Transcription Start Sites Of Genes Research Articles

EPB41L4A-AS1 is required to maintain basal autophagy to modulates Aβ clearance

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by the deposition of β-amyloid (Aβ) plaques. Aβ is generated from the cleavage of the amyloid precursor protein by β and γ-secretases and cleared by neuroglial cells mediated autophagy. The imbalance of the intracellular Aβ generation and clearance is the causative factor for AD pathogenesis. However, the exact underlying molecular mechanisms remain unclear. Our previous study reported that EPB41L4A-AS1 is an aging-related long non-coding RNA (lncRNA) that is repressed in patients with AD. In this study, we found that downregulated EPB41L4A-AS1 in AD inhibited neuroglial cells mediated-Aβ clearance by decreasing the expression levels of multiple autophagy-related genes. We found that EPB41L4A-AS1 regulates the expression of general control of amino acid synthesis 5-like 2, an important histone acetyltransferase, thus affecting histone acetylation, crotonylation, and lactylation near the transcription start site of autophagy-related genes, ultimately influencing their transcription. Collectively, this study reveals EPB41L4A-AS1 as an AD-related lncRNA via mediating Aβ clearance and provides insights into the epigenetic regulatory mechanism of EPB41L4A-AS1 in gene expression and AD pathogenesis.

Exploration of Genome-Wide Recombination Rate Variation Patterns at Different Scales in Pigs.

Meiotic recombination is a prevalent process in eukaryotic sexual reproduction organisms that plays key roles in genetic diversity, breed selection, and species evolution. However, the recombination events differ across breeds and even within breeds. In this study, we initially computed large-scale population recombination rates of both sexes using approximately 52 K SNP genotypes in a total of 3279 pigs from four different Chinese and Western breeds. We then constructed a high-resolution historical recombination map using approximately 16 million SNPs from a sample of unrelated individuals. Comparative analysis of porcine recombination events from different breeds and at different resolutions revealed the following observations: Firstly, the 1Mb-scale pig recombination maps of the same sex are moderately conserved among different breeds, with the similarity of recombination events between Western pigs and Chinese indigenous pigs being lower than within their respective groups. Secondly, we identified 3861 recombination hotspots in the genome and observed medium- to high-level correlation between historical recombination rates (0.542~0.683) and estimates of meiotic recombination rates. Third, we observed that recombination hotspots are significantly far from the transcription start sites of pig genes, and the silico-predicted PRDM9 zinc finger domain DNA recognition motif is significantly enriched in the regions of recombination hotspots compared to recombination coldspots, highlighting the potential role of PRDM9 in regulating recombination hotspots in pigs. Our study analyzed the variation patterns of the pig recombination map at broad and fine scales, providing a valuable reference for genomic selection breeding and laying a crucial foundation for further understanding the molecular mechanisms of pig genome recombination.

Epigenetic mechanisms regulate sex differences in cardiac reparative functions of bone marrow progenitor cells

Historically, a lower incidence of cardiovascular diseases (CVD) and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients. However, meta-analysis data from these trials suggest a better functional outcome in postmenopausal women as compared with aged-matched men. Mechanisms governing sex-specific cardiac reparative activity in BMSCs, with and without the influence of sex hormones, remain unexplored. To discover these mechanisms, Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by in vivo functional assessments of cardiac repair. F-EPCs and OVX EPCs show a lower inflammatory profile and promote enhanced cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in M-EPCs compared with F-EPCs and OVX-EPCs. Our study unveiled that functional sex differences in EPCs are, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering the sex of donor cells for progenitor-based tissue repair.

Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine-mediated regulation of c-Fos.

CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME influences Treg cell differentiation is not understood. Here, we measured 630 metabolites in the TME and found that serine and palmitic acid, substrates required for the synthesis of sphingolipids, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos. Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits antitumor immunity.

Molecular phenotyping of small cell lung cancer using targeted cfDNA profiling of transcriptional regulatory regions.

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.

Abstract LB156: Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression

Abstract Head and neck cancer is a complex malignancy with its major anatomical subsite, cancer of the oral cavity, ranking among the most deadly and disfiguring cancers. Oral cancer presents primarily as HPV-negative oral squamous cell carcinoma (OSCC) whose etiology is associated with tobacco and alcohol use and for which there are limited treatments. Whereas immunotherapies, such as anti-PD-1 antibodies, have become a promising therapeutic option, only ~20% of head neck cancer patients benefit from anti-PD-1 immunotherapy. We and others have shown that non-mutational epigenetic reprogramming mediated by the β-catenin/CBP complex promotes aggressive cell phenotypes in OSCC by establishing open chromatin at the transcription start sites of genes associated with cancer stemness. Accordingly, pharmacological inhibition of this complex dramatically inhibits OSCC evolution in a syngeneic mouse model of OSCC. Here, we evaluated whether the inhibition of β-catenin/CBP epigenetic activity would enhance the effects of anti-PD-1 immunotherapy. We adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis that utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumors of C57BL/6J mice. After tumors developed for 7 days, mice were blindly separated into treatment cohorts (n=13, each) to receive vehicle control (PBS), monotherapy (either 10 mg/kg anti-PD-1 EOD IP, or 100 mg/kg E7386, a Wnt signaling pathway modulator, QD oral gavage) or combination therapy (anti-PD-1 + E7386). Tumor isografts were harvested 11 days post initiation of treatments and processed for histopathology, immunohistochemistry, and immunofluorescence analyses. Measurements of tumor volumes showed that combination therapy resulted in a statistically significant reduction by ~80% compared to control mice and that it surpassed tumor decrease by either monotherapy. These results were confirmed by histopathology analyses. Evaluation of tumor tissues by immunofluorescence revealed statistically significant decreases in Cbp, H3K4me3, Keratin 14, and Podoplanin expression in response to E7386 alone and in combination with anti PD-1. Collectively our results suggest that a combination therapy comprising anti PD-1 treatment with E7386-targeting β-catenin/CBP epigenetic signaling is likely to impede OSCC evolution to advanced disease. Supported by Eisai, Co Ltd Research Award (MAK), NIH 5R01 DE030350 (MAK, SM, XV) and 5R01 DE031413 (MVB). Citation Format: Emily Fisher, Anthony Spinella, Junji Matsui, Kenichi Nomoto, Xaralabos Varelas, Manish Bais, Maria Kukuruzinska. Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB156.

Targeting histone demethylases JMJD3 and UTX: selenium as a potential therapeutic agent for cervical cancer

BackgroundThe intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease.ResultSelenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets.ConclusionsOur study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

Identification of a chromatin-bound ERRα interactome network in mouse liver

ObjectivesEstrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver. MethodsRIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes. ResultsCharacterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration. ConclusionsOur work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.

Abstract 5626: Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma

Abstract A major obstacle in the efficacy of targeted therapies of oncogene-driven tumors are drug-tolerant persister cells (DTPs) that build the basis for the outgrowth of drug-resistant clones and ultimately limit patient survival. During treatment, DTPs enter a reversible senescent state to survive therapy while cell death is induced in non-DTPs. Here, using RNA-Seq and proteomic analyses, we identified drug-induced TGFβ2 secretion in DTPs derived from different oncogene-driven lung cancer cell lines. As expected, TGFβ2 induces epithelial-to-mesenchymal transition (EMT) that over time promotes the ability of cells to survive targeted therapy. Using CRISPR/Cas9-mediated loss-of-function and reconstitution experiments we show that downstream of TGFβ2, expression of the transcription factor SNAI2 (SLUG) is essential for the outgrowth of DTPs during targeted treatment. Unsupervised RNA-Seq data analyses in combination with Cut&Tag profiling revealed that EMT signaling is in part a SLUG regulated process but that is also independent of direct SLUG binding at the transcription start sites (TSS) of EMT signature genes. In contrast, we observed high occupancy of SLUG at TSS of genes involved in the regulation of cell cycle, being repressed in DTPs and at TSS of genes that regulate sphingolipid metabolism and MAPK signaling, being induced in DTPs. In line with our previous findings, a motif-based analysis of the Cut&Tag data (TOBIAS) furthermore uncovered a tight connection between DTP outgrowth and inflammatory signaling induced through IRF7, IRF4 or STAT1 activation. In vivo, EGFR-mutant SLUG deficient cells showed a significantly prolonged tumor onset and a higher response rate to osimertinib treatment. Overall, we uncover a major role of TGFβ2/SLUG-mediated EMT signaling that may be induced indirectly through SLUG-dependent reprogramming of cell cycle directed and metabolic processes in drug tolerant cells. These insights may offer unique therapeutic opportunities to limit the outgrowth of drug resistant tumors in cancer patients. Citation Format: Jenny Ostendorp, Hannah Lea Tumbrink, Pascal Hunold, Michaela Hoehne, Philipp Jurmeister, Anastasia Dekker, Felix Heisel, Johannes Brägelmann, Frederick Klauschen, Robert Haensel-Hertsch, Martin L. Sos. Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma [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 5626.

Abstract 974: DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation

Abstract Cell-free DNA (cfDNA) in the bloodstream is increasingly gaining attention as a diagnostic tool for the early detection of cancer. Nevertheless, the characteristics and sources of cfDNA fragmentation in the blood remain poorly understood. In this study, we sought to unravel the impact of DNA methylation and gene expression on the naturally occurring genome-wide fragmentation of cfDNA. We performed a comprehensive analysis of plasma samples from 969 individuals, including 182 individuals diagnosed with cancer (pancreatic, colorectal, ovarian, lung and breast cancer). In healthy individual cfDNA fragmentation patterns like DNA motifs at cfDNA fraqgments ends, recurrent cfDNA fragment start sites, cfDNA coverage and cfDNA fragment size were compared to publicly available data on cfDNA methylation and gene expression from myeloid cells. We discovered that the ends of cfDNA fragments, particularly those containing CGs or CCGs, displayed a distinct pattern of enrichment or depletion, respectively, at methylated CpG positions throughout the genome. This phenomenon is consistent with structural models suggesting an increased interaction of CG fragment ends with nucleosomes. cfDNA fragments were more prevalent (up to 3.7 fold) and of larger sizes (up to 4-5 base larger) when derived from regions of methylated CpGs or transcriptional start sites of genes that were not actively expressed. Differences in cfDNA fragmentation reflected specific biological pathways associated with their tissue of origin (like E2f transcription factors and blood cell metabolism genes in cfDNA from healthy individuals). Through analyses of cfDNA from patients with cancer, we identified a connection between tumor-related hypomethylation, increased gene expression, and a global reduction in cfDNA fragment size. This connection may offer an explanation for the generally smaller cfDNA fragments found in individuals with cancer. We found that cancer-specific methylation at CpG sites from pancreatic cancers were associated with widespread changes in cfDNA fragment ends, particularly for patients with pancreatic (test statistic = Welch two sample t-test; p<2.2e-16) and other cancers. These findings establish a direct link between epigenetic modifications and cfDNA fragmentation that may have implications for non-invasive detection of cancer. Citation Format: Michaël Noë, Akshaya V. Annapragada, Zachariah H. Foda, Jamie E. Medina, Dimitrios Mathios, Stephen Cristiano, Christopher Cherry, Daniel C. Bruhm, Noushin Niknafs, Vilmos Adleff, Leonardo Ferreira, Hari Easwaran, Stephen Baylin, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation [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 974.

Translating genetic findings to epigenetics: identifying the mechanisms associated with aging after high-radiation exposure on earth and in space.

Exposure to radiation is a health concern within and beyond the Earth's atmosphere for aircrew and astronauts in their respective austere environments. The biological effects of radiation exposure from a multiomics standpoint are relatively unexplored and stand to shed light on tailored monitoring and treatment for those in these career fields. To establish a reference variable for genetic damage, biological age seems to be closely associated with the effect of radiation. Following a genetic-based study, this study explores the epigenetic landscape of radiation exposure along with its associative effects on aging processes. We imported the results of the genetics-based study that was a secondary analysis of five publicly available datasets (noted as Data1). The overlap of these genes with new data involving methylation data from two datasets (noted as Data2) following similar secondary analysis procedures is the basis of this study. We performed the standard statistical analysis on these datasets along with supervised and unsupervised learning to create preranked gene lists used for functional analysis in Ingenuity Pathway Analysis (IPA). There were 664 genes of interest from Data1 and 577 genes from Data2. There were 40 statistically significant methylation probes within 500 base pairs of the gene's transcription start site and 10 probes within 100 base pairs, which are discussed in depth. IPA yielded 21 significant pathways involving metabolism, cellular development, cell death, and diseases. Compared to gold standards for gestational age, we observed relatively low error and standard deviation using newly identified biomarkers. We have identified 17 methylated genes that exhibited particular interest and potential in future studies. This study suggests that there are common trends in oxidative stress, cell development, and metabolism that indicate an association between aging processes and the effects of ionizing radiation exposure.

Abstract 3668: Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients

Abstract Plasma cell-free DNA (cfDNA) originates from apoptotic cells in the blood, providing a real-time insight into the biology of various cell types. The fragment coverage pattern of cfDNA across the genome is known to reflect the distinct chromatin status of originating cells. While extensive research has focused on fragments derived from cancer, known as circulating tumor DNA (ctDNA), for inference about cancer biology, cfDNA fragments from healthy cells have received little attention. Our study explores the potential of cfDNA to decipher the relative abundance of distinct blood cell types, particularly immune system cell types. We collected a cohort of 273 bladder (BC) and colorectal cancer (CRC) patients and 45 healthy controls for plasma whole genome sequencing and assessed the fragment coverage profile at transcription start sites (TSS) of protein coding genes. A significant negative correlation was observed between TSS coverage and whole blood gene expression (Spearman’s rho = -0.65). Furthermore, TSS coverage revealed a bimodal distribution, characterized by modes corresponding to high and low gene expression levels in blood cells. This may suggest the cfDNA coverage profile as a binary expression status classifier to distinguish between expressed and unexpressed genes in blood cells. We further evaluated TSS with differential coverage between age groups of healthy individuals and relapse status of cancer patients. Notably, out of the TSS that significantly differentiated middle-aged (aged 40-65) from older individuals (aged 65+), 31 were also able to distinguish relapse from non-relapse patients. Among these TSS were CD36, SLAMF7, TABBP and PDCD1, known to be involved in immune related pathways. Finally, we evaluated coverage at cell-type specific TSS and found dendritic cells (P-value: CRC = 8e-06, BC = 5.1e-3), monocytes (CRC = 1.6e-04, BC = 2.5e-3), and NK cells (CRC = 8.5e-04, BC = 0.026) to exhibit a significant increase in coverage in the cancer cohorts compared to healthy controls. Conversely, T-cells showed a decrease in coverage (CRC = 0.041, BC = 0.062). In conclusion, cfDNA coverage profile may identify TSS with differential coverage between patient groups and characterize a shift in blood cell type composition as a response to cancer. Future work may show if fragment coverage quantifies the relative contribution of cfDNA from distinct immune cell types. We anticipate our analysis to illuminate the utility of plasma cfDNA as a biomarker for cancer patient’s immune competency, enabling improved patient stratification for treatment, leveraging their distinct immune capabilities. Citation Format: Laura Andersen, Amanda Frydendahl, Tenna Vesterman Henriksen, Christina Demuth, Mads Heilskov Rasmussen, Iver Nordentoft, Karin Birkenkamp-Demtröder, Jakob Skou Pedersen, Søren Besenbacher, Lars Dyrskjøt, Claus L. Andersen, Nicolai J. Birkbak. Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients [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 3668.

Abstract 5028: Detection of non-small cell lung and bladder cancer signatures in peripheral blood using a novel antibody-free chromatin capture assay

Abstract Immune checkpoint inhibition (ICI) via anti-PD-1/PD-L1 has improved clinical outcomes for multiple cancers, including bladder and non-small cell lung cancer (NSCLC). Yet, the response rates remain low highlighting a significant knowledge gap in the mechanisms driving response and resistance. While the expression of PD-L1 has been established as a predictive biomarker of ICI response, other features, including molecular tumor burden, tumor microenvironment (TME), and the host’s immune mechanisms may contribute to overall response. The complex interactions between a tumor and the immune system underscore the need for a more comprehensive approach to explore novel biological and immunological insights of cancer progression. Circulating tumor DNA assays, while integral to contemporary decision-making in oncology, fall short in delineating the transcriptional programs that dictate cancer phenotypes and their evolution over the disease trajectory. To bridge this gap, we have developed a plasma-based active chromatin capture method that enables the comprehensive epigenetic and transcriptomic interrogation of both tumor and immune components. In this study, we quantified epigenetic profiles from plasma samples (n=100) of patients with bladder cancer (n=20) and NSCLC (n=24) undergoing ICI therapy, along with samples (n=70) from healthy individuals (n=5). As expected, the total cfDNA yield (ng) of lung (1.7x, p=2.3e-08) and bladder (1.98x, p=1.6e-11) cancer samples were higher than those of healthy individuals. More Interestingly, the longer cfDNA fragments derived from regulatory-active chromatin, also showed a significantly higher abundance in plasma samples from both lung (2.0x, p=2.5e-09) and bladder (3.2x, p=3.2e-14) cancer patients. Furthermore, the patterns of epigenetic regulations across cancer and healthy conditions showed distinct and stable clusters, consistent between gene bodies and transcription start sites, highlighting the epigenetic changes that may be driving cancer progression. We also observed a common enrichment of immune epigenetic signatures in both lung and bladder cancer samples, likely attributed to shared mechanisms associated with the modulation of the tumor microenvironment and immune interactions. Understanding these changes at an epigenetic level could provide insights into novel therapeutic approaches that target the tumor microenvironment and the immune interactions. Citation Format: Kevin Lai, Katharine Dilger, Rachael Cunningham, Timothy Barnes, Khiet Truong, Kathy Lam, Rhea Boquiren, Maggie C. Louie, Diana Abdueva. Detection of non-small cell lung and bladder cancer signatures in peripheral blood using a novel antibody-free chromatin capture assay [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 5028.

Histone H3K4ac, as a marker of active transcription start sites and enhancers, plays roles in histone eviction and RNA transcription

The lysine 4 of histone H3 (H3K4) can be methylated or acetylated into four states: H3K4me1, H3K4me2, H3K4me3, or H3K4ac. Unlike H3K4 methylation, the genome-wide distribution and functional roles of H3K4ac remain unclear. To understand the relationship of acetylation with methylation at H3K4 and to explore the roles of H3K4ac in the context of chromatin, we analyzed H3K4ac across the human genome and compared it with H3K4 methylation in K562 cells. H3K4ac was positively correlated with H3K4me1/2/3 in reciprocal analysis. A decrease in H3K4ac through the mutation of the histone acetyltransferase p300 reduced H3K4me1 and H3K4me3 at the H3K4ac peaks. H3K4ac was also impaired by H3K4me depletion in the histone methyltransferase MLL3/4-mutated cells. H3K4ac peaks were enriched at enhancers in addition to the transcription start sites (TSSs) of genes. H3K4ac of TSSs and enhancers was positively correlated with mRNA and eRNA transcription. A decrease in H3K4ac reduced H3K4me3 and H3K4me1 in TSSs and enhancers, respectively, and inhibited the eviction of histone H3 from them. The mRNA transcription of highly transcribed genes was affected by the reduced H3K4ac. Interestingly, H3K4ac played a redundant role with regard to H3K27ac in eRNA transcription. These results indicate that H3K4ac serves as a marker of both active TSSs and enhancers and plays a role in histone eviction and RNA transcription by leading to H3K4me1/3.

Global repair is the primary nucleotide excision repair subpathway for the removal of pyrimidine-pyrimidone (6-4) damage from the Arabidopsis genome

Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).

Transcriptional Control of Subcutaneous Adipose Tissue by the Transcription Factor CTCF Modulates Heterogeneity in Fat Distribution in Women.

Determining the mechanism driving body fat distribution will provide insights into obesity-related health risks. We used functional genomics tools to profile the epigenomic landscape to help infer the differential transcriptional potential of apple- and pear-shaped women's subcutaneous adipose-derived stem cells (ADSCs). We found that CCCTC-binding factor (CTCF) expression and its chromatin binding were increased in ADSCs from pear donors compared to those from apple donors. Interestingly, the pear enriched CTCF binding sites were located predominantly at the active transcription start sites (TSSs) of genes with active histone marks and YY1 motifs and were also associated with pear enriched RNAPII binding. In contrast, apple enriched CTCF binding sites were mainly found at intergenic regions and when identified at TSS, they were enriched with the bivalent chromatin signatures. Altogether, we provide evidence that CTCF plays an important role in differential regulation of subcutaneous ADSCs gene expression and may influence the development of apple vs. pear body shape.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

Analysis of the selenoprotein P (rs7579) gene polymorphism and expression in patients with chronic pancreatitis combined with hypothyroidism

Background. The specific role and place of genetic factors in the development of chronic pancreatitis and hypothyroidism, which determine the activity of glutathione antioxidant protection, have not been clarified. They are interconnected with changes in the fat and carbohydrate metabolism, and also affect the transport and signaling pathways of key nutrients for the work of the immune, endocrine and nervous systems. The purpose of the study is to investigate the selenoprotein P (SEPP1) (rs7579) gene polymorphism and expression in patients with chronic pancreatitis combined with hypothyroidism. Materials and methods. Forty-nine patients with chronic pancreatitis and hypothyroidism and 30 practically healthy individuals passed the screening stage. The SEPP1 (rs7579) gene polymorphism was determined by the polymerase chain reaction. Results. The study showed that out of 98 isolated alleles in patients with chronic pancreatitis and hypothyroidism and 60 alleles of the control group, the G allele of the SEPP1 gene (rs7579, 25191G/A) dominated over the A allele: in the examined patients — by 34.7 % (χ2 = 23.59; p < 0.001), in the practically healthy group — by 53.34 % (χ2 = 34.13; p < 0.001). At the same time, the relative frequency of individual genotypes, as well as wild-type and mutant alleles, probably did not differ between the experimental and control groups. The A allele of the SEPP1 gene (rs7579) slightly increases the risk of chronic pancrea­titis in the studied population, but non-significantly [risk ratio (RR) = 1.43; RR 95% CI (confidence interval): 0.91–2.26; odds ratio (OR) = 1.65; OR 95% CI: 0.88–3.08; p = 0.115]. Conclusions. In patients with chronic pancreatitis who are residents of Northern Bukovyna, mutation of the SEPP1 gene (rs7579, 25191G/A) in the homozygous state occurs with a frequency of 10.2 %, while it is absent in practically healthy people. In both groups, the G allele dominates over the A allele: in the examined patients — by 34.7 % (χ2 = 23.59; p < 0.001), in controls — by 53.34 % (χ2 = 34.13; p < 0.001). SEPP1 gene polymorphism (rs7579, 25191G/A) does not determine the risk of chronic pancreatitis in the population. However, the A allele increases the risk of hypothyroidism in chro­nic pancreatitis patients twice [OR = 2.0; OR 95% CI: 1.09–3.66; p = 0.023], with the lowest chances of its appearance in carriers of the G allele [OR = 0.50; OR 95% CI: 0.27–0.91; p = 0.023]. Mapping of the expression quantitative trait loci on both sides of the SEPP1 gene (rs7579) transcription start site evidenced 152 statistically significant cis-variants of rs7579 of the SEPP1 gene (SELENOP) associations with the expression of 20 genes in 35 different organs and tissues and 22 phenotypic traits.

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

Cell-Free DNA Extracted from CSF for the Molecular Diagnosis of Pediatric Embryonal Brain Tumors

Background: Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: The CSF cfDNA of pediatric patients with medulloblastoma (n = 18), ATRT (n = 3), ETMR (n = 1), CNS NB FOXR2 (n = 2) and pediatric EBT NOS (n = 1) (mean cfDNA concentration 48 ng/mL; range 4–442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n = 15), a mean of 83 (range 1–160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1–62) or CSF-specific SNVs (mean 5; range 0–25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.