Alterations In Chromatin Structure Research Articles

ING1 inhibits Twist1 expression to block EMT and is antagonized by the HDAC inhibitor vorinostat

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.

SEMA7a primes integrin α5β1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5β1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Fibrosis-the tale of H3K27 histone methyltransferases and demethylases.

Fibrosis, or excessive scarring, is characterized by the emergence of alpha-smooth muscle actin (αSMA)-expressing myofibroblasts and the excessive accumulation of fibrotic extracellular matrix (ECM). Currently, there is a lack of effective treatment options for fibrosis, highlighting an unmet need to identify new therapeutic targets. The acquisition of a fibrotic phenotype is associated with changes in chromatin structure, a key determinant of gene transcription activation and repression. The major repressive histone mark, H3K27me3, has been linked to dynamic changes in gene expression in fibrosis through alterations in chromatin structure. H3K27-specific homologous histone methylase (HMT) enzymes, Enhancer of zeste 1 and 2 (EZH1, EZH2), which are the alternative subunits of the Polycomb Repressive Complex 2 (PRC2) and demethylase (KDM) enzymes, Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and Lysine demethylase 6B (KDM6B), are responsible for regulating methylation status of H3K27me3. In this review, we explore how these key enzymes regulate chromatin structure to alter gene expression in fibrosis, highlighting them as attractive targets for the treatment of fibrosis.

A review on the epigenetics modifications to nanomaterials in humans and animals: Novel epigenetic regulator

Abstract In the nanotechnology era, nanotechnology applications have been intensifying their prospects to embrace all the vigorous sectors persuading human health and animal. The safety and concerns regarding the widespread use of engineered nanomaterials (NMs) and their potential effect on human health still require further clarification. Literature elucidated that NMs exhibited significant adverse effects on various molecular and cellular alterations. Epigenetics is a complex process resulting in the interactions between an organism’s environment and genome. The epigenetic modifications, including histone modification and DNA methylation, chromatin structure and DNA accessibility alteration, regulate gene expression patterns. Disturbances of epigenetic markers induced by NMs might promote the sensitivity of humans and animals to several diseases. Also, this paper focuses on the epigenetic regulators of some dietary nutrients that have been confirmed to stimulate the epigenome and, more exactly, DNA histone modifications and non-histone proteins modulation by acetylation, and phosphorylation inhibition, which counteracts oxidative stress generations. The present review epitomizes the recent evidence of the potential effects of NMs on histone modifications, in addition to in vivo and in vitro cytosine DNA methylation and its toxicity. Furthermore, the part of epigenetic fluctuations as possible translational biomarkers for uncovering untoward properties of NMs is deliberated.

DIPG-47. DISCOVERING A MOLECULAR BASIS FOR VULNERABILITY OF H3K27M-DMG TO HISTONE DEACETYLASE INHIBITORS

Abstract Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29), we detected a 20-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Acetylation and deacetylation of wtH3 alter chromatin structure as part of normal regulation of gene expression. The methionine substitution for Lysine in H3K27M removes this histone from participating in acetylation/deacetylation regulated gene expression. Concomitantly, H3K27M induces a marked reduction in global acetylation of histone tails on wtH3. In the native state of H3K27M DMG with hypoacetylated H3, we posit that the specific pattern of nucleosome integration with genes responsible for cell survival/death underlies the differential vulnerability of DMGs to HDACi. Additionally, the nucleosome integration pattern with cell survival/death genes may be based on the relative abundance of wtH3wt versus H3K27M- histones. Following Quis treatment (48 hrs) in both preclinical models, total wtH3ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes K27 acetylated H3 histone turnover. Comparison of differentially expressed genes (DEGs) from control and Quis-treated PBT22 and PBT29 will seed gene ontology analysis focusing on chromatin remodeling, cell death, and growth arrest pathways accounting for differential vulnerability. Findings will be validated by measuring transcripts and proteins (qRT-PCR and ELISA or mass spectrometry) from analytes from a larger panel of DMG cell lines. The data depicts DMG preclinical models with large differential sensitivity to Quis. The long-term goal is to discover a molecular profile of DMGs predictive of the vulnerability to HDACi. The discovery exercise is also likely to yield insight into the development of resistance to HDAC inhibitors.

Molecular profile of SWI/SNF complex in Chinese patients with ovarian cancer.

e17538 Background: The SWI/SNF chromatin remodeling complexes are involved in cancer initiation and progression. ARID1A, ARID1B, and SMARCA4, the essential ATPase subunit of the SWI/SNF chromatin remodeling complex, play important roles in altering chromatin structure for various cellular functions, including transcription, DNA synthesis, and repair. In ovarian cancer (OC), mutations in several SWI/SNF complexes were found, but the molecular and clinical characteristics of SWI/SNF complexes in Chinese ovarian cancer patients have never been reported. Herein, we explored the profiles of SWI/SNF mutation in the Chinese population. Methods: Genetic mutations were reviewed in OC patients who underwent hybridization capture-based next-generation sequencing (NGS). SWI/SNF mutant tumors (MT) were compared to wild-type (WT), and other co-mutations were also evaluated. The pathogenicity of germline mutations was categorized based on American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Among 461 OC patients, mutation in at least one evaluated SWI/SNF gene was identified in 23% of cases. The higher frequency of SWI/SNF alterations were ARID1A (41.03%), ARID1B (13.33%), SMARCA4 (11.28%). The co-mutated genes mainly were TP53, PIK3CA, PTEN. In addition, the MT group tended to have more BRCA1/2 somatic mutations (16.34% vs. 7.84%, χ2＝5.42, p < 0.05) and less BRCA1/2 germline pathogenic mutations (8.7% vs. 17.2%, χ2＝4.40, p < 0.05) compared to WT group. There was a significant statistical difference in TMB value that the MT group was higher than the WT group (11.1 vs. 2.7, p < 0.001). In the MT group, the TMB of patients co-mutated with TP53, PIK3CA, or PTEN mutations were 10.2, 17.3, and 32.3, respectively. The proportion of SWI/SNF alterations in MSI-H tumors was higher than those without mutations (10.0% vs. 0.8%, χ2＝19.29, p < 0.001). Conclusions: SWI/SNF mutant tumors tended to be associated with higher TMB (11.1 vs. 2.7), a higher proportion of MSI-H (10% vs. 0.8%), and more BRCA1/2 somatic mutations ( 16.34% vs. 7.84%) in OC patients. Patients with SWI/SNF alterations combined with PTEN mutation have extremely high TMB (TMB=32.3), indicating they may benefit more from immunotherapy.

Abstract A006: Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss

Abstract KRAS-targeted inhibition yielded promising, yet far from ideal, clinical responses, revealing that cancer cells easily bypass KRAS loss. Therefore, we aim to understand how mutant KRAS cancer cells tolerate so well the loss of a key oncogene to which they were addicted to. KRAS expression was silenced through siRNAs in colorectal cancer (CRC) cell lines that carry KRAS mutations. The proteome characterization was obtained by mass spectrometry and the expression of significantly altered proteins was validated by western blotting. Chromatin states were investigated using electron microscopy. Upon KRAS inhibition, there was a significant reduction in the cell number, accompanied by changes in cell cycle, thus supporting KRAS-dependency. Proteomics analysis revealed that KRAS inhibition-tolerant cells upregulated several proteins associated with the extracellular exosome or nuclear compartments. Molecular function and biological process gene ontology terms revealed an up-regulation of proteins mainly associated with binding activities (RNA, protein, nucleosome, and core promoter binding) as well as gene expression regulation, mRNA splicing and processing, and nucleosome assembly and repositioning. In addition, the proteomics data also revealed an upregulation of proteins associated with active chromatin states. Moreover, KRAS-silenced persister cells also presented alterations in some histone post-translational modifications and in chromatin packing, both suggesting that transcription is impacted. Overall, our results suggest an epigenetic mechanism underlying tolerance to KRAS inhibition that involves chromatin structural changes and transcription alterations, which we are currently pursuing. Citation Format: Flavia Martins, Ana L. Machado, Patricia D. Carvalho, Joana Carvalho, Rune Matthiesen, Vadim Backman, Sergia Velho. Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A006.

Genome-Wide Identification and Characterization of the Soybean Snf2 Gene Family and Expression Response to Rhizobia.

Sucrose nonfermenting 2 (Snf2) family proteins are the core component of chromatin remodeling complexes that can alter chromatin structure and nucleosome position by utilizing the energy of ATP, playing a vital role in transcription regulation, DNA replication, and DNA damage repair. Snf2 family proteins have been characterized in various species including plants, and they have been found to regulate development and stress responses in Arabidopsis. Soybean (Glycine max) is an important food and economic crop worldwide, unlike other non-leguminous crops, soybeans can form a symbiotic relationship with rhizobia for biological nitrogen fixation. However, little is known about Snf2 family proteins in soybean. In this study, we identified 66 Snf2 family genes in soybean that could be classified into six groups like Arabidopsis, unevenly distributed on 20 soybean chromosomes. Phylogenetic analysis with Arabidopsis revealed that these 66 Snf2 family genes could be divided into 18 subfamilies. Collinear analysis showed that segmental duplication was the main mechanism for expansion of Snf2 genes rather than tandem repeats. Further evolutionary analysis indicated that the duplicated gene pairs had undergone purifying selection. All Snf2 proteins contained seven domains, and each Snf2 protein had at least one SNF2_N domain and one Helicase_C domain. Promoter analysis revealed that most Snf2 genes had cis-elements associated with jasmonic acid, abscisic acid, and nodule specificity in their promoter regions. Microarray data and real-time quantitative PCR (qPCR) analysis revealed that the expression profiles of most Snf2 family genes were detected in both root and nodule tissues, and some of them were found to be significantly downregulated after rhizobial infection. In this study, we conducted a comprehensive analysis of the soybean Snf2 family genes and demonstrated their responsiveness to Rhizobia infection. This provides insight into the potential roles of Snf2 family genes in soybean symbiotic nodulation.

Abstract 4719: Alterations of chromatin structure and TGF-β signaling in ARID1A-mutated colon cancer cells

Abstract ARID1A, a subunit of the SWI/SNF complex which regulates gene expression by remodeling chromatin structure, is often mutated in various cancer and is known as a tumor suppressor gene. Many studies revealed the loss of ARID1A promotes tumor progression by activating PI3K-Akt signaling, but other pathways have not yet been fully elucidated. By analyzing RNA sequencing data and ATAC-seq data with ARID1A wild-type cell lines HCT116 and isogenic ARID1A Q456*/Q456* which has truncation mutations for ARID1A, we investigated the alterations of epigenomic and transcriptomic characteristics according to the loss of ARID1A. The overall openness of chromatin decreased in ARID1A Q456*/Q456* cells. Since SWI/SNF complex regulates chromatin structure and gene expression accordingly, we analyzed genes whose change of RNA expression and openness of chromatin in the same direction. The results showed 109 upregulated genes and 715 downregulated genes compared to wild type cell line with Padj < 0.05, |log2FC| > 0.58 from both RNA sequencing and ATAC-seq. To determine which gene sets are activated in the ARID1A-deficient cell line, the differentially expressed genes with consistent chromatin openness were subjected to Gene Ontology analysis. The upregulated genes in the ARID1A-deficient cell line were enriched in ‘Cell proliferation’ and ‘TGF-β signaling’. In addition, the expression of representative genes associated with TGF-β signaling increased in ARID1A-deficient cells. Our data suggests that loss-of-mutation in ARID1A significantly changes the chromatin structure, resulting in alterations of gene expression associated with cell proliferation and TGF-β signaling. Citation Format: Jiwon Kim, Giyong Jang, Soo-Jin Lee, Sang Eun Lee, Sung-Yup Cho. Alterations of chromatin structure and TGF-β signaling in ARID1A-mutated colon cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4719.

Abstract 4740: Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons

Abstract Diffuse Midline Glioma of the pons (DMG) is a lethal, aggressive heterogeneous brain stem tumor. Median overall survival is less than a year, with radiation as the only standard treatment. Recently, mutations in DMGs have arisen as potential therapeutic targets, specifically a mutation in one of the histone H3 genes, resulting in methionine substituted for lysine at site 27 (H3K27M). H3K27M induces a marked reduction in global acetylation of histone tails, altering chromatin structure and causing aberrant gene expression. Histone deacetylase inhibitors, HDACis, are epigenetic drugs that show anticancer activity. In our studies, Quisinostat (Quis) is used to preserve histone acetylation. Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29) we detected a 100-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Following Quis treatment (48 hrs) in both preclinical models, total H3K27ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes turnover of K27 acetylated H3 histone. We are pursuing studies to test whether sensitivity to HDACi in DMGs is determined by a shift in relative or total abundance of respective H3wt- and H3K27M- histones. We posit that changes in H3K27ac manifest as shifts in nucleosome integration with genes responsible for cell survival/death. This project will profile differentially expressed genes (DEGs) from Quis-treated PBT22 and PBT29. Additionally, total and relative abundance of H3 proteins (wt and mt, me, me2, me3, and ac) from treated cells will be determined. Gene ontology analysis will focus on pathways accounting for chromatin remodeling, cell death, and growth arrest. RNA (qRT-PCR) and proteins (western blot) from analytes from a larger panel of DMG cell lines and neural stem cells treated with Quis will be used to validate the findings. Overall, the data depicts DMG preclinical models with large differential sensitivity to Quis, which may be partially due to different oncoprints between the models. The markedly different sensitivity of these models enables mechanistic study of the consequences of elevated abundance of histone 3 acetylation. The long term goal is to discover a molecular profile of DMGs indicative of the vulnerability to HDACi. Citation Format: Danyelle Paine, Nanyun Tang, Yue Hao, Matt Biery, Carrie Meyers, Alyssa Noll, Nicholas Vitanza, Michael Berens. Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4740.

Abstract 3606: HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

Abstract Melanomas are heterogeneous and adopt multiple transcriptional states that can confer an invasive phenotype and resistance to therapy. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity and tumor progression. Here we identify stress-induced HDAC8 activity as the driver of a neural crest stem cell (NCSC)-like transcriptional state that increased the formation of melanoma brain metastases (MBM). Exposure of melanocytes and melanoma cells to multiple different stresses led to HDAC8 activation, a switch to a NCSC gene expression signature and the adoption of an amoeboid, invasive phenotype. This cell state enhanced the survival of melanoma cells under shear stress conditions and increased the formation of metastases in the brain. Single cell RNA-seq analyses showed that HDAC8 expression was correlated with the NCSC cell state in clinical MBM specimens. ATAC-Seq and ChIP-Seq analysis showed HDAC8 to alter chromatin structure by increasing H3K27ac and accessibility at c-Jun binding sites without changing global histone acetylation. The increased accessibility of Jun binding sites was paralleled by decreased H3K27ac and accessibility at MITF binding sites and loss of melanoma-lineage gene expression. Mass spectrometry-based acetylomics demonstrated that HDAC8 deacetylated the histone acetyltransferase (HAT) EP300 leading to its enzymatic inactivation. This, in turn, led to an increased binding of EP300 to Jun-transcriptional sites and decreased binding to MITF-transcriptional sites. Increased expression of EP300 decreased invasion and increased the sensitivity of melanoma cells to multiple stresses while inhibition of EP300 function increased invasion and resistance to stress. We identified HDAC8 as a novel mediator of transcriptional co-factor inactivation and chromatin accessibility that increases MBM development. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Chao Zhang, Robert Macaulay, Daphne Dupéré-Richér, Bin Fang, John Koomen, Jiannong Li, Ann Chen, Edward Seto, Jonathan Licht, Keiran Smalley. HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3606.

Epigenetic reprogramming in pancreatic premalignancy and clinical implications.

Pancreatic cancer (PC) is the most lethal human cancer, with less than 10% 5-year survival. Pancreatic premalignancy is a genetic and epigenomic disease and is linked to PC initiation. Pancreatic premalignant lesions include pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and mucinous cystic neoplasm (MCN), with pancreatic acinar-to-ductal metaplasia (ADM) as the major source of pancreatic premalignant lesions. Emerging evidence reveals that an epigenetic dysregulation is an early event in pancreatic tumorigenesis. The molecular mechanisms of epigenetic inheritance include chromatin remodeling; modifications in histone, DNA, and RNA; non-coding RNA expression; and alternative splicing of RNA. Changes in those epigenetic modifications contribute to the most notable alterations in chromatin structure and promoter accessibility, thus leading to the silence of tumor suppressor genes and/or activation of oncogenes. The expression profiles of various epigenetic molecules provide a promising opportunity for biomarker development for early diagnosis of PC and novel targeted treatment strategies. However, how the alterations in epigenetic regulatory machinery regulate epigenetic reprogramming in pancreatic premalignant lesions and the different stages of their initiation needs further investigation. This review will summarize the current knowledge of epigenetic reprogramming in pancreatic premalignant initiation and progression, and its clinical applications as detection and diagnostic biomarkers and therapeutic targets in PC.

Abnormal Cellular Phenotypes Induced by Three TMPO/LAP2 Variants Identified in Men with Cardiomyopathies.

A single missense variant of the TMPO/LAP2α gene, encoding LAP2 proteins, has been associated with cardiomyopathy in two brothers. To further evaluate its role in cardiac muscle, we included TMPO in our cardiomyopathy diagnostic gene panel. A screening of ~5000 patients revealed three novel rare TMPO heterozygous variants in six males diagnosed with hypertrophic or dilated cardiomypathy. We identified in different cellular models that (1) the frameshift variant LAP2α p.(Gly395Glufs*11) induced haploinsufficiency, impeding cell proliferation and/or producing a truncated protein mislocalized in the cytoplasm; (2) the C-ter missense variant LAP2α p.(Ala240Thr) led to a reduced proximity events between LAP2α and the nucleosome binding protein HMGN5; and (3) the LEM-domain missense variant p.(Leu124Phe) decreased both associations of LAP2α/β with the chromatin-associated protein BAF and inhibition of the E2F1 transcription factor activity which is known to be dependent on Rb, partner of LAP2α. Additionally, the LAP2α expression was lower in the left ventricles of male mice compared to females. In conclusion, our study reveals distinct altered properties of LAP2 induced by these TMPO/LAP2 variants, leading to altered cell proliferation, chromatin structure or gene expression-regulation pathways, and suggests a potential sex-dependent role of LAP2 in myocardial function and disease.

Epigenetics and Methylmercury-Induced Neurotoxicity, Evidence from Experimental Studies

MeHg is an environmental neurotoxin that can adversely affect the development of the nervous system. The molecular integrity of chromatin in the nucleus is an important target of MeHg. Low levels of MeHg trigger epigenetic mechanisms that may be involved in long-lasting and transgenerational neurotoxicity after exposure. Emerging evidence has shown that these mechanisms include histone modification, siRNA, and DNA methylation. The MeHg-induced inhibition of neurodifferentiation and neurogenesis are mechanistically associated with epigenetic alterations in critical genes, such as neurotrophin brain-derived neurotrophic factor (BDNF). Further, MeHg exposure has been shown to alter the activity and/or expression of the upstream regulators of chromatin structure, including histone deacetylases (HDACs) and DNA methyltransferase (DNMTs), which may trigger permanent alterations in histone modifications and DNA methylation. MeHg-exposure also alters several species of miRNA that are associated with neurodevelopment. Genetic studies in the C. elegans model of MeHg-induced toxicity proposes a potential interplay between exogenous RNAi and antioxidant defense. In this review, we discuss the molecular basis for MeHg exposure-induced alterations in chromatin structure and the roles of histone modifications, siRNA, and DNA methylation in MeHg-induced neurotoxic effects.

Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates.

The rate of RNA polymerase II (RNAPII) transcriptional elongation plays a critical role in mRNA biogenesis, from transcription initiation to alternative splicing. As RNAPII moves along the DNA, it must read the DNA sequences wrapped up as chromatin. Thus, the structure of chromatin impedes the movement and speed at which RNAPII moves, presenting a crucial regulation to gene expression. Therefore, factors that bind and regulate the structure of chromatin will impact the rate of RNAPII elongation. We previously showed that PARP1 (poly-ADP-ribose polymerase 1) is one of such factors that bind and alter chromatin dynamics. We also showed that its alteration of chromatin structure modulates RNAPII processivity during transcriptional elongation. Here, we aim to understand how PARP1 alters RNAPII elongation kinetics genome wide.

Abstract B024: Deconvolution by scRNAseq, scATACseq and functional genomics of impact of a relevant high risk diet on stem cells and homeostasis

Abstract Dietary patterns are major determinants of sporadic colon cancer incidence, the vast majority of the disease. NWD1 is a unique purified rodent diet that establishes mouse exposure to key nutrients recapitulating levels linked to higher risk in human, and is the only mouse model that develops sporadic intestinal and colon tumors, reflecting the etiology, incidence, frequency and lag with developmental age of the human disease. NWD1 fed mice reflect human metabolic syndrome with chronic inflammation, exhibit elevated Wnt signaling throughout the small and large intestinal mucosa and altered lineage marker expression, but Lgr5hi cells are suppressed in their lineage tracing, accumulation of mutations, and efficiency in tumor initiation. Alternate cells are mobilized to maintain the mucosa with increased efficiency to cause tumors. Deconvolving impact of NWD1 using scRNAseq, scATACseq and functional genomics provided novel insight into mechanisms elevating risk for sporadic tumors. NWD1 extensively, rapidly, and reversibly reprogrammed Lgr5hi stem cells, down-regulating Ppargc1a expression that altered mitochondrial structure and function and suppressed Oxphos and the TCA cycle. Ppargc1a genetic knockout in Lgr5hi cells recapitulated the dietary effects, and NWD1 or Ppargc1a inactivation repressed developmental maturation of Lgr5hi cell progeny as cells progressed through progenitor cell compartments. In compensation, mobilized Bmi1+, Ascl2hi cells maintained and adapted the mucosa. In parallel, NWD1 altered Ppargc1a chromatin structure in stem cells, and also for genes adapting enterocytes to the diet, independently confirming NWD1 epigenetic reprogramming of stem and differentiated cells. Importantly, pathways of antigen processing and presentation were elevated, especially in mature enterocytes, a pathogenic mechanism causing human chronic low-level pro-tumorigenic inflammation in IBD, also present in NWD1 fed mice. Fundamental conclusions: 1) plasticity of cells to function as stem cells mediates physiological adaptation of the mucosa. Thus, the human mucosa is in constant flux in response to its environment, supporting historic concepts of homeostasis by Claude Bernard and Walter Cannon as a continual process of tissue adaptation to its environment, not a single optimized state. 2) our data and the literature suggest that failure to recapitulate key human nutritional exposures contributes to why human stem cells reach crypt clonality in 6 years, 50-fold longer than the 6 weeks in the mouse. Thus, better modeling of human nutritional exposures in the mouse more accurately reflects human mechanisms of homeostasis and pathogenesis. 3) Complexity of cell reprogramming reported for the earliest premalignant human colon tumors is already present in the mucosa at nutritional risk for tumor development. 4) Oncogenic mutations provide a competitive advantage to intestinal stem cells in tumorigenesis, but the competition takes place on a playing field sculpted by the nutritional environment, a major determinant of who wins. Citation Format: Jiahn Choi, Xusheng Zhang, Wenge Li, Michele Houston, Karina Peregrina, Robert Dubin, Kenny Ye, Leonard H. Augenlicht. Deconvolution by scRNAseq, scATACseq and functional genomics of impact of a relevant high risk diet on stem cells and homeostasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B024.

A review on the epigenetics modifications to nanomaterials in humans and animals: novel epigenetic regulator

Abstract In the nanotechnology era, nanotechnology applications have been intensifying their prospects to embrace all the vigorous sectors persuading human health and animal. The safety and concerns regarding the widespread use of engineered nanomaterials (NMA) and their potential effect on human health still require further clarification. Literature elucidated that NMA exhibited significant adverse effects on various molecular and cellular alterations. Epigenetics is a complex process resulting in the interactions between an organism’s environment and genome. The epigenetic modifications, including histone modification and DNA methylation, chromatin structure and DNA accessibility alteration, regulate gene expression patterns. Disturbances of epigenetic markers induced by NMA might promote the sensitivity of humans and animals to several diseases. Also, this paper focus on the epigenetic regulators of some dietary nutrients that have been confirmed to stimulate the epigenome and, more exactly, DNA histone modifications and non-histone proteins modulation by acetylation, and phosphorylation inhibition, which counteracts oxidative stress generations. The present review epitomizes the recent evidence of the potential effects of NMA on histone modifications, in addition to in vivo and in vitro cytosine DNA methylation and its toxicity. Furthermore, the part of epigenetic fluctuations as possible translational biomarkers for uncovering untoward properties of NMA is deliberated.

Genome-Wide Methylation and Haplotype-Resolved Aberrant Somatic Hypermutation Patterns in B-Cell Lymphomas

Introduction Diffuse large B-cell lymphoma (DLBCL) is a genetically, phenotypically, and clinically heterogenous disease. Multiple molecular classification systems using either genomic aberrations or gene expression profiles are used to delineate subgroups with shared biology. The cell of origin (COO) gene expression-based classification system stratifies DLBCLs into germinal center B-cell-like (GCB) and activated B-cell-like subgroups of DLBCL. More recently, the double-hit signature (DHITsig) has been used to further stratify GCB DLBCLs into DHITsig+ and DHITsig-, where the former is characterized by a gene expression profile reminiscent of the GC dark zone and inferior outcomes. Currently, patterns of somatic driver mutations can only partially explain the expression of DHITsig in some tumors. Genetic features such as DNA methylation have been shown to be associated with transcriptional repression and altered chromatin structure. We hypothesized that resolving DNA methylation patterns genome-wide using long read whole genome sequencing (LR-WGS) may reveal epigenetic differences associated with differential gene expression profiles within the DHITsig+ and DHITsig- subgroups and in the context of histologic transformation from follicular lymphoma. Methods Whole genome sequencing data of 10 patient samples and 4 cell line GCB DLBCL samples were generated using both short read (SR-WGS; Illumina) and LR-WGS (Oxford Nanopore Technologies, PromethION). The complementary approach combines somatic variant calling from highly accurate SR data with DNA methylation calling from LR data. GCB samples were selected based on the NanoString DLBCL90 assay and further subdivided into DHITsig+ (N=4) and DHITsig- (N=3). RNA sequencing data of 9 patient samples and 3 GCB DLBCL cell lines were generated and analyzed using Salmon and DESeq2. Simple somatic mutations from SR-WGS data were called using an ensemble of somatic variant callers, which confirmed all cases to belong to the EZB genetic subgroup. Methylated bases were called with Nanopolish followed by a differential methylation analysis using the DSS package in R to identify differences in methylation between both subgroups. Differentially methylated regions (DMRs) were annotated for GC B-cell specific superenhancers, CpG islands, and genes. Using WhatsHap and Nanomethphase, heterozygous single nucleotide polymorphisms (SNPs) from SR and LR data were used to phase LRs into haplotypes. Haplotype analyses were performed to determine the phase of aberrant somatic hypermutation (aSHM) and methylation patterns in the context of proximal translocation breakpoints. Results Most DMRs were found to be associated with regulatory regions such as promoters, superenhancers, and CpG islands. We found methylation differences between DHITsig+ and DHITsig- subgroups in 1430 superenhancers known to be active in GC B-cells. Within these superenhancer regions, DHITsig+ tumors were typically hypomethylated relative to DHITsig-. Several DHITsig classifier genes, including WNK2, TNFSF8, and IL21R, had methylation patterns inverted relative to the typical expression pattern (i.e. low methylation with high expression). Similarly, among 633 differentially methylated promoters, several including TCF3 and WNK2 demonstrated the same inverted methylation pattern. CpG islands showed distinct patterns of methylation in both subgroups that were inversely associated with expression, this also included genes differentially expressed between DHITsig+ and DHITsig- tumors. The DHITsig- group had increased levels of methylation of DHITsig genes in both promoters and gene bodies suggesting a functional role for gene body methylation. Finally, we observed that a high density of mutations near the BCL2 transcription start site consistently phased into the same haplotype as the BCL2-IGH translocation in five tumours, demonstrating the allele specificity of aSHM associated with these events (Panel B). Conclusions These results demonstrate differences in methylation between the subgroups. The association of hypomethylated superenhancers, promoters, and CpG islands with genes over-expressed in DHITsig+ suggests these methylation patterns may contribute to the DHITsig phenotype. These results may aid in improving GCB DLBCL patient classification and reveal factors contributing to its heterogeneity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cohesin-STAG2 Mutations Alter Chromatin Structure and Gene Regulation in Human Acute Myeloid Leukemia

Cohesin-STAG2 Mutations Alter Chromatin Structure and Gene Regulation in Human Acute Myeloid Leukemia

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.