Modulation Of Chromatin Structure Research Articles

Ketogenic diet modifies ribosomal protein dysregulation in KMT2D Kabuki syndrome

BackgroundKabuki syndrome (KS) is a genetic disorder caused by DNA mutations in KMT2D, a lysine methyltransferase that methylates histones and other proteins, and therefore modifies chromatin structure and subsequent gene expression. Ketones, derived from the ketogenic diet, are histone deacetylase inhibitors that can ‘open’ chromatin and encourage gene expression. Preclinical studies have shown that the ketogenic diet rescues hippocampal memory neurogenesis in mice with KS via the epigenetic effects of ketones. MethodsSingle-cell RNA sequencing and mass spectrometry-based proteomics were used to explore molecular mechanisms of disease in individuals with KS (n=4) versus controls (n=4). FindingsPathway enrichment analysis indicated that loss of function mutations in KMT2D are associated with ribosomal protein dysregulation at an RNA and protein level in individuals with KS (FDR <0.05). Cellular proteomics also identified immune dysregulation and increased abundance of other lysine modification and histone binding proteins, representing a potential compensatory mechanism. A 12-year-old boy with KS, suffering from recurrent episodes of cognitive decline, exhibited improved cognitive function and neuropsychological assessment performance after 12 months on the ketogenic diet, with concomitant improvement in transcriptomic ribosomal protein dysregulation. InterpretationOur data reveals that lysine methyltransferase deficiency is associated with ribosomal protein dysfunction, with secondary immune dysregulation. Diet and the production of bioactive molecules such as ketone bodies serve as a significant environmental factor that can induce epigenetic changes and improve clinical outcomes. Integrating transcriptomic, proteomic, and clinical data can define mechanisms of disease and treatment effects in individuals with neurodevelopmental disorders. FundingThis study was supported by the Dale NHMRC Investigator Grant (APP1193648) (R.D), Petre Foundation (R.D), and The Sydney Children’s Hospital Foundation/Kids Research Early and Mid-Career Researcher Grant (E.T).

Linking Aging to Cancer: The Role of Chromatin Biology.

Epigenetic changes have been established to be a hallmark of aging, which implies that aging science requires collaborating with the field of chromatin biology. DNA methylation patterns, changes in relative abundance of histone post-translational modifications (PTMs), and chromatin remodeling are the central players in modifying chromatin structure. Aging is commonly associated with an overall increase in chromatin instability, loss of homeostasis and decondensation. However, numerous publications have highlighted that the link between aging and chromatin changes is not nearly as linear as previously expected. This complex interplay of these epigenetics elements during the lifetime of an organism likely contribute to cellular senescence, genomic instability, and disease susceptibility. Yet, the causal links between these phenomena still need to be fully unraveled. In this perspective article, we discuss potential future directions of aging chromatin biology.

P22 A single-cell map of the epigenomic landscape in keratinocytes exposed to the cytokine milieu of atopic dermatitis

Abstract Introduction and aims In atopic dermatitis (AD), induction of inflammatory responses regulated by cytokines such as of thymic stromal lymphopoietin exacerbate the disrupted integrity of the skin barrier. Cytokines, such as interleukin (IL)-4, IL-13 and IL-22, present in the skin milieu, bind to their corresponding receptors on the cell surface of keratinocytes, activating multiple signal transduction pathways that lead to changes in the gene expression profile of keratinocytes, in part, by modulating chromatin structure. Understanding the intricate interplay between cytokine-mediated signalling pathways in keratinocytes and the resulting epigenetic changes is essential for elucidating the pathogenesis of AD. In this study, we utilized high-throughput technology to examine the effects of cytokines on modulation of the epigenetic landscape at the keratinocyte single-cell level, and subsequent effects on the gene expression. Methods We utilized the reconstructed human epidermis model, an in vitro three-dimensional human epidermis model consisting of a polycarbonate membrane and keratinocytes mimicking the structure of native human epidermis. Following exposure to AD cytokine(s), the model gene expression and chromatin accessibility of target cells were analysed using single-cell multiomics approaches. Results Our preliminary data show that IL-4, IL-13 and IL-22 reduce the expression levels of the barrier-related gene filaggrin in keratinocytes. Conclusions Unlike genetic changes, epigenetic alterations are dynamic and can be restored to their normal state, which has major implications for the development of therapeutics for various diseases. This study will provide valuable insights into the epigenetic mechanisms underlying AD and present potential targets for epigenetic-related therapeutic interventions. These interventions can be used in combination with available compounds to reduce the recurrence of AD and improve the treatment efficiency.

KAT8 beyond Acetylation: A Survey of Its Epigenetic Regulation, Genetic Variability, and Implications for Human Health.

Lysine acetyltransferase 8, also known as KAT8, is an enzyme involved in epigenetic regulation, primarily recognized for its ability to modulate histone acetylation. This review presents an overview of KAT8, emphasizing its biological functions, which impact many cellular processes and range from chromatin remodeling to genetic and epigenetic regulation. In many model systems, KAT8's acetylation of histone H4 lysine 16 (H4K16) is critical for chromatin structure modification, which influences gene expression, cell proliferation, differentiation, and apoptosis. Furthermore, this review summarizes the observed genetic variability within the KAT8 gene, underscoring the implications of various single nucleotide polymorphisms (SNPs) that affect its functional efficacy and are linked to diverse phenotypic outcomes, ranging from metabolic traits to neurological disorders. Advanced insights into the structural biology of KAT8 reveal its interaction with multiprotein assemblies, such as the male-specific lethal (MSL) and non-specific lethal (NSL) complexes, which regulate a wide range of transcriptional activities and developmental functions. Additionally, this review focuses on KAT8's roles in cellular homeostasis, stem cell identity, DNA damage repair, and immune response, highlighting its potential as a therapeutic target. The implications of KAT8 in health and disease, as evidenced by recent studies, affirm its importance in cellular physiology and human pathology.

Unraveling transcriptomic signatures and dysregulated pathways in systemic lupus erythematosus across disease states

ObjectivesThis study aims to elucidate the transcriptomic signatures and dysregulated pathways in patients with Systemic Lupus Erythematosus (SLE), with a particular focus on those persisting during disease remission.MethodsWe conducted bulk RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from a well-defined cohort comprising 26 remission patients meeting the Low Lupus Disease Activity State (LLDAS) criteria, 76 patients experiencing disease flares, and 15 healthy controls. To elucidate immune signature changes associated with varying disease states, we performed extensive analyses, including the identification of differentially expressed genes and pathways, as well as the construction of protein-protein interaction networks.ResultsSeveral transcriptomic features recovered during remission compared to the active disease state, including down-regulation of plasma and cell cycle signatures, as well as up-regulation of lymphocytes. However, specific innate immune response signatures, such as the interferon (IFN) signature, and gene modules involved in chromatin structure modification, persisted across different disease states. Drug repurposing analysis revealed certain drug classes that can target these persistent signatures, potentially preventing disease relapse.ConclusionOur comprehensive transcriptomic study revealed gene expression signatures for SLE in both active and remission states. The discovery of gene expression modules persisting in the remission stage may shed light on the underlying mechanisms of vulnerability to relapse in these patients, providing valuable insights for their treatment.

Single-cell 3D genome structure reveals distinct human pluripotent states

BackgroundPluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive.ResultsBy single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes.ConclusionsOur data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Altered Histone Deacetylase Versus Histone Acetyltransferase Activity in Peripheral Blood Mononuclear Cells from Apparently Healthy Young Adults with Prior Exposure to Adverse Childhood Experiences

Introduction: Adverse childhood experiences (ACEs) are severe psychosocial stressors which occur in early developmental periods and promote later life cardiometabolic disease in a dose-dependent manner. ACE-related risk may be mediated in part by epigenetic alterations, particularly in myeloid lineage immune cells. Histone acetylation is a dynamic epigenetic process that regulates gene expression via modification of chromatin structure and is dictated by the opposed actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). An appropriate balance between HDAC and HAT activity is critical for maintaining cellular function, and an imbalance has been implicated in the premature development of human diseases including cardiometabolic disease. Glucocorticoids (e.g., cortisol) are well-described regulators of immune function and can influence histone acetylation through glucocorticoid receptors. Thus, we hypothesized that HDAC:HAT activity would be altered in young adults with prior ACE exposure, and that HDAC:HAT activity would be associated with salivary cortisol concentrations. Purpose: We examined HDAC:HAT activity in peripheral blood mononuclear cells (PBMCs) in apparently healthy young adults with versus without prior exposure to ACEs and explored whether circulating cortisol was associated with HDAC:HAT activity. Methods: PBMCs were isolated from whole blood collected within 1 hour of wakening from 27 young adult women who reported high (≥ 4 ACEs) ACE exposure (ACE4+; mean ± SD; age = 21 ± 3 y, BMI = 25.7 ± 4.5 kg/m2) and from 14 young adult women who reported no ACE exposure (CON; age = 21 ± 3 y, BMI = 26.1 ± 6 kg/m2). Salivary cortisol was also collected within 1 hour of wakening and 8 hours later. Nuclear proteins were isolated from PBMCs and HDAC and HAT activities were quantified by commercially available assay kits. Salivary cortisol was quantified via ELISA. Independent samples t-tests were used to examine differences in HDAC:HAT, HDAC, and HAT activity. Pearson correlation coeffcients were used to examine the relation of HDAC:HAT activity with morning and afternoon cortisol concentrations, and the diurnal cortisol slope in the complete sample. Results: HDAC:HAT activity was reduced in ACE4+ versus CON (140.4 ± 52 vs. 189.6 ± 42 arbitrary units, p = 0.004). This difference was primarily driven by reduced HDAC activity (13,706 ± 4,696 vs. 18,060 ± 3,641 ng/h/mg, p = 0.004) in ACE4+ versus CON, whereas HAT activity was not different (99.7 ± 11 vs. 96.1 ± 9 ng/h/mg, p = 0.30). HDAC:HAT activity was not associated with morning (r=0.27, p = 0.07) or afternoon (r=-0.23, p = 0.16) cortisol, but was associated with the diurnal cortisol slope (r=-0.37, p = 0.02). Discussion: Young adults with prior ACE exposure have an altered balance between HDAC and HAT activity in PBMCs that appears to be driven by lower HDAC activity, and which is negatively related to diurnal cortisol slope. These findings warrant further exploration, particularly regarding the specific HDACs implicated, their role in reduced cellular function as it relates to disease risk, and whether these can be modulated to improve function among those with ACE exposure. Further, our data support future examination of the role of glucocorticoid signaling in regulating histone acetylation in this population. This study was supported by the Center for Integrative Research on Childhood Adversity (Grant #P20GM109097), through the National Institute of General Medical Sciences (to NDMJ and TKT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [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 5592.

Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusarium fujikuroi

BackgroundFusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as ‘bakanae’ or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin.ResultsThrough the combination of diverse ‘-omics‘ methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism.ConclusionIn this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 isone of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Beyond the tail: the consequence of context in histone post-translational modification and chromatin research.

The role of histone post-translational modifications (PTMs) in chromatin structure and genome function has been the subject of intense debate for more than 60 years. Though complex, the discourse can be summarized in two distinct - and deceptively simple - questions: What is the function of histone PTMs? And how should they be studied? Decades of research show these queries are intricately linked and far from straightforward. Here we provide a historical perspective, highlighting how the arrival of new technologies shaped discovery and insight. Despite their limitations, the tools available at each period had a profound impact on chromatin research, and provided essential clues that advanced our understanding of histone PTM function. Finally, we discuss recent advances in the application of defined nucleosome substrates, the study of multivalent chromatin interactions, and new technologies driving the next era of histone PTM research.

Identification and partial characterization of new cell density-dependent nucleocytoplasmic shuttling proteins and open chromatin

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.

High Mobility Group A1 Chromatin Regulators Drive Regenerative Capacity during Stress Hematopoiesis through Modulation of TNF-α and Aging Transcriptional Networks

Introduction: Chromatin structure and epigenetic modifications are key determinants of regenerative function and cell fate decisions, although mechanisms governing these properties in hematopoiesis are only beginning to emerge. The High Mobility Group A1 ( HMGA1) chromatin regulator gene is induced by growth factors and highly expressed during embryogenesis and in hematopoietic stem cells (HSCs), but silenced in differentiated tissues ( Cancer Res 2018,78:1890). In leukemia and solid tumors, HMGA1 is re-expressed where it activates enhancers that induce stem cell transcriptional networks, leading to aberrant proliferation and differentiation ( Blood 2022,139:2797; JCI 2023 133:e151601). HMGA1 also drives self-renewal by amplifying Wnt networks in intestinal stem cells ( Nature Comm 2017,8:15008). However, its role in normal hematopoiesis is unknown. We therefore sought to: 1) Define HMGA1 function in HSC, and, 2) Test the hypothesis that modulating HMGA1 epigenetic networks enhances regenerative function. Methods: First, we compared HMGA1 expression across hematopoietic stem and progenitor cells (HSPC) and differentiated progeny in humans and mice. Next, we generated mice with global Hmga1 knock-out ( Hmga1-KO) or Hmga1 deficiency confined to HSC (vav- Hmga1-KO) to assess HSPC function in clonogenic assays, competitive bone marrow transplantation (cBMT), and following irradiation. To elucidate underlying mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. To assess HMGA1 function in human CD34 + cells, we used CRISPR to inactivate HMGA1. Results: HMGA1 is expressed in HSC, multipotent progenitors (MPP), and megakaryocyte-erythroid progenitors (MEP), with decreasing levels after differentiation, suggesting that HMGA1 functions in stem and progenitors. However, steady state hematopoiesis is unperturbed in both Hmga1-KO and vav- Hmga1-KO mice up to 18 months. By contrast, HSPC lacking Hmga1 show decreases in total colony forming units (CFU), but only after serial replating, suggesting that Hmga1 deficiency impairs long-term HSC (LT-HSC) regenerative function. Accordingly, in cBMT assays, Hmga1-KO HSC are outcompeted by HSC with both Hmga1 alleles, with most pronounced effects after serial transplantation. Serial cBMT from young donors (6 weeks) show decreases in LT-HSC, short-term HSC (ST-HSC), and progenitors from Hmga1 KO HSPC compared to control HSPC. Results were similar with older donors (4 months, 18 months). Following irradiation, vav- Hmga1-KO mice have impaired platelet recovery, although the frequency of HSPC in bone marrow were similar to controls. HSPC function in clonogenic assays is also impaired after irradiation, as evidenced by decreases in total CFU, GM-CFU and megakaryocyte (Mk)-CFU. To determine how HMGA1 modulates cell state, we compared scRNAseq in BM-derived LSK cells from adult mice (4 months old) with intact Hmga1 to those with Hmga1-KO. The transcriptomes in HSPC lacking Hmga1 were distinct from those with both Hmga1 alleles intact. The Hmga1 KO HSPC show a decrease in quiescent HSC (marked by high Procr expression), with a relative expansion in more proliferative HSC and early progenitors. In human CD34 + cells, CRISPR-mediated HMGA1 inactivation decreases total CFU, GM-CFU, and Mk-CFU. To elucidate underlying mechanisms, we applied gene set enrichment analyses to scRNAseq results which revealed that Hmga1 activates TNF-a signaling via NF-κB, a pathway that maintains HSC survival and regeneration following inflammatory stress. Hmga1 also represses aging-related transcriptional networks. Further, TNFa partially rescues HSC with Hmga1 deficiency in serial clonogenicity assays. Conclusions: HMGA1 deficiency impairs HSC regenerative capacity under conditions of stress hematopoiesis by depleting quiescent HSC. Mechanistically, HMGA1 induces transcriptional networks involved in survival signals, including TNFa -NF-κB networks, while repressing transcriptional networks activated with aging. Further, TNF-apartially rescues regenerative capacity in clonogenicity assays. Our findings highlight HMGA1 as a critical factor in stress hematopoiesis and suggest that modifying its function could enhance regenerative capacity and repress aging pathways following BMT or irradiation.

Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias

Introduction Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018). Aims: The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC). Methods Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) &amp;gt; 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software. Results Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C &amp; KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant. AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency &amp;gt; 0.001. The average Phred score was 19.8; 12/20 patients had Phred score &amp;gt; 20, suggesting that most mutations are in the top 1% of the deleterious variants. Conclusion We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.

Unraveling the allosteric inhibition mechanism of PARP-1 CAT and the D766/770A mutation effects via Gaussian accelerated molecular dynamics and Markov state model

PARP-1 (Poly (ADP-ribose) polymerase 1) is a nuclear enzyme and plays a key role in many cellular functions, such as DNA repair, modulation of chromatin structure, and recombination. Developing the PARP-1 inhibitors has emerged as an effective therapeutic strategy for a growing list of cancers. The catalytic structural domain (CAT) of PARP-1 upon binding the inhibitor allosterically regulates the conformational changes of helix domain (HD), affecting its identification with the damaged DNA. The typical type I (EB47) and III (veliparib) inhibitors were able to lengthening or shortening the retention time of this enzyme on DNA damage and thus regulating the cytotoxicity. Nonetheless, the basis underlying allosteric inhibition is unclear, which limits the development of novel PARP-1 inhibitors. Here, to investigate the distinct allosteric changes of EB47 and veliparib against PARP-1 CAT, each complex was simulated via classical and Gaussian accelerated molecular dynamics (cMD and GaMD). To study the reverse allosteric basis and mutation effects, the complexes PARP-1 with UKTT15 and PARP-1 D766/770A mutant with EB47 were also simulated. Importantly, the markov state models were built to identify the transition pathways of crucial substates of allosteric communication and the induction basis of PARP-1 reverse allostery. The conformational change differences of PARP-1 CAT regulated by allosteric inhibitors were concerned with to their interaction at the active site. Energy calculations suggested the energy advantage of EB47 in inhibiting the wild-type PARP-1, compared with D766/770A PARP-1. Secondary structure results showed the change of two key loops (αB-αD and αE-αF) in different systems. This work reported the basis of PARP-1 allostery from both thermodynamic and kinetic views, providing the guidance for the discovery and design of more innovative PARP-1 allosteric inhibitors.

Enhanced Transcriptional Signature and Expression of Histone-Modifying Enzymes in Salivary Gland Tumors.

Salivary gland tumors (SGTs) are rare and complex neoplasms characterized by heterogenous histology and clinical behavior as well as resistance to systemic therapy. Tumor etiology is currently under elucidation and an interplay of genetic and epigenetic changes has been proposed to contribute to tumor development. In this work, we investigated epigenetic regulators and histone-modifying factors that may alter gene expression and participate in the pathogenesis of SGT neoplasms. We performed a detailed bioinformatic analysis on a publicly available RNA-seq dataset of 94 ACC tissues supplemented with clinical data and respective controls and generated a protein-protein interaction (PPI) network of chromatin and histone modification factors. A significant upregulation of TP53 and histone-modifying enzymes SUV39H1, EZH2, PRMT1, HDAC8, and KDM5B, along with the upregulation of DNA methyltransferase DNMT3A and ubiquitin ligase UHRF1 mRNA levels, as well as a downregulation of lysine acetyltransferase KAT2B levels, were detected in ACC tissues. The protein expression of p53, SUV39H1, EZH2, and HDAC8 was further validated in SGT tissues along with their functional deposition of the repressive histone marks H3K9me3 and H3K27me3, respectively. Overall, this study is the first to detect a network of interacting proteins affecting chromatin structure and histone modifications in salivary gland tumor cells, further providing mechanistic insights in the molecular profile of SGTs that confer to altered gene expression programs.

OR22-06 Functional Characterization Of X-linked Genes In Regulating Epigenetic Landscape In Endocrine-resistant Breast Cancer

Abstract Disclosure: E.I. Ramos: None. B. Yang: None. M. Sedano: None. V. Reid: None. K. Diwa: None. A. Hidalgo: None. A. Beard: None. J. Patel: None. S.S. Gadad: None. Studies show that genes transcribed from chromosome X play critical roles in several biological processes. X-linked genes are tightly regulated and expressed in tissues protected from immune surveillance. However, several of them get dysregulated in diseases such as cancer. Intriguingly, genome-wide analyses showed their presence in the previously considered gene deserts in the human genome. Their direct role in estrogen transcription in tamoxifen-resistant breast cancer and biological outcomes are still unknown. To this end, we leveraged the power of functional genomics, including RNA with 4-thiouridine (4sU) coupled with RNA-sequencing and CRISPR-based genetic screening, to identify X-linked genes with a potential role in tamoxifen-directed estrogen-regulated transcription. Through a series of phenotypic assays, we selected a poorly studied transcript, “H19X,” with limited protein-coding potential; RNA-In-Situ-Hybridization revealed its localization to the nucleus. In the current study, we have identified the H19X-controlled estrogen-dependent gene regulatory mechanisms in tamoxifen-resistant estrogen receptor-positive (ER+) breast cancer cells. We manipulated the levels of H19X in tamoxifen-resistant ER+ breast cancer cells to generate a comprehensive list of direct transcripts using 4sU-RNA-seq. Interestingly, H19X regulates the expression of estrogen-driven intergenic, divergent, and antisense transcripts whose promoters are bound by ER-alpha (ERα) binding sites. Mechanistically, ‘Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)’ revealed that H19X modulates chromatin structure at target regions to control gene expression. In addition, ‘chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing ChIP-seq’ (H3K27ac, H3K27me3) identified a critical role for H19X in controlling the epigenetic landscape in tamoxifen-resistant ER+ breast cancer cells. Collectively, our study sheds light on the molecular functions of X-linked genes and allows us to identify new diagnostic and therapeutic targets in tamoxifen-resistant ER+ breast cancer.S.S.G. is supported by grants from a) The Cancer Prevention and Research Institute of Texas (CPRIT; RR170020); b) Lizanell and Colbert Coldwell foundation; c) The Edward N. and Margaret G. Marsh Foundation; d) The American Cancer Society Presentation: Saturday, June 17, 2023

Advances in direct detection of lysine methylation and acetylation by nuclear magnetic resonance using 13C-enriched cofactors.

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [1H, 13C]-HSQC and 13C direct-detect NMR measurements. Finally, we present 13C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.

The role of epigenetic modifications in the formation of heterogeneous phenotypes in diabetes mellitus (a literature review)

This review article provides a summary and update on the role of epigenetic mechanisms in predisposition and progression of diabetes, analyzes the data concerning the cause-and-effect relationship between epigenetic changes and the emergence of distinct metabolic phenotypes. Extensive genetic research has enabled the isolation of a group of genes associated with a high risk of developing diabetes. However, numerous data point to the key role of so-called epigenetic modifications in the interaction between genes and the environment, which arise during ontogenesis based on the existing genotype under the influence of external factors. These modifications do not affect the primary DNA sequence, but influence gene expression through chemical modification and alteration of the secondary structure of DNA molecules and chromatin. Epigenetic mechanisms can program pathological phenotypes in subsequent generations. The main molecular mechanisms of epigenetic modifications are DNA methylation, histone and miRNA modification. Changes in the expression of genes that ensure the synthesis of key enzymes and regulatory molecules lead to disruption in the main signaling metabolic pathways. Deregulation of genes responsible for inflammatory, atherosclerotic and other pathological processes, in particular, leads to endothelial dysfunction and development of diabetic complications, such as cardiovascular diseases, diabetic nephropathy, retinopathy, neuropathy. Hyperglycemia, oxidative stress, inflammatory factors are known as mediators in the pathogenesis of type 2 diabetes and its complications. Since epigenetic modifications are reversible, the methylation process can be influenced by exercise, dietary, lifestyle changes and pharmacological agents such as methyl group donors. For example, S-adenosylmethionine, through participation in methylation reactions, can modulate the folate cycle function and production of homocysteine, an endothelium-toxic substance. Thus, the study of molecular modifications in chromatin structure and the features of activation and inhibition of various signaling pathways is a pressing task, the resolution of which will enable a deeper understanding of the pathogenesis of diabetes and the development of approaches to correct metabolic disorders.

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.