentation and cytokine production, and neutrophils, the most abundant leukocyte type serving as a “first line of defense” against infection. Excitingly, we and others have observed strong sex-related differences in the transcriptional and functional phenotypes of these cells and have observed sex-dimorphic “omic” trajectories for these cells with aging. Based on our data and published literature, it is likely that mechanisms involving both gonadal hormones and sex chromosomes may fine-tune different aspects of immunity and, thus, overall health and lifespan.

Impact of Betamethasone Pretreatment on Engrafment of Cord Blood-Derived Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) transplantation is crucial to cure hematologic malignancies. Umbilical cord blood (UCB) is a source of stem cells, but 90% of UCB units are discarded due to low cellularity. Improving the engraftment capacities of CD34+ stem cells would allow the use of UCB that were so far rejected. Betamethasone induces long-term transcriptomic and epigenomic changes in immune cells through glucocorticoid receptor. We hypothesize that discarded UCB could be used owing to improvements induced by betamethasone. Isolated CD34+ HSC from UCB were exposed to the synthetic glucocorticoids betamethasone and fluticasone for 20 h, and cell phenotype was determined before transplantation. NSG mice were sub-lethally irradiated (1 Gy or 2 Gy) 6 h before intravenously transferring 2–5 × 105 CD34+ HSC. The peripheral blood engraftment levels and the leukocyte subsets were followed up for 20 weeks using flow cytometry. At end point, the engraftment and leukocyte subsets were determined in the spleen and bone marrow. We demonstrated that betamethasone has surprising effects in recovering immune system homeostasis. Betamethasone and fluticasone increase CXCR4 and decrease HLA class II and CD54 expression in CD34+ HSCs. Both glucocorticoids-exposed cells showed a similar engraftment in 2 Gy-irradiated NSG mice. Interestingly, betamethasone-exposed cells showed enhanced engraftment in 1 Gy-irradiated NSG mice, with a trend to increase regulatory T cell percentage when compared to control. Betamethasone induces alterations in CD34+ HSCs and improve the engraftment, leading to a faster immune system recovery, which will contribute to engrafted cells survival.

Regional mutational signature activities in cancer genomes.

Cancer genomes harbor a catalog of somatic mutations. The type and genomic context of these mutations depend on their causes and allow their attribution to particular mutational signatures. Previous work has shown that mutational signature activities change over the course of tumor development, but investigations of genomic region variability in mutational signatures have been limited. Here, we expand upon this work by constructing regional profiles of mutational signature activities over 2,203 whole genomes across 25 tumor types, using data aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium. We present GenomeTrackSig as an extension to the TrackSig R package to construct regional signature profiles using optimal segmentation and the expectation-maximization (EM) algorithm. We find that 426 genomes from 20 tumor types display at least one change in mutational signature activities (changepoint), and 306 genomes contain at least one of 54 recurrent changepoints shared by seven or more genomes of the same tumor type. Five recurrent changepoint locations are shared by multiple tumor types. Within these regions, the particular signature changes are often consistent across samples of the same type and some, but not all, are characterized by signatures associated with subclonal expansion. The changepoints we found cannot strictly be explained by gene density, mutation density, or cell-of-origin chromatin state. We hypothesize that they reflect a confluence of factors including evolutionary timing of mutational processes, regional differences in somatic mutation rate, large-scale changes in chromatin state that may be tissue type-specific, and changes in chromatin accessibility during subclonal expansion. These results provide insight into the regional effects of DNA damage and repair processes, and may help us localize genomic and epigenomic changes that occur during cancer development.

Abstract A007: Integrative multiomic analysis reveals NOTCH signaling is derepressed by loss of PRC2 in malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis Type 1 syndrome (NF1) is a cancer predisposition syndrome caused by inheritance of one loss of function allele of the NF1 gene. NF1 patients can develop malignant peripheral nerve sheath tumors (MPNST), a deadly soft tissue sarcoma. MPNSTs develop after somatic loss of the wild-type NF1 allele, resulting in an increase in Ras-GTP activated signaling. This malignant transformation is still not completely understood, but loss of TP53 or CDKN2A/2B function and the polycomb repressor complex 2 (PRC2) are common events during the transition to MPNST. SUZ12, EED and EZH2 are core components of PRC2, which is responsible for trimethylation of Histone H3 at lysine 27 (H3K27me3), a repressive epigenetic mark that silences genes through formation of heterochromatin. We hypothesized loss of PRC2 has direct and indirect effects on gene expression resulting in MPNSTs. PRC2 loss may result in altered topologically associated domains, which can affect access of promoters by distal enhancers. Altered gene expression leads to deregulation of cell differentiation and proliferation controls, promoting the transition to MPNSTs. The purpose of this study is to identify epigenomic vulnerabilities of MPNSTs using multi-omics to elucidate more effective treatments. We have engineered NF1-deficient human Schwann cells with or without concomitant loss of function SUZ12 or EED mutations. We found major epigenomic changes in the histone code of SUZ12 mutants including complete loss of H3K27me3 with concomitant gain in H3K27 acetylation. SUZ12-deficient cells also become hypersensitive to histone deacetylase inhibitors. RNA sequencing has revealed many differentially expressed genes when SUZ12 and NF1 are lost in our engineered cell lines. Preliminary results show 92 differentially expressed genes that are common to PRC2-deficient MPNSTs and engineered cell lines. 824 genes are common between our engineered cell lines where 686 of these are derepressed when SUZ12 or EED are lost with NF1. We also identified an increase in differentially expressed genes when PRC2 is lost with NF1 versus NF1 loss alone. Comparing these to genes expressed in MPNST patient samples, we have identified potential drivers of MPNST generation. Pathway enrichment analysis on differentially expressed genes indicates many upregulated cancer related pathways when PRC2 is lost. We found NOTCH and Sonic Hedgehog signaling are common to all comparisons. NOTCH signaling has been implicated in Schwann cell development. These are currently being validated via Western blot. Proteomics data will also be compared to the findings. Citation Format: Minu Bhunia, Christopher M. Stehn, Mahathi Madala, Kyle Williams, Alex Larsson, Tyler Jubenville, Suganth Suppiah, Gelareh Zadeh. Integrative multiomic analysis reveals NOTCH signaling is derepressed by loss of PRC2 in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A007.

Single-base resolution methylome of different ecotype from Pyrus betulaefolia reveals epigenomic changes in response to salt stress

• Drawing the cytosine methylation map across the whole genome of Pyrus betulaefolia roots. • The changes of methylomes in two ecotypes after salt exposed dependent on their salt tolerance abilities. • DNA demethylation induced higher expression levels of some Ca 2+ sensor genes in P. betulaefolia roots. • The higher K + /Na + ratio in the roots of salt-tolerance ecotype is relationship with Ca 2+ sensor genes transcription causing by hypomethylation. The root is the first organ to feel salt stress in woody fruit trees. Understanding the potential epigenetic mechanism induced by salt stress in roots of Pyrus betulaefolia is important for pear breeding using diverse genetic resources. With the help of whole-genome bisulfite sequencing, cytosine methylation was mapped at single-base resolution across the whole genome of P. betulaefolia roots. The P. betulaefolia root genome revealed nearly 11.40%, 39.93%, 22.73%, and 4.17% methylation across all sequenced C sites and in the CG, CHG, and CHH sequence contexts, respectively. Then, the changes in the methylome of the roots relating to salt stress were comparatively analysed in two ecotypes with different salt-stress tolerances. After a global methylome and transcriptome combined analysis, gene body methylation demonstrated an obvious negative correlation with transcription levels, whereas promoter-methylated genes presented lower expression levels than promoter-unmethylated genes. Furthermore, the methylation profiles of Ca 2+ sensor genes in roots of P. betulaefolia . were described, and some were demethylated under salt-stress conditions. In detail, the up-regulation of Ca 2+ sensors and their downstream genes, PbCDPK20.1, PbCDPK20.2, PbAKT1 , and PbHAK25 , resulted from salt-induced promoter methylation changes . Finally, K + accumulation was promoted, and a higher K + /Na + ratio was maintained in roots under salt-stress compared with normal conditions. Additionally, the application of the DNA methylation inhibitor 5-azacytidine in vitro induced DNA demethylation and promoted K + enrichment, whereas the accelerator methyl trifluoromethanesulfonate produced the opposite effects. In conclusion, our result revealed the relationship of DNA methylation and gene expression in P. betulaefolia during salt stress, which will increase our understanding of the epigenetic mechanisms of woody trees.

Epigenomic landscape study reveals molecular subtypes and EBV-associated regulatory epigenome reprogramming in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) latent infection is associated with genome-wide epigenomic changes in several malignancies, but its role in epigenetic dysregulation remains unclear in nasopharyngeal carcinoma (NPC). To investigate EBV-associated epigenetic dysregulation, we performed a multi-omics study by integrating whole-genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-Seq) data. In addition to the known global DNA hypermethylated subtype, we discovered a novel subtype with global hypomethylation in EBV+NPC. The consistent EBV-specific differentially methylated regions (EBV-DMRs) in the human genome were identified from both subtypes and associated with loss of CTCF binding (P<2.2e-16). Importantly, CTCF is a master chromatin regulator and CTCF protein was reduced in 45% of NPC cases, especially in those with advanced NPC (Stage IV vs. others: 62% vs. 38%, P=0.034). This result links EBV with chromatin changes. The ATAC-Seq data suggest regulatory epigenome reprogramming through chromatin accessibility changes in EBV+NPC with altered CTCF binding and the switch of transcription factor binding from differentiation-associated KLF/SP family to the innate and adaptive immunity-related NF-ĸB and IRF families. Detailed chromatin accessibility analysis identified a potential EBV target gene CD74, which mediated EBV-specific cell-cell communications in the tumor microenvironment (TME) and was strongly correlated with Tcell exhaustion (r2=0.55). Our study reveals the unexpected epigenetic heterogeneity, providing insights into NPC pathogenesis and highlighting the involvement of host factors in virus-associated epigenetic changes. EBV infection is associated with epigenome reprogramming and may promote immune evasion. This study was funded by the Hong Kong Research Grants Council grant (AoE/M-06/08) to MLL, General Research Fund (17103218 and 17102619) and seed funding for basic research (201611159158) to WD, and General Research Fund (17119618) to HC.

Single‐nucleus open chromatin accessibility landscape of Pick’s and Alzheimer’s disease

AbstractBackgroundPick’s disease (PiD), a behavioral variant of frontotemporal dementia, is one of the common neurodegenerative dementia that is characterized by tau lesions. Despite the distinct tau aggregation observed in Pick’s disease compared with the tauopathy of Alzheimer’s disease (AD), the similarity in cognitive and behavioral impairments during the progression of the disease make their diagnosis challenging. What determines the differences and similarities between PiD and AD progression, especially at the epigenetic level, are largely unknown. We believe that recent advancement in single‐cell epigenomic profiling methods, Assay for Transposase‐Accessible Chromatin (ATAC) combined with high throughput sequencing, will enable us to map the chromatin‐regulatory landscapes of disease brains at a single‐cell resolution and enable us to understand underlying molecular changes in these diseases.MethodIn the present study, we have isolated single nuclei from the frontal cortex region and performed single‐nucleus ATAC sequencing (snATAC‐seq) of 198,722 nuclei to generate cell‐type specific chromatin accessibility profiles of postmortem brains of PiD and AD patients and to uncover their cellular heterogeneity and similarity. We used cellranger to process the paired‐end FastQ reads of each sample to obtain its chromatin accessibility count matrix. Next, we utilized ArchR and Signac pipelines to generate 501‐bp fixed‐width peak regions and performed downstream differential accessible regions analysis. Quality control was also performed to remove the contribution of low‐quality nuclei.ResultWe constructed chromatin accessibility profiles from 19,690 nuclei from PiD patients, 59,148 nuclei from AD patients, and 119,884 nuclei from healthy controls. We applied UMAP dimensionality reduction and clustered nuclei through open chromatin region to the batch‐corrected epigenomic datasets. We identified seven distinct cell types, including astrocyte, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, OPC, and pericyte/endothelial cells, and we applied pseudotime trajectory analysis to characterize disease‐associated cell state in both AD and PiD at the epigenomics level.ConclusionOur data identified the cell‐type‐specific open chromatin accessible regions in AD and PiD brains. Although the causative molecular mechanisms of AD and PiD remain unknown, our work helps to identify shared epigenomic changes in AD and PiD, especially in regards to cell‐type‐specific genomic loci with disease risk.

Generation and multiomic profiling of a TP53/CDKN2A double-knockout gastroesophageal junction organoid model.

Inactivation of the tumor suppressor genes tumor protein p53 (TP53) and cyclin-dependent kinase inhibitor 2A (CDKN2A) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated TP53 and CDKN2A knockout (TP53/CDKN2AKO) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2AKO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

Эпигеномные изменения в лейкоцитах периферической крови у пациентов с гипертонической болезнью и дисциркуляторной энцефалопатией

В настоящее время эпигеномный ландшафт, лежащий в основе гипертонической болезни и дисциркуляторной энцефалопатии, мало изучен. Идентификация эпигенетического паттерна в комплексе с генетическим паспортом индивида может предоставить дополнительную информацию об этиологии заболеваний и риске развития осложнений, а также о выборе стратегии и тактики лечения. В представленной работе проведена количественная оценка уровня полногеномного метилирования ДНК и степени компактизации хроматина в лейкоцитах периферической крови у пациентов с гипертонической болезнью и дисциркуляторной энцефалопатией и здоровых добровольцев из Санкт-Петербурга. У пациентов с гипертонической болезнью выявлены гендерные различия в риске развития дисциркуляторной энцефалопатии. По сравнению с контролем при гипертонической болезни повышены уровень метилирования ДНК и степень компактизации хроматина. В исследованных группах у мужчин и женщин обнаружена различная эпигеномная реакция лейкоцитов на лекарственную терапию при данных заболеваниях. Currently, the epigenomic landscape underlying hypertension and dyscirculatory encephalopathy is poorly understood. Identification of the epigenetic pattern in combination with genetic passport can provide additional information about the etiology of diseases and the risk of complications, as well as the choice of strategy and tactics of treatment. We carried out a quantitative assessment of the level of genome-wide DNA methylation and the degree of chromatin compaction in peripheral blood leukocytes in patients with hypertension and dyscirculatory encephalopathy, and also in healthy volunteers from St. Petersburg. Gender differences in the risk of developing dyscirculatory encephalopathy were revealed in patients with hypertension. It was found that the level of DNA methylation and the degree of chromatin compaction are increased in hypertension (compared to control). In the studied groups, men and women showed different epigenomic reactions of leukocytes to drug therapy in these pathologies.

Genome-wide DNA methylation study identifies significant epigenomic changes associated with internalized stigma in adults with non-specific chronic low back pain.

Non-specific chronic low back pain (cLBP) represents a common musculoskeletal condition with no identifiable cause. It cannot be diagnosed with conventional neuroimaging techniques such as computerized tomography (CT). The diagnostic uncertainty that characterizes non-specific cLBP can lead to stigmatizing responses from others that can become internalized Among individuals with non-specific cLBP, internalized stigma is associated with greater pain intensity and disability. Yet, no study has examined the biological mechanism linking high internalized stigma to worse outcomes in individuals with non-specific cLBP. We aimed to identify differentially methylated loci (DML), enrichment pathways, and associated network interactions among individuals with non-specific cLBP experiencing low vs. high internalized stigma. We examined DNA methylation in whole blood samples from 48 adults, ages 19-85, using reduced representation bisulfite sequencing (RRBS). After controlling for age, sex, race, and multiple testing, differentially methylated loci (DML) differed in adults with low vs. high internalized stigma by at least 10% and q < 0.01 in 3,665 CpG sites: 2,280 hypomethylated and 1,385 hypermethylated. Gene ontology (GO) analyses of the annotated genes from these sites revealed significant enrichment of 274 biological processes, 29 cellular components, and 24 molecular functions (adjusted p < 0.05). The top enriched molecular functions regulate protein binding and DNA binding of transcription factor activity. Pathway analyses indicated that many functional genomic pathways, including Hippo Signaling, Melanogenesis, and Pathways in Cancer, were enriched with differentially methylated genes. Also, there was a significant interaction between relevance pathways such as P53, mTOR, PI3K-Akt, and Wnt signaling pathways. These pathways have previously been associated with neuroinflammation, neurodegeneration, and stress-related conditions. Thus, findings point to possible stress-induced DNAm changes as the link between high levels of internalized stigma and worse outcomes in adults with non-specific cLBP.

Myocardial effects of a low-protein diet in experimental kidney dysfunction

THE AIM: to evaluate the effect of low protein diet supplemented with ketosteril on morphological and epigenomic changes in the myocardium of Wistar rats with simulated kidney dysfunction . MATERIALS AND METHODS. The work was performed on male Wistar rats subjected to 5/6 nephrectomy (NE). The first group after NE received a standard diet (20.16% animal protein ), the second – low protein diet (LPD), including 10% ketosteril . Control rats received a standard diet . After 4 months , blood pressure (BP) and left ventricular mass index (LVMI) were assessed in rats , and a histological examination of the myocardium was performed . In the myocardium , the relative expression levels of NF- kB , miRNA-21, miRNA-133, and miRNA-203 were determined . RESULTS. After 4 months in rats with NE on a standard diet , an increase in blood pressure , an increase in the mass index of the LV myocardium was recorded . MBD with the inclusion of 10% Ketosteril slowed down the growth of systolic blood pressure and the development of LV myocardial hypertrophy in rats with kidney dysfunction . At the histological level , the use of LPD provided a decrease in the degree of hypertrophy of cardiomyocytes and degenerative changes in cardiomyocytes . Animals treated with LPD had less pronounced diffuse and perivascular fibrosis compared with animals fed normal food . The use of MBD slowed down the increase in the relative level of expression of the NFκB gene and miRNA-21 in the myocardium of rats with NE and promoted an increase in the expression level of miRNA-133 and miRNA-203 compared to the indices of animals with NE that received standard food . CONCLUSION: long-term use of LPD with the use of ketoanalogues of essential amino acids can have a potential cardioprotective effect in CKD, slowing down the growth of blood pressure , an increase in LV myocardial mass and the formation of structural changes in the myocardium . It is possible that a decrease in the expression of NF- kB and miRNA-21, as well as an increase in the expression of miRNA-203 and 133 in the myocardium , can play a significant role in this .

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Tumor genomic landscape in smokers compared to non-smoker patients with locally advanced or metastatic urothelial carcinoma

By Nishita Tripathi, Nicolas Sayegh & 10 more. This study shows a need a future need of investigation of epigenomic changes and transcriptomic profiling to further elucidate the effect of smoking on bladder cancer.