Epigenetic Analysis Research Articles

Abstract 7552: Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome

Abstract Introduction: Colorectal pre-cancers in Lynch Syndrome (LS) display a distinct immune profile thus presenting a unique opportunity to develop novel immune-interception approaches to halt carcinogenesis. Epigenetic modulation by EZH2 of immune-related genes has been implicated in many cancers including colorectal but it has yet to be explored in LS carcinogenesis. The main objective of this study is to test the effect of EZH2 inhibition on immune cell profiling, colorectal carcinogenesis, gene expression, and histone methylation, using ex vivo organoids and a LS mouse model. Methods: A mouse model of LS colorectal carcinogenesis (Villin-Cre;Msh2fl/fl;TgfβRII-KI; herein referred to as VCMsh2THu) and ex vivo colonic organoids were utilized to assess the effects of the EZH2 inhibitor GSK503. Mice were treated for 9-weeks with GSK503 with downstream studies exploring polyp multiplicity, changes to the immune landscape, and epigenetic analyses to derive mechanistic insights into EZH2 regulation in LS. Results: GSK503 significantly altered transcriptomic changes in VCMsh2THu organoids with tissue-specific tropism for dysplastic organoids as compared to organoids prepared from adjacent normal mucosa. GSK503 significantly increased CD4+and CD8+ T cells in splenocytes and colonic mucosa of treated mice compared to controls in vitro and in vivo. A preventive-dose (200μg/kg) of GSK503 for 9 weeks significantly reduced adenoma multiplicity in the large intestine of VCMsh2THu mice with significant changes in the immune landscape of treated mice compared to controls. The data suggests GSK503 significantly enriched both total and activated cytotoxic T-lymphocytes CD8+/CD137+), stromal macrophages (CD68+), and activated helper T-lymphocytes (CD4+/CD134+) in the large intestine of VCMsh2THu mice. Transcriptome and molecular analyses using IHC, Western blots, and computational tools showed activation of immune and apoptotic markers with concomitant reduction of H3K27 methylation levels in colonic crypts. ChIPseq analyses revealed decreased levels of H3K27me3 and H3K4me1 (enhancer), a slight increased levels of H3K27ac (enhancer) marks with no changes in levels of H3K4me3 in treated mice, indicating that EZH2 inhibition yields global reprograming of poised promoters and active enhancers in the LS mouse genome. Conclusions: Collectively, our findings reveal that GSK503 has a promising potential as an immuno-interception modality establishing the framework for launching phase I clinical trials with EZH2 inhibitors for LS carriers. Citation Format: Fahriye Duzagac, Charles M. Bowen, Laura Reyes-Uribe, Mahira Zaheer, Jacklyn Thompson, Nan Deng, Melissa Taggart, Abhinav Jain, Winfried Edelmann, Krishna M. Sinha, Eduardo Vilar. Inhibition of histone methyltransferase EZH2 for immune-interception of colorectal cancer in Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7552.

Abstract 7651: Evaluating epigenetic markers for naive, memory and overall B cells for disease diagnosis and prognosis in patients with B-CLL

Abstract Chronic lymphocytic leukemia (CLL) is a hematological cancer classified as low-malignancy non-Hodgkin lymphoma that accounts for approximately a quarter of all leukemias. Since only some patients will develop aggressive cancer and require treatment, accurate disease prognosis is crucial. A wide range of parameters with prognostic value have been described, including age, clinical stage, mutational status, protein markers, and flow cytometry levels of CD19+CD38+ B cells above 20%. Here, we investigated epigenetic markers for B cell types including a marker for overall B cells, naïve B cells, memory B cells and IgM positive B cells in a cohort of more than 40 patients with B-CLL. When comparing to a cohort of more than 100 healthy donors, the epigenetic B cell marker identified all patients with 100% sensitivity and specificity. In addition, the epigenetic B cell markers for naïve and memory B cells identified with high sensitivity and excellent specificity a subgroup of B-CLL patients with CD19+CD38+ B cell levels above 20% which is associated with an unfavorable prognosis. The results suggest that the epigenetic analysis of B cells in capillary blood may be a promising tool for B-CLL diagnosis and disease prognosis. Citation Format: Konstantin Schildknecht, Kati Bourquain, Jeannette Werner, Janika Schulze, Steffi Walter, Eva Raschke, Deborah Phippard, Angelina Bisconte, Barbara Seliger, Claudia Wickenhauser, Sven Olek. Evaluating epigenetic markers for naive, memory and overall B cells for disease diagnosis and prognosis in patients with B-CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7651.

Retracted: Multiomics Analysis of Genetics and Epigenetics Reveals Pathogenesis and Therapeutic Targets for Atrial Fibrillation.

[This retracts the article DOI: 10.1155/2021/6644827.].

The Impact of Sample Storage on Blood Methylation: Towards Assessing Myelin Gene Methylation as a Biomarker for Progressive Multiple Sclerosis.

One of the major challenges in multiple sclerosis (MS) is to accurately monitor and quantify disability over time. Thus, there is a pressing need to identify new biomarkers for disease progression. Peripheral blood DNA methylation has been demonstrated to be an easily accessible and quantifiable marker in many neurodegenerative diseases. In this study, we aimed to investigate whether methylation patterns that were previously determined in chronic inactive white matter lesions of patients with progressive MS are also reflected in the blood, and whether the latter can serve as a biomarker for disease progression in MS. While our initial analysis revealed differences in the blood methylation state of important myelin-related genes between patients with progressive MS and controls, these findings could not be validated in other independent patient cohorts. Subsequent investigation suggests that sample storage can selectively influence DNA methylation patterns, potentially hindering accurate epigenetic analysis. Therefore, sample storage time should be taken into consideration during the initial sample selection stage in biomarker studies.

Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.

Epigenetic control of microglial immune responses.

Microglia, the major population of brain-resident macrophages, are now recognized as a heterogeneous population comprising several cell subtypes with different (so far mostly supposed) functions in health and disease. A number of studies have performed molecular characterization of these different microglial activation states over the last years making use of "omics" technologies, that is transcriptomics, proteomics and, less frequently, epigenomics profiling. These approaches offer the possibility to identify disease mechanisms, discover novel diagnostic biomarkers, and develop new therapeutic strategies. Here, we focus on epigenetic profiling as a means to understand microglial immune responses beyond what other omics methods can offer, that is, revealing past and present molecular responses, gene regulatory networks and potential future response trajectories, and defining cell subtype-specific disease relevance through mapping non-coding genetic variants. We review the current knowledge in the field regarding epigenetic regulation of microglial identity and function, provide an exemplary analysis that demonstrates the advantages of performing joint transcriptomic and epigenomic profiling of single microglial cells and discuss how comprehensive epigenetic analyses may enhance our understanding of microglial pathophysiology.

Nonpathogenic leaf-colonizing bacteria elicit pathogen-like responses in a colonization density-dependent manner.

Leaves are colonized by a complex mix of microbes, termed the leaf microbiota. Even though the leaf microbiota is increasingly recognized as an integral part of plant life and health, our understanding of its interactions with the plant host is still limited. Here, mature, axenically grown Arabidopsis thaliana plants were spray inoculated with six diverse leaf-colonizing bacteria. The transcriptomic changes in leaves were tracked over time and significant changes in ethylene marker (ARL2) expression were observed only 2-4 days after spray inoculation. Whole-transcriptome sequencing revealed that 4 days after inoculation, leaf transcriptional changes to colonization by nonpathogenic and pathogenic bacteria differed in strength but not in the type of response. Inoculation of plants with different densities of the nonpathogenic bacterium Williamsia sp. Leaf354 showed that high bacterial titers resulted in disease phenotypes and led to severe transcriptional reprogramming with a strong focus on plant defense. An in silico epigenetic analysis of the data was congruent with the transcriptomic analysis. These findings suggest (1) that plant responses are not rapid after spray inoculation, (2) that plant responses only differ in strength, and (3) that plants respond to high titers of nonpathogenic bacteria with pathogen-like responses.

Abstract P13: Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity

Abstract IG translocations occur in various B-cell neoplasms and result in the deregulation of (onco)genes due to their juxtaposition with regulatory elements in the immunoglobulin loci, predominantly the immunoglobulin heavy chain (IGH) locus (14q32). Some of these IGH translocations are pathognomonic for distinct disease entities, like those involving MYC, BCL2 or CCND1 in Burkitt, Follicular or Mantle Cell Lymphomas, respectively. In Chronic Lymphocytic Leukemia (CLL), the BCL3 gene (19q13), a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family, is a known partner gene in these translocations. Two subsets of IG::BCL3 translocation-positive B-cell neoplasms have been described that exhibit differences in chromosomal aberrations, IGHV mutation status, and histopathology [Martín-Subero et al. 2007]. In CLL, IG::BCL3 translocation is linked to a younger age at diagnosis, a more aggressive clinical course, and an atypical tumor cell phenotype [Michaux et al. 1997; Au et al. 2002]. A total of 89 B-cell neoplasms, primarily diagnosed as CLL, displaying IG::BCL3 translocation detected by fluorescence in situ hybridization (FISH), were examined. Analysis of DNA methylation and copy number aberrations (CNA) was conducted for 86 IGH::BCL3-translocated B-cell neoplasms using 850K EPIC BeadChip array data. Evaluation of chromosomal aberrations typical for CLL was moreover performed by FISH and the IGHV mutation status was determined. IGH::BCL3 breakpoint junctions were sequenced in 23 samples using targeted capture and whole-genome sequencing approaches. RNA expression of BCL3 was assessed using the HTG mRNA pan B-cell panel. In UMAP analysis of DNA methylation, CLLs with IG::BCL3 translocation exhibited segregation from other CLLs, irrespective of IGH translocations. Supervised analysis revealed global hypomethylation of B-cell neoplasms with IG::BCL3 translocation compared to other CLL. A total of 66/69 CLLs with IG::BCL3 translocation showed an unmutated IGHV status (96%). Copy number determination using BeadChip data (n=86) and FISH analysis (n=85) showed a significantly higher incidence of trisomy 12 in IG::BCL3-translocated cases compared to the general CLL population (p=0.011). Breakpoint sequencing indicated IG::BCL3 junctions to be recurrently associated with aberrant class-switch recombination at the IGH locus, involving IGHA (n=11/23), IGHG (n=5/23) and IGHM segments (n=5/23). Breakpoints at the BCL3 locus displayed two groups. 80 cases (all CLL) were located upstream of BCL3 and two other B-cell neoplasms (NHL, DLBCL) were located downstream of BCL3. BCL3 expression analysis in 15 IG::BCL3-translocated CLLs confirmed its transcriptional upregulation compared to the general CLL population. Our genetic and epigenetic analyses support the view that CLL with IG::BCL3 translocation may constitute a genetically and epigenetically distinct subtype of B-cell neoplasms, diverging from the typical CLL. Citation Format: Cosima Drewes, Cristina López, Nnamdi Okeke, Sina Hillebrecht, Petra Schütz, Anja Fischer, Anja Mottok, Susanne Bens, Christof Schneider, Stephan Stilgenbauer, Eugen Tausch, Reiner Siebert. Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P13.

Complex regulatory networks influence pluripotent cell state transitions in human iPSCs

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Loss of NSD2 causes dysregulation of synaptic genes and altered H3K36 dimethylation in mice.

Background: Epigenetic disruptions have been implicated in neurodevelopmental disorders. NSD2 is associated with developmental delay/intellectual disability; however, its role in brain development and function remains unclear. Methods: We performed transcriptomic and epigenetic analyses using Nsd2 knockout mice to better understand the role of NSD2 in the brain. Results and discussion: Transcriptomic analysis revealed that the loss of NSD2 caused dysregulation of genes related to synaptic transmission and formation. By analyzing changes in H3 lysine 36 dimethylation (H3K36me2), NSD2-mediated H3K36me2 mainly marked quiescent state regions and the redistribution of H3K36me2 occurred at transcribed genes and enhancers. By integrating transcriptomic and epigenetic data, we observed that H3K36me2 changes in a subset of dysregulated genes related to synaptic transmission and formation. These results suggest that NSD2 is involved in the regulation of genes important for neural function through H3K36me2. Our findings provide insights into the role of NSD2 and improve our understanding of epigenetic regulation in the brain.

Integrative Epigenetic and Molecular Analysis Reveals a Novel Promoter for a New Isoform of the Transcription Factor TEAD4.

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

Digital methylation-specific PCR: New applications for liquid biopsy.

Epigenetic analysis is a fundamental part of understanding pathophysiological processes with potential applications in diagnosis, prognosis, and assessment of disease susceptibility. Epigenetic changes have been widely studied in chronic obstructive pulmonary disease (COPD), but currently, there is no molecular marker used to improve the treatment of patients. Furthermore, this progressive disease is a risk factor for the development of more severe COVID-19. Methylation-specific polymerase chain reaction (MSP-PCR) plays an important role in the analysis of DNA methylation profiles, and it is one of the most widely used techniques. In this context, the combination of MSP-PCR with emerging PCR technologies, such as digital PCR (dPCR), results in more accurate analyses of the DNA methylation profile of the genes under study. In this study, we propose the application of the MSP-dPCR technique to evaluate the methylation profile of the ADAM33 gene from saliva samples and lung tissue biopsies of patients with COPD and COVID-19. MSP-dPCR generated a measurable prediction of gene methylation rate, with the potential application of this combined technology for diagnostic and prognostic purposes. It has also proven to be a powerful tool for liquid biopsy applications.

Smart selection of soil microbes for resilient and sustainable viticulture.

The grapevine industry is of high economic importance in several countries worldwide. Its growing market demand led to an acceleration of the entire production processes, implying increasing use of water resources at the expense of environmental water balance and the hydrological cycle. Furthermore, in recent decades climate change and the consequent expansion of drought have further compromised water availability, making current agricultural systems even more fragile from ecological and economical perspectives. Consequently, farmers' income and welfare are increasingly unpredictable and unstable. Therefore, it is urgent to improve the resilience of vineyards, and of agro-ecosystems in general, by developing sustainable and environmentally friendly farming practices by more rational biological and natural resources use. The PRIMA project PROSIT addresses these challenges by characterizing and harnessing grapevine-associated microbiota to propose innovative and sustainable agronomic practices. PROSIT aims to determine the efficacy of natural microbiomes transferred from grapevines adapted to arid climate to commonly cultivated grapevine cultivars. In doing so it will test those natural microbiome effects on drought tolerance. This multidisciplinary project will utilize in vitro culture techniques, bioimaging, microbiological tests, metabolomics, metabarcoding and epigenetic analyses. These will be combined to shed light on molecular mechanisms triggered in plants by microbial associations upon water stress. To this end it is hoped that the project will serve as a blueprint not only for studies uncovering the microbiome role in drought stress in a wide range of species, but also for analyzing its effect on a wide range of stresses commonly encountered in modern agricultural systems.

Enzyme-Free Dynamic DNA Reaction Networks for On-Demand Bioanalysis and Bioimaging.

ConspectusThe pursuit of in-depth studying the nature and law of life activity has been dominating current research fields, ranging from fundamental biological studies to applications that concern synthetic biology, bioanalysis, and clinical diagnosis. Motivated by this intention, the spatiotemporally controlled and in situ analysis of living cells has been a prospective branch by virtue of high-sensitivity imaging of key biomolecules, such as biomarkers. The past decades have attested that deoxyribonucleic acid (DNA), with biocompatibility, programmability, and customizable features, is a competitive biomaterial for constructing high-performance molecular sensing tools. To conquer the complexity of the wide extracellular-intracellular distribution of biomarkers, it is a meaningful breakthrough to explore high-efficiently amplified DNA circuits, which excel at operating complex yet captivating dynamic reaction networks for various bioapplications. In parallel, the multidimensional performance improvements of nucleic acid circuits, including the availability, detection sensitivity, and reliability, are critical parameters for realizing accurate imaging and cell regulation in bioanalysis.In this Account, we summarize our recent work on enzyme-free dynamic DNA reaction networks for bioanalysis from three main aspects: DNA circuitry functional extension of molecular recognition for epigenetic analysis and regulation, DNA circuitry amplification ability improvement for sensitive biomarker detection, and site-specific activation of DNA circuitry systems for reliable and accurate cell imaging. In the first part, we have designed an epigenetically responsive deoxyribozyme (DNAzyme) circuitry system for intracellular imaging and gene regulation, which enriches the possible analyzed species by chemically modifying conventional DNAzyme. For example, an exquisite N6-methyladenine (m6A)-caged DNAzyme was built for achieving the precise FTO (fat mass and obesity-associated protein)-directed gene regulation. In addition, varieties of DNAzyme-based nanoplatforms with self-sufficient cofactor suppliers were assembled, which subdued the speed-limiting hardness of DNAzyme cofactors in live-cell applications. In the second part, we have developed a series of hierarchically assembled DNA circuitry systems to improve the signal transduction ability of traditional DNA circuits. First, the amplification ability of the DNAzyme circuit has been significantly enhanced via several heterogeneously or homogeneously concatenated circuitry models. Furthermore, a feedback reaction pathway was integrated into these concatenated circuits, thus dramatically increasing the amplification efficiency. Second, considering the complex cellular environment, we have simplified the redundancy of multicomponents or reaction procedures of traditional cascaded circuits, relying on the minimal component complexity and merely one modular catalytic reaction, which guaranteed high cell-delivering uniformity while fostering reaction kinetics and analysis reliability. In the third part, we have constructed in-cell-selective endogenous-stimulated DNA circuitry systems via the multiply guaranteed molecular recognitions, which could not only eliminate the signal leakage, but could also retain its on-site and multiplex signal amplification. Based on the site-specific activation strategy, more circuitry availability in cellular scenarios has been acquired for reliable and precise biological sensing and regulation. These enzyme-free dynamic DNA reaction networks demonstrate the purpose-to-concreteness engineering for tailored multimolecule recognition and multiple signal amplification, achieving high-gain signal transduction and high-reliability targeted imaging in bioanalysis. We envision that the enzyme-free dynamic DNA reaction network can contribute to more bioanalytical layouts, which will facilitate the progression of clinical diagnosis and prognosis.

RETRACTED ARTICLE: The predictive value of BTG1 for the response of newly diagnosed acute myeloid leukemia to decitabine

BackgroundDecitabine has been widely used to treat acute myeloid leukemia (AML); however as AML is a heterogeneous disease, not all patients benefit from decitabine. This study aimed to identify markers for predicting the response to decitabine.MethodsAn intersection of in vitro experiments and bioinformatics was performed using a combination of epigenetic and transcriptomic analysis. A tumor-suppressor gene associated with methylation and the response to decitabine was screened. Then the sensitivity and specificity of this marker in predicting the response to decitabine was confirmed in 54 samples from newly diagnosed AML patients treated with decitabine plus IA regimen in a clinical trial (ChiCTR2000037928).ResultsIn vitro experiments showed that decitabine caused hypomethylation and upregulation of BTG1, while downregulation of BTG1 attenuated the inhibitory effect of decitabine. In newly diagnosed AML patients who received decitabine plus IA regimen, the predictive value of BTG1 to predict complete remission (CR) was assigned with a sensitivity of 86.7% and a specificity of 100.0% when BTG1 expression was < 0.292 (determined using real-time quantitative PCR), with area under the curve (AUC) = 0.933, P = 0.021. The predictive value of BTG1 to predict measurable residual disease (MRD) negativity was assigned with a sensitivity of 100.0% and a specificity of 80.0% when BTG1 expression was < 0.292 (AUC = 0.892, P = 0.012). Patients were divided into low and high BTG1 expression groups according to a cutoff of 0.292, and the CR rate of the low-expression group was significantly higher than that of the high-expression group (97.5% vs. 50%, P < 0.001).ConclusionsLow expression of BTG1 was associated with CR and MRD negativity in newly diagnosed AML patients treated with a decitabine-containing regimen, suggesting that BTG1 is a potential marker for predicting the response to decitabine in newly diagnosed AML.Clinical trial registration: ChiCTR2000037928.

Evaluating histone modification analysis of individual preimplantation embryos

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT).ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing.ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection

BackgroundDetecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge.MethodsTo address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq. MESA enables simultaneous inference of four epigenetic modalities: cfDNA methylation, nucleosome occupancy, nucleosome fuzziness, and windowed protection score for regions surrounding gene promoters and polyadenylation sites.ResultsWhen applied to 690 cfDNA samples from 3 colorectal cancer clinical cohorts, MESA’s novel modalities, which include nucleosome fuzziness, and genomic features, including polyadenylation sites, improve cancer detection beyond the traditional epigenetic markers of promoter DNA methylation.ConclusionsTogether, MESA stands as a major advancement in the field by utilizing comprehensive and complementary epigenetic profiles of cfDNA for effective non-invasive cancer detection.

The bioinformatic analysis of CFTR in essential hypertension

The CFTR gene is associated with cystic fibrosis, a genetic disease primarily affecting the respiratory and digestive systems. Essential hypertension is a common cardiovascular disorder characterized by high blood pressure. In this bioinformatic analysis, various databases and tools were used to investigate the potential mechanisms underlying this association. Gene expression data and epigenetic analyses were used to identify the biological processes between CFTR and hypertension. Additionally, genetic variants within CFTR were analysed for potential effects on hypertension susceptibility. The results of this analysis suggest that CFTR may not play a role in hypertension. Moreover, hypertension has no special epigenetic feather. Further studies are needed to confirm the mechanisms of such a phenomenon.

XRCC1: a potential prognostic and immunological biomarker in LGG based on systematic pan-cancer analysis.

X-ray repair cross-complementation group 1 (XRCC1) is a pivotal contributor to base excision repair, and its dysregulation has been implicated in the oncogenicity of various human malignancies. However, a comprehensive pan-cancer analysis investigating the prognostic value, immunological functions, and epigenetic associations of XRCC1 remains lacking. To address this knowledge gap, we conducted a systematic investigation employing bioinformatics techniques across 33 cancer types. Our analysis encompassed XRCC1 expression levels, prognostic and diagnostic implications, epigenetic profiles, immune and molecular subtypes, Tumor Mutation Burden (TMB), Microsatellite Instability (MSI), immune checkpoints, and immune infiltration, leveraging data from TCGA, GTEx, CELL, Human Protein Atlas, Ualcan, and cBioPortal databases. Notably, XRCC1 displayed both positive and negative correlations with prognosis across different tumors. Epigenetic analysis revealed associations between XRCC1 expression and DNA methylation patterns in 10 cancer types, as well as enhanced phosphorylation. Furthermore, XRCC1 expression demonstrated associations with TMB and MSI in the majority of tumors. Interestingly, XRCC1 gene expression exhibited a negative correlation with immune cell infiltration levels, except for a positive correlation with M1 and M2 macrophages and monocytes in most cancers. Additionally, we observed significant correlations between XRCC1 and immune checkpoint gene expression levels. Lastly, our findings implicated XRCC1 in DNA replication and repair processes, shedding light on the precise mechanisms underlying its oncogenic effects. Overall, our study highlights the potential of XRCC1 as a prognostic and immunological pan-cancer biomarker, thereby offering a novel target for tumor immunotherapy.

Role of varicocele repair in the era of assisted reproductive technologies: Lessons from 2000 cases of microsurgical varicocele repair.

In an era of advanced maternal age, there is less conclusive evidence regarding the treatment outcomes of varicocele repair for assisted reproductive technology (ART). Progress in basic research on varicocele is notable whereas there are many clinically relevant points to discuss. Based on our experience with more than 2000 cases of microsurgical varicocele repair, we focused on the effectiveness of varicocele repair, pathophysiology, surgical approaches, contributions to ART, sperm DNA fragmentation, and varicocele-associated azoospermia in this review with the aim of identifying clearer directions for basic and clinical research on varicocele. Microsurgical low ligation for varicocele repair is expected to remain the gold standard for surgical therapy. Based on the findings from a number of systematic reviews and meta-analyses, negative opinions regarding the efficacy of microsurgical varicocele repair in male infertility treatment have become virtually nonexistent. However, the majority of evidence regarding surgical indications and effectiveness pertains to improvements in semen parameters or non-ART pregnancy rates. Further understandings regarding to pathophysiology of varicocele will likely be gained through comprehensive genetic, transcriptomic, and epigenetic analyses using blood and testicular samples from humans and we hope to develop new diagnostic methods and pharmacotherapy.