Epigenetic Regulation Of Gene Expression Research Articles

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

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

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

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

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

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

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

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

Reprogramming tendon healing: a guide to novel molecular tools.

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

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

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

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

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

Role of sirtuins in epigenetic regulation and aging control.

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

Involvement of transposable elements in Alzheimer's disease pathogenesis.

Alzheimer's disease affects an average of 5 % of the population with a significant increase in prevalence with age, suggesting that the same mechanisms that underlie aging may influence this pathology. Investigation of these mechanisms is promising for effective methods of treatment and prevention of the disease. Possible participants in these mechanisms are transposons, which serve as drivers of epigenetic regulation, since they form species-specific distributions of non-coding RNA genes in genomes in evolution. Study of miRNA involvement in Alzheimer's disease pathogenesis is relevant, since the associations of protein-coding genes (APOE4, ABCA7, BIN1, CLU, CR1, PICALM, TREM2) with the disease revealed as a result of GWAS make it difficult to explain its complex pathogenesis. Specific expression changes of many genes were found in different brain parts of Alzheimer's patients, which may be due to global regulatory changes under the influence of transposons. Experimental and clinical studies have shown pathological activation of retroelements in Alzheimer's disease. Our analysis of scientific literature in accordance with MDTE DB revealed 28 miRNAs derived from transposons (17 from LINE, 5 from SINE, 4 from HERV, 2 from DNA transposons), the expression of which specifically changes in this disease (decreases in 17 and increases in 11 microRNA). Expression of 13 out of 28 miRNAs (miR-151a, -192, -211, -28, -31, -320c, -335, -340, -378a, -511, -576, -708, -885) also changes with aging and cancer development, which indicates the presence of possible common pathogenetic mechanisms. Most of these miRNAs originated from LINE retroelements, the pathological activation of which is associated with aging, carcinogenesis, and Alzheimer's disease, which supports the hypothesis that these three processes are based on the primary dysregulation of transposons that serve as drivers of epigenetic regulation of gene expression in ontogeny.

Catalytic-dependent and -independent roles of TET3 in the regulation of specific genetic programs during neuroectoderm specification

The ten-eleven-translocation family of proteins (TET1/2/3) are epigenetic regulators of gene expression. They regulate genes by promoting DNA demethylation (i.e., catalytic activity) and by partnering with regulatory proteins (i.e., non-catalytic functions). Unlike Tet1 and Tet2, Tet3 is not expressed in mouse embryonic stem cells (ESCs) but is induced upon ESC differentiation. However, the significance of its dual roles in lineage specification is less defined. By generating TET3 catalytic-mutant (Tet3m/m) and knockout (Tet3–/–) mouse ESCs and differentiating them to neuroectoderm (NE), we identify distinct catalytic-dependent and independent roles of TET3 in NE specification. We find that the catalytic activity of TET3 is important for activation of neural genes while its non-catalytic functions are involved in suppressing mesodermal programs. Interestingly, the vast majority of differentially methylated regions (DMRs) in Tet3m/m and Tet3–/– NE cells are hypomethylated. The hypo-DMRs are associated to aberrantly upregulated genes while the hyper-DMRs are linked to downregulated neural genes. We find the maintenance methyltransferase Dnmt1 as a direct target of TET3, which is downregulated in TET3-deficient NE cells and may contribute to the increased DNA hypomethylation. Our findings establish that the catalytic-dependent and -independent roles of TET3 have distinct contributions to NE specification with potential implications in development.

Abstract LB158: Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy

Abstract Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines and comparison of the effects of each drug combination with the effect of the individual drugs are shown in Table. The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes. Table. Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines. Comparison of the effects of each drug combination with the effect of the individual drugs (Table shows the p-values for each comparison.) Colony formation BxPC-3 PL45 Capan-1 Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano+TLZ 0.00048 0.0052 2.40E-06 2.70E-06 0.0051 0.021 Pano+Ola 0.0035 0.078 0.0011 0.00094 0.0016 0.022 SAHA+TLZ 1.80E-05 0.17 0.0029 0.012 0.0014 0.019 SAHA+Ola 0.00018 0.014 0.0016 0.0068 0.0066 0.029 Pano+TLZ+DAC 4.20E-05 0.00026 0.0015 1.70E-05 1.20E-05 2.70E-06 0.00052 0.00088 0.0064 Pano+Ola+DAC 3.40E-05 0.00049 0.0013 7.10E-07 1.70E-06 2.20E-06 0.00068 0.0028 0.0053 SAHA+TLZ+DAC 0.00016 0.00016 0.0011 1.90E-05 3.70E-06 9.30E-07 0.00018 0.00017 0.011 SAHA+Ola+DAC 4.90E-07 0.00035 0.001 0.00022 0.00026 0.00084 0.00016 0.0016 0.0035 Drug-mediated inhibition of cell proliferation Pano+TLZ+DAC 3.57E-05 3.89E-07 1.30E-07 2.91E-05 3.76E-06 1.80E-08 0.0239 ND 0.0092 Pano+Ola+DAC 7.76E-05 1.43E-07 2.52E-07 6.59E-07 1.03E-08 2.22E-07 0.0013 0.21 0.0034 SAHA+TLZ+DAC 3.04E-06 0.00034 1.17E-05 4.91E-08 4.45E-06 1.74E-07 0.0013 1.75E-01 0.007 SAHA+Ola+DAC 0.00012 0.0032 0.000068 9.64E-09 1.53E-08 9.35E-07 0.0007 0.18 0.0054 Abbreviations: DAC: decitabine; ND: not determined; Ola: olaparib; Pano: panobinostat; SAHA: vorinostat; TLZ: talazoparib P ≤ 0.05 is considered statistically significant. Citation Format: Apostolia M. Tsimberidou, Benigno C. Valdez, Bin Yuan, Yago Nieto, Mehmet Baysal, Abhijit Chakraborty, Borje S. Andersson. Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy [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 LB158.

Epigenetic regulation of heart failure.

The studies on chromatin-modifying enzymes and how they respond to different stimuli within the cell have revolutionized our understanding of epigenetics. In this review, we provide an overview of the recent studies on epigenetic mechanisms implicated in heart failure. We focus on the major mechanisms and the conceptual advances in epigenetics as evidenced by studies in humans and mouse models of heart failure. The significance of epigenetic modifications and the enzymes that catalyze them is also discussed. New findings from the studies of histone lysine demethylases demonstrate their significance in regulating fetal gene expression, as well as their aberrant expression in adult hearts during HF. Similarly, the relevance of histone deacetylases inhibition in heart failure and the role of HDAC6 in cardio-protection are discussed. Finally, the role of LMNA (lamin A/C), a nuclear membrane protein that interacts with chromatin to form hundreds of large chromatin domains known as lamin-associated domains (LADs), and 3D genome structure in epigenetic regulation of gene expression and heart failure is discussed. Epigenetic modifications provide a mechanism for responding to stress and environmental variation, enabling reactions to both external and internal stimuli, and their dysregulation can be pathological as in heart failure. To gain a thorough understanding of the pathological mechanisms and to aid in the development of targeted treatments for heart failure, future research on studying the combined effects of numerous epigenetic changes and the structure of chromatin is warranted.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

Abstract 5019: Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers

Abstract Early detection of tumor activity in clinically asymptomatic patients has the potential to improve quality of life and prevent unnecessary exposure to toxic therapies. DNA methylation is one of the most studied, stable, and fundamental epigenetic marks and represents a prominent alteration in most cancers. Analysis of DNA methylation, particularly in liquid biopsies has been introduced in cancer diagnosis and risk stratification. Despite indisputable achievements, translation into clinical practice is lagging behind, primarily due to 1) challenges associated with the analysis of DNA methylation, 2) the fragmented nature of cell-free DNA (cfDNA), aggravated by bisulfite treatment, 3) lack of clinical validation of reported cfDNA methylation marks, and 4) the paucity of knowledge about the functional consequences of cfDNA methylation marks within tumors. Using a sample pooling strategy, complemented with robust whole genome and reduced representation methylation sequencing, we establish a pan-cancer cfDNA methylation resource encompassing 12 cancer entities with diverse clinical phenotypes and treatment modalities. We built this compendium using a robustly performing whole genome and reduced representaion enzymatic methylation sequencing of more than 150 cfDNA and tumor DNA. We complement our data by analyzing DNA methylation data from 15 cancer entities (more than 7,000 patients) within the TCGA. We developed an enzymatic digital PCR (dPCR) approach for pan-cancer but also entity-specific analysis of cfDNA methylation at single base-resolution. Using this dPCR assay, we analyzed cfDNA and tumor DNA samples from more than 400 patients and compared enzymatic digestion with bisulfite treatment. We demonstrate that this resource can allow for the identification of entity-agnostic and specific markers, and that cfDNA methylation patterns are mirrored in tumor samples. The approach can be applied for methylation profiling of yet unanalyzed entities. Finally, we demonstrate unconventional epigenetic regulation of gene expression by methylated DNA-binding transcription factors whose activities are tissue- and context-specific and dosage-dependent. This work, therefore, provides a reference resource to identify minimally invasive, entity-specific and pan-cancer markers applicable for diagnostics, surveillance and patient stratification. It also sets the stage for further investigation of tissue- and context-specific epigenetic regulation of gene expression in health and disease by methylated DNA-binding transcription factors. Citation Format: Smiths Sengkwawoh Lueong, Martin Metzenmacher, Gregor Zaun, Gina Lauren Mayer, Erik Jan Hemmer, Katharina Lückerath, Kelsey Pomykala, Balazs Hegedüs, Peter A. Horn, Marija Trajkovic-Arsic, Tibor Szarvas, Renáta Váraljai, Corinna Keup, Ingeborg Tinhofer, Stephen George, Sabine Kasimir-Bauer, Samuel Peña-Llopis, Alexander Roesch, Cornelius Kürten, Lukas Boosfeld, Kirsten Bruderek, Christopher Darr, Thomas Hilser, Viktor Grünwald, Sven Brandau, Boris Hadaschik, Hans Neubauer, Irene Irene Esposito Irene Esposito, Tanja Fehm, Csilla Oláh, Anita Csizmarik, Fabinshy Thangarajah, Laura Reetz, Ghanam Jamal, Basant Kumar Thakur, Halime Kalkavan, Alexander Schramm, Martin Schuler, Jens T. Siveke. Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers [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 5019.

Abstract 334: Flexible and high-throughput microwell-based single-cell capture for multiomic ATAC-seq and RNA-seq profiling

Abstract The simultaneous profiling of chromatin accessibility and gene expression is an active area of interest for researchers studying how epigenetic changes regulate gene expression at the single-cell level. Here, we demonstrate the feasibility of single nuclei assay for transposase-accessible chromatin (snATAC) seq either with or without simultaneous gene expression profiling using the microwell-based BD Rhapsody™ HT Xpress System. Taking advantage of an eight-lane microwell cartridge, the BD Rhapsody™ HT Xpress System can support the snATAC seq assay with nuclei input ranging from 1,000 to 400,000 nuclei in a single experiment. To demonstrate the flexible throughput capabilities, 1,000 to 50,000 nuclei from cryopreserved peripheral blood mononuclear cells (PBMCs) were tagmented and loaded onto a single lane of the BD Rhapsody™ HT Xpress System. The snATAC assay libraries were prepared and sequenced, revealing multiple high-quality ATAC-seq metrics: high counts of unique ATAC fragments, high fraction of reads in peak regions (FRiP), and high transcriptional start site (TSS) enrichment score. The data were obtained consistently across the wide range of input nuclei with use of the gentle and robust nuclei loading capabilities of the BD Rhapsody™ HT Xpress System. Moreover, after capturing ATAC fragments, the BD Rhapsody™ Enhanced Cell Capture Beads are stable and can be stored at 4 °C for up to 3 months, allowing researchers the flexibility in sequencing subsets of nuclei or lanes for QC purposes before sequencing the entire library and returning to samples for complete sequencing. Furthermore, to demonstrate the consistency of both open chromatin profiling and mRNA gene expression as a combination (multiomic snATAC + snRNAseq), firstly, we compared snRNAseq data from a whole transcriptome assay (WTA) with snRNAseq from an ATAC+mRNA multiomic assay using the same sample. This strategy enabled us to show that there is negligible gene expression differences between snRNAseq from the BD whole transcriptome assay and snRNAseq from the multiomic scATAC and scRNA assay. Likewise, the snATACseq assay and snATACseq assay with simultaneous whole transcriptome profiling shows that chromatin accessibility profiling is equivalent with and without gene expression profiling. Enabling this new scATAC assay on the BD Rhapsody™ HT Xpress System can advance the study of epigenetic regulation of gene expression with a robust and flexible workflow. This will allow researchers to analyze both open chromatin and mRNA at varying nuclei inputs with single-cell resolution to understand the underlying molecular mechanisms in gene expression regulatory pathways. This assay can be readily extended to broader samples to gain greater understanding of disease states in immunology and immuno-oncology studies. For Research Use Only. Not for use in diagnostic or therapeutic procedures. © 2023 BD. All rights reserved. Citation Format: Rosary Nguyen, Hongduan Huang, Quyen Bao, Punya Narayan, Hye-Won Song, Elham Hatami, Zhiqi Zhang, Thomas McCarthy, Youngsook Kim, Ruifang Li, Chelsea Gordon, Larry Wang, Aruna Ayer. Flexible and high-throughput microwell-based single-cell capture for multiomic ATAC-seq and RNA-seq profiling [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 334.

Abstract 3483: SIRT7 as a potential therapeutic target for drug resistance in lung adenocarcinoma: Insights from histone modification-related genes

Abstract Background: Lung cancer is one of the leading causes of cancer-related deaths worldwide. Understanding the molecular mechanisms underlying its development and progression is crucial for the development of targeted therapies. In recent years, histone modification has emerged as a key player in the epigenetic regulation of gene expression, and its involvement in lung cancer has gained significant attention. Histone modifications, including methylation, acetylation, phosphorylation, and ubiquitination, influence chromatin structure and, consequently, gene transcription. Aberrant histone modifications can lead to dysregulated gene expression patterns, contributing to the initiation and progression of lung cancer. In this study, we investigated the expression patterns of 55 histone modification-related genes and identified an epigenetic tumor regulator as a potential therapeutic target. Methods: We obtained 55 histone modification-related genes from dbHiMo (http://hme.riceblast.snu.ac.kr/). Transcriptomic (n= 576), clinical data (n=706) and survival data (n=641) of lung adenocarcinoma (LUAD) were downloaded from XenaTCGA database (https://xenabrowser.net/). The transcriptomic and IC50 values with 87 lung cancer cell lines and 310 drugs were downloaded from DepMap portal (https://depmap.org/portal/). RNA-seq data of GSE197555 was downloaded from GEO database (https://www.ncbi.nlm.nih.gov/geo/). All statistical analysis was performed using R. Results: Among a total of 55 genes related to histone modifications, 6 genes including KAT2A, SIRT7, CARM1, KDM5B, HDAC1 and PRMT3 were significantly overexpressed in tumor tissues in LUAD transcriptome data from TCGA. Out of these 6 genes, 3 (KAT2A, SIRT7 and CARM1) showed significant differences in survival analysis with disease-free interval in the adjuvant therapy-received TCGA LUAD dataset. All three demonstrated that as their expression levels increased, the prognosis became worse. The expression levels of SIRT7 and KAT2A were highly positively correlated with the IC50 value of trametinib, which is MEK inhibitor. Among these two genes, the expression level of SIRT7 was up-regulated in trametinib-treated group in resistance to MEK inhibitor in the H460 cell line. However, sensitive to MEK inhibitor in A549, the SIRT7 expression level was down-regulated in the trametinib-treated group. Conclusion: The histone modification-related gene SIRT7 was found to be associated with resistance to trametinib. In this study, these findings suggest that SIRT7 may serve as a potential therapeutic target related to drug resistance in combination therapy with dabrafenib and trametinib. Citation Format: Kyoung-Ho Pyo, YoungJoon Park, Gang-Taik Lee, Juwon Ahn, Su-Jin Choi, Qi Kejia, Taeku Lee, Youngtaek Kim, Joon Yeon Hwang, Byoung Chul Cho. SIRT7 as a potential therapeutic target for drug resistance in lung adenocarcinoma: Insights from histone modification-related genes [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 3483.

Abstract 7022: Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. Traditionally, sodium bisulfite conversion was used to distinguish 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) from cytosines. However, this method damages DNA and introduces significant sequencing bias. NEBNext® Enzymatic Methyl-seq (EM-seq™) is an enzymatic conversion approach that minimizes DNA damage therefore enabling longer insert sizes, lower duplication rates and a more accurate quantification of methylation in DNA samples with inputs ranging from 10 ng to 200 ng. An improved enzymatic conversion method, EM-seq v2, has a more streamlined workflow and an expanded DNA input range of 100 pg to 200 ng. Libraries made using NA12878 genomic DNA show improved yields, consistent global methylation and an even GC bias representation. The increased library yields subsequently reduces sequencing duplications and costs. Sequencing costs are an important consideration when performing whole genome methylome analysis as this often needs high sequencing depth to attain meaningful coverage to accurately call methylation. Reduced representation libraries, a commonly used method to investigate DNA methylation, relies on enrichment of CpGs within libraries digested using MspI. These CpGs are commonly found in regulatory regions. Although lacking comprehensive CpG coverage, for many researchers this method provides enough cost effective CpG methylation coverage for their studies. Reduced Representation EM-seq libraries (RREM-seq) and Reduced Representation Bisulfite libraries (RRBS) were made using 200 ng to 100 pg of NA12878 DNA. EM-seq adaptors were ligated to MspI digested DNA followed by conversion using either EM-seq v2 or sodium bisulfite. Libraries were PCR amplified and sequenced on an Illumina NovaSeq platform. RREM-seq resulted in higher yields and reproducible results regardless of input amounts compared to RRBS libraries. RREM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. RREM-seq libraries have superior sequencing metrics resulting in robust methylation profiling. Focusing on a subset of the genome generates higher coverage DNA methylation data at a lower DNA sequencing cost. Citation Format: Vaishnavi Panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Ariel Erijman, Bradley Langhorst, Louise Williams. Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes [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 7022.

Abstract 2342: BARTsc: A transcription factor analysis suite for single-cell omics data

Abstract In Eukaryotic cells, the expression of a small set of transcription factors (TFs) is usually sufficient to set up a cell-type-specific gene expression program. The underlying complicated epigenetic mechanisms that explain how cells change their states in response to intra- and extra-cellular signals in different scenarios remain to be fully understood. Importantly, epigenetic regulation of gene expression has been considered a central regulatory mechanism of cell fate determination and cellular plasticity. Insights into it will also profoundly impact our understanding of misregulation of gene expression in diseases such as cancer. However, the functional TFs of many cell types remain unclear, which greatly hinders the study of epigenetic regulatory mechanisms. The rapid development of single-cell technology is a promising opportunity that enables us to infer functional TFs for different cell types. Some computational methods have been published in this area. However, existing methods have various pitfalls. For instance, some methods use transcription factor binding motifs (TFBMs) as the reference of TF binding, overlooking widespread non-specific binding. Some other methods hold strict criteria for training data and thus can only predict a small number of TFs. To overcome these issues, we propose to leverage publicly available high-quality ChIP-seq data to predict TFs functional in single-cell omics data. Here, we present BARTsc, a comprehensive suite for a series of TF analysis functionalities, including inference of signature TFs of given cell types/clusters, cross-cell-type/cluster TF activity comparative analysis, and TF clustering based on their cross cell-type distributions. The input of BARTsc can be either scRNA-seq, scATAC-seq, or scMultiome data. We demonstrate that BARTsc outperforms several existing tools in both sensitivity and specificity in identifying key cell type regulators in single-cell datasets of well-studied systems. Applying BARTsc to a scRNA-seq dataset for cancer-associated fibroblasts (CAFs), we identified distinct regulatory activities of some TFs in different subtypes of CAFs, but the TF genes are universally expressed in these subtypes. In addition, by time course BARTsc analysis, we identified a list of TFs that may either promote or suppress CAF differentiation, offering a new approach to find potential candidate therapeutic targets. Citation Format: Hongpan Zhang, Jingyi Wang, Zhenjia Wang, Chongzhi Zang. BARTsc: A transcription factor analysis suite for single-cell omics data [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 2342.

Abstract 7008: NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution

Abstract DNA methylation is a critical epigenetic regulator of gene expression during development and in diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), can be detected by sequencing libraries generated using existing methods: NEBNext® EM-seq™ or bisulfite sequencing. However, these methods cannot differentiate between 5mC and 5hmC. Interest in the role of 5hmC in regulating gene expression and as a cancer biomarker has increased, resulting in the need for robust methods for the identification of specific 5hmC sites. Methods currently exist to enable discrimination of 5mC and 5hmC (for example, oxBS-seq and TAB-seq), however these are based on modifications of bisulfite sequencing, and suffer from reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC, termed NEBNext® Enzymatic 5hmC-seq (E5hmC-seqTM). E5hmC-seq libraries were generated for 0.1 ng to 200 ng DNA isolated from human brain and E14 mouse embryonic stem cells. Libraries were prepared using NEBNext® Ultra IITM reagents followed by two enzymatic steps to detect 5hmC. In the first step, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. This conversion allows discrimination of 5hmC from cytosine and 5mC. Finally, libraries were PCR amplified using NEBNext® Q5U® and sequenced on the Illumina platform. The E5hmC-seq libraries have similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. Moreover, E5hmC-seq libraries were well-correlated between inputs and replicates at higher sequencing depths. Fully hydroxymethylated T4147 phage DNA was used as an internal control, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for a period of 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. E5hmC-seq libraries provide accurate measurements of 5hmC across a wide input range with expected insert sizes and minimal GC bias. Additionally, subtracting E5hmC-seq data from EM-seq data, which detects both 5mC and 5hmC, enables the precise location of individual 5mC and 5hmC sites. The ability to discriminate between 5mC and 5hmC will allow key insights to be made into the role of cytosine modifications in development and cancer. Citation Format: Garrett M. Cammarata, Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matt A. Campbell, Nan Dai, Bradley W. Langhorst, Louise Williams. NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution [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 7008.

O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression

Histone 2A monoubiquitination (uH2A) underscores a key epigenetic regulation of gene expression. In this report, we show that the deubiquitinase for uH2A, ubiquitin-specific peptidase 16 (USP16), is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation involves the installation of the O-GlcNAc moiety to Ser/Thr residues. It crosstalks with Ser/Thr phosphorylation, affects protein-protein interaction, alters enzyme activity or protein folding, and changes protein subcellular localization. In our study, we first confirmed that USP16 is glycosylated on Thr203 and Ser214, as reported in a previous chemoenzymatic screen. We then discovered that mutation of the O-GlcNAcylation site Thr203, which is adjacent to deubiquitination-required Cys204, reduces the deubiquitination activity toward H2AK119ub invitro and in cells, while mutation on Ser214 had the opposite effects. Using USP16 Ser552 phosphorylation-specific antibodies, we demonstrated that O-GlcNAcylation antagonizes cyclin-dependent kinase 1-mediated phosphorylation and promotes USP16 nuclear export. O-GlcNAcylation of USP16 is also required for deubiquitination of Polo-like kinase 1, a mitotic master kinase, and the subsequent chromosome segregation and cytokinesis. In summary, our study revealed that O-GlcNAcylation of USP16 at Thr203 and Ser214 coordinates deubiquitination of uH2A and Polo-like kinase 1, thus ensuring proper cell cycle progression.