Changes In Histone Methylation Research Articles

Amphetamine Exposure during Embryogenesis Alters Expression and Function of Tyrosine Hydroxylase and the Vesicular Monoamine Transporter in Adult C. elegans.

Amphetamines (Amph) are psychostimulants broadly used as physical and cognitive enhancers. However, the long-term effects of prenatal exposure to Amph have been poorly investigated. Here, we show that continuous exposure to Amph during early development induces long-lasting changes in histone methylation at the C. elegans tyrosine hydroxylase (TH) homolog cat-2 and the vesicular monoamine transporter (VMAT) homologue cat-1 genes. These Amph-induced histone modifications are correlated with enhanced expression and function of CAT-2/TH and higher levels of dopamine, but decreased expression of CAT-1/VMAT in adult animals. Moreover, while adult animals pre-exposed to Amph do not show obvious behavioral defects, when challenged with Amph they exhibit Amph hypersensitivity, which is associated with a rapid increase in cat-2/TH mRNA. Because C. elegans has helped reveal neuronal and epigenetic mechanisms that are shared among animals as diverse as roundworms and humans, and because of the evolutionary conservation of the dopaminergic response to psychostimulants, data collected in this study could help us to identify the mechanisms through which Amph induces long-lasting physiological and behavioral changes in mammals.

Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells

Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.0001), peaking at 15 min. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p < 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [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 444.

Epigenetic Profiling of PTPN11 Mutant JMML Hematopoietic Stem and Progenitor Cells Reveals an Aberrant Histone Landscape.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

Induction of Hibernation and Changes in Physiological and Metabolic Indices in Pelodiscus sinensis.

Pelodiscus sinensis (P. sinensis) is a commonly cultivated turtle species with a habit of hibernation. To study the changes in histone expression and methylation of P. sinensis during hibernation induction, a model was established by artificial induction. Physiological and metabolic indices were measured, and the expression and localization of histone (H1, H2A, H2B, H3, and H4) and methylation-related genes (ASH2L, KMT2A, KMT2E, KDM1A, KDM1B, and KDM5A) were measured by quantitative PCR, immunohistochemistry, and Western blot analysis. The results indicated that the metabolism, antioxidation index, and relative expression of histone methyltransferase were significantly decreased (p < 0.05), whereas the activity and expression of histone demethyltransferase were significantly increased (p < 0.05). Although our results showed significant changes in physiological and gene expression after hibernation induction, we could not confirm that P. sinensis entered deep hibernation. Therefore, for the state after cooling-induced hibernation, cold torpor might be a more accurate description. The results indicate that the P. sinensis can enter cold torpor through artificial induction, and the expression of histones may promote gene transcription. Unlike histones expressed under normal conditions, histone methylation may activate gene transcription during hibernation induction. Western blot analysis revealed that the ASH2L and KDM5A proteins were differentially expressed in the testis at different months (p < 0.05), which may perform a role in regulating gene transcription. The immunohistochemical localization of ASH2L and KDM5A in spermatogonia and spermatozoa suggests that ASH2L and KDM5A may perform a role in mitosis and meiosis. In conclusion, this study is the first to report changes in histone-related genes in reptiles, which provides insight for further studies on the physiological metabolism and histone methylation regulation of P. sinensis during the hibernation induction and hibernation period.

Abstract 3697: Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels

Abstract Introduction: Cancer cells often develop a program of metabolic adaptability and epigenetic remodelling to promote gene expression changes causing cancerous cell proliferation and invasion. The metabolic-epigenetic axis as a an underlying mechanism of carcinogenesis could lead to new approaches to cancer treatment. Nicotinamide N-Methyl Transferase (NNMT) is a metabolic enzyme that catalyzes the transfer of a methyl group from S-Adenosyl methionine (SAM) to nicotinamide, thereby decreasing the overall methyl pools for RNA, histone and DNA methylation. High NNMT expression has been previously associated with a poor outcome in TNBC. Hypothesis: We hypothesized that high versus low NNMT activity and resulting oncogenic gene expression in TNBC may alter histone methylation is association with altered metabolic pathway activation. Methods: We used two TNBC cell lines models, MDA-MB 231 and SUM159, to study the association between high versus low NNMT activity and metabolic pathway activation. Surprisingly, we identified gene expression pathways suggestive of increased free cholesterol biosynthesis gene expression with low NNMT, and then used biochemical assays and filipin to measure free cholesterol in both total high and low NNMT TNBC cell lysates and relative cholesterol expression in the High vs Low NNMT cell membranes. Results: In vitro, NNMT knockdown resulted in increased cholesterol synthetic pathway enzyme expression in both TNBC cell lines. In vivo studies also showed that protein expression of cholesterol synthesis enzymes was also increased in NNMT-depleted MDA-MB 231 xenografted tumors. Interestingly, the increased cholesterol level upon NNMT depletion was mainly localized at the plasma membrane via Filipin III staining, suggesting a possible association with less cellular membrane fluidity and a decreased oncogenic phenotype. In ongoing experiments, we are evaluating whether changes in histone methylation in high vs low NNMT cells are associated with differential expression of cholesterol biosynthetic genes, and free cholesterol production. We hypothesize that NNMT might epigenetically decrease the methylation of histones regulating cholesterol production in TNBC. Citation Format: Woei-Yaw Chee, Deniz Nesli Dolcen, Lynda Bennett, Suzanne D. Conzen. Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels. [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 3697.

Sodium Valproate Modulates the Methylation Status of Lysine Residues 4, 9 and 27 in Histone H3 of HeLa Cells.

Valproic acid/sodium valproate (VPA), a well-known anti-epileptic agent, inhibits histone deacetylases, induces histone hyperacetylation, promotes DNA demethylation, and affects the histone methylation status in some cell models. Histone methylation profiles have been described as potential markers for cervical cancer prognosis. However, histone methylation markers that can be studied in a cervical cancer cell line, like HeLa cells, have not been investigated following treatment with VPA. In this study, the effect of 0.5 mM and 2.0 mM VPA for 24 h on H3K4me2/me3, H3K9me/me2 and H3K27me/me3 signals as well as on KMT2D, EZH2, and KDM3A gene expression was investigated using confocal microscopy, Western blotting, and RT-PCR. Histone methylation changes were also investigated by Fourier-transform infrared spectroscopy (FTIR). We found that VPA induces increased levels of H3K4me2/me3 and H3K9me, which are indicative of chromatin activation. Particularly, H3K4me2 markers appeared intensified close to the nuclear periphery, which may suggest their implication in increased transcriptional memory. The abundance of H3K4me2/me3 in the presence of VPA was associated with increased methyltransferase KMT2D gene expression. VPA induced hypomethylation of H3K9me2, which is associated with gene silencing, and concomitant with the demethylase KDM3A, it increased gene expression. Although VPA induces increased H3K27me/me3 levels, it is suggested that the role of the methyltransferase EZH2 in this context could be affected by interactions with this drug. Histone FTIR spectra were not affected by VPA under present experimental conditions. Whether our epigenetic results are consistent with VPA affecting the aggressive tumorous state of HeLa cells, further investigation is required.

Fixed Cell Immunofluorescence for Quantification of Hypoxia-Induced Changes in Histone Methylation.

Hypoxia and its signalling pathway play a key role in human physiology and a variety of diseases. Alterations in histone methylation coordinate transcriptional responses to hypoxia. Here, we detail a fixed cell immunofluorescence method for quantifying hypoxia-induced changes in histone methylation, exemplified by the measurement of H3K27me3.

The Changes of Histone Methylation Induced by Adolescent Social Stress Regulate the Resting-State Activity in mPFC.

Early-life stress can lead to sustained alterations in regional resting-state brain functions, but the underlying molecular mechanism remains unclear. Stress can also induce sustained changes in epigenetic modifications across brain regions, which are not limited to a few genes; rather, they often tend to produce global levels of change. The functional implication of these changes also remains to be elucidated. We hypothesize that global epigenetic changes may partly modulate the resting-state functions of brain regions to influence behavior. To test this hypothesis, we used an adolescent social stress (ASS) model in mice and examined the relationship between epigenetic modifications and regional resting-state brain activity using resting-state functional magnetic resonance imaging (rs-fMRI). The results showed that, compared to the control mice, the stressed mice showed increased anxiety and social avoidance behaviors and greater levels of dimethylation of histone H3 at lysine 9 (H3K9me2) in the medial prefrontal cortex (mPFC). In addition, the resting-state activity represented by the amplitude of low-frequency fluctuation (ALFF) was significantly lower in the mPFC of stressed mice. To verify the relationship of H3K9me2 and ALFF, the specific inhibition of H3Kme2 was performed by using the drug UNC0642, which reversed the anxiety behavior induced by ASS and significantly increase the ALFF value of mPFC in both normal and ASS animals. Our study is the first to report an association between histone modifications and rs-fMRI findings, providing a new perspective for understanding of the significance of regional brain epigenetic changes and a possible molecular explanation for rs-fMRI findings.

The Role of Epigenetics in Neuroinflammatory-Driven Diseases

Neurodegenerative disorders are characterized by the progressive loss of central and/or peripheral nervous system neurons. Within this context, neuroinflammation comes up as one of the main factors linked to neurodegeneration progression. In fact, neuroinflammation has been recognized as an outstanding factor for Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and multiple sclerosis (MS). Interestingly, neuroinflammatory diseases are characterized by dramatic changes in the epigenetic profile, which might provide novel prognostic and therapeutic factors towards neuroinflammatory treatment. Deep changes in DNA and histone methylation, along with histone acetylation and altered non-coding RNA expression, have been reported at the onset of inflammatory diseases. The aim of this work is to review the current knowledge on this field.

Female athlete triad affects rat intestinal morphology and sucrase-isomaltase expression.

Female athletes follow a strict diet and perform rigorous exercise to boost their performance, which induces health issues called the female athlete triad (FAT), defined as the combination of disordered eating, amenorrhoea and low bone mineral density. It is known to have a significant effect on bones. However, its effects on the small intestine, which is responsible for nutrient uptake into the body, remain unclear. In this study, we created an animal model of FAT to examine its effects on digestive and absorptive molecules in the small intestine. Thirty 5-week-old female Sprague-Dawley (sd) rats with an initial body weight of about 147 g were divided into control (Con, n = 7), exercise (Ex, n = 7), food restriction (FR, n = 8) and exercise plus food restriction (FAT, n = 8) groups. The rats were subjected to 4 weeks of wheel running (Ex, FAT) and 50-40 % food restriction (FR, FAT) to examine the effects on bone and typical digestive enzymes and transporters in the jejunum. Two-way ANOVA and the Kruskal-Wallis test were used for statistical analysis of normal and non-normal data, respectively. Four weeks of exercise and food restriction decreased bone weight (vs. other group P < 0·01) and bone breaking power (vs. other group P < 0·01). Villus height decreased in the jejunum (vs. other group P < 0·01), but the expression of typical macronutrients digestive enzyme and absorptive molecules remained unchanged. In contrast, sucrase-isomaltase gene (v. Ex P = 0·02) and protein expression were increased (vs. other group P < 0·05). The study findings show that FAT affects sucrase-isomaltase without histone methylation changes.

Chromatin condensation regulates endothelial cell adaptation to shear stress.

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS.

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection

ABSTRACT Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host–parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

Reciprocal regulation of phosphatidylcholine synthesis and H3K36 methylation programs metabolic adaptation.

Phospholipid biosynthesis plays a role in mediating membrane-to-histone communication that influences metabolic decisions. Upon nutrient deprivation, phospholipid methylation generates a starvation signal in the form of S-adenosylmethionine (SAM) depletion, leading to dynamic changes in histone methylation. Here we show that the SAM-responsive methylation of H3K36 is critical for metabolic adaptation to nutrient starvation in the budding yeast Saccharomyces cerevisiae. We find that mutants deficient in H3K36 methylation exhibit defects in membrane integrity and pyrimidine metabolism and lose viability quickly under starvation. Adjusting the synthesis of phospholipids potently rewires metabolic pathways for nucleotide synthesis and boosts the production of antioxidants, ameliorating the defects resulting from the loss of H3K36 methylation. We further demonstrate that H3K36 methylation reciprocally regulates phospholipid synthesis by influencing redox balance. Our study illustrates an adaptive mechanism whereby phospholipid synthesis entails a histone modification to reprogram metabolism for adaptation in a eukaryotic model organism.

Histone Acetylation and Methylation Underlie Oligodendroglial and Myelin Susceptibility in Schizophrenia.

Schizophrenia is a complex neuropsychiatric disorder affected by both genetic and epigenetic factors. Except for neuronal dysfunction, oligodendroglial abnormalities also contribute to the disease pathogenesis, characterized by a robust dysregulation of oligodendrocyte and myelin related genes. Accumulating evidence shows that histone modifications play important roles in transcriptional regulation of the genes crucial for oligodendrocyte differentiation and myelination. Specifically, the histone acetylation and methylation were two well-recognized histone modification abnormalities in the schizophrenic brains. In this mini-review, we will describe the dynamic changes of histone acetylation and methylation in schizophrenia, which may coordinate and induce deleterious epigenetic memory in oligodendroglial cells, and further lead to oligodendrocyte and myelin deficits. Precise modulation of histone modification status in oligodendroglial cells needs to secure the balance of epigenetic marks, which may revise the therapeutic strategy for the white matter etiology of neuropsychiatric disorders.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Precision Oncology in Lower-Grade Gliomas: Promises and Pitfalls of Therapeutic Strategies Targeting IDH-Mutations.

Simple SummaryMutations of isocitrate dehydrogenase (IDH) genes are the distinctive genetic feature of lower-grade gliomas (LGGs). Tumor-associated IDH1/2 mutations result in a loss of normal enzymatic function and the abnormal production of 2-hydroxyglutarate (2-HG), which acts as an oncometabolite causing widespread changes in histone and DNA methylation and altering cellular metabolism. In the present review, we examine the “truncal” role of IDH mutations in gliomagenesis, giving hints on the different therapeutic strategies targeting IDH1/2-mutated gliomas. We analyze in detail, preclinical and, when available, data from clinical trials of specific inhibitors blocking the mutant enzyme, IDH-targeted immunotherapeutic approaches, and agents exploiting cellular metabolic and epigenetic vulnerabilities associated with the IDH mutant phenotype.Mutations in isocitrate dehydrogenase (IDH)1 and its homolog IDH2 are considered an earliest “driver” genetic event during gliomagenesis, representing now the molecular hallmark of lower-grade gliomas (LGGs). IDH-mutated genes encode for a neomorphic enzyme that converts α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2-HG), which accumulates to high concentrations and alters cellular epigenetics and metabolism. Targeting IDH mutations is the first attempt to apply “precision oncology” in LGGs. Two distinct strategies have been proposed so far and are under intense clinical investigation: (i) reducing the amount of intratumoral 2-HG by directly blocking the function of mutant IDH enzyme; (ii) exploiting the selective epigenetic and metabolic cellular vulnerabilities as a consequence of 2-HG accumulation. The present review describes the physiopathological mechanisms by which IDH mutations lead to tumorigenesis, discussing their prognostic significance and pivotal role in the gliomas diagnostic classification system. We critically review preclinical evidence and available clinical data of first-generation mutant-selective IDH inhibitors and novel IDH-targeted vaccines. Finally, as an alternative and attractive approach, we present the rationale to take advantage of selective 2-HG related epigenetic and metabolic weaknesses. The results of ongoing clinical trials will help us clarify the complex scenario of IDH-targeted therapeutic approaches in gliomas.

Differential Histone Posttranslational Modifications Induced by DNA Hypomethylating Agents.

IntroductionThe prototype DNA hypomethylating agents 5-azacytidine (5AC) and decitabine (DAC) are currently FDA-approved for treatment of blood and bone marrow disorders like myelodysplastic syndrome. 5AC and DAC are considered similar drugs and were shown to induce histone modifications that modulate gene expression. The aim of this study is to compare the effect of both drugs on histone acetylation and methylation at multiple histone amino acids residues.MethodsMass spectrometry was used to compare the effect of both drugs on 95 different histone posttranslational modifications (PTMs) in leukemia cells. ChIP-Seq analysis was used to compare the impact of both drugs on the genome-wide acetylation of the H3K9 mark using primary leukemia cells from six de-identified AML patients.ResultsBoth DAC and 5AC induced histone PTMs in different histone isoforms like H1.4, H2A, H3, H3.1, and H4. Changes in both histone methylation and acetylation were observed with both drugs; however, there were distinct differences in the histone modifications induced by the two drugs. Since both drugs were shown to increase the activity of the HDAC SIRT6 previously, we tested the effect of 5AC on the acetylation of H3K9, the physiological substrate SIRT6, using ChIP-Seq analysis and compared it to the previously published DAC-induced changes. Significant H3K9 acetylation changes (P< .05) were detected at 925 genes after 5AC treatment vs only 182 genes after DAC treatment. Nevertheless, the gene set modified by 5AC was different from that modified by DAC with only ten similar genes modulated by both drugs.ConclusionDespite similarity in chemical structure and DNA hypomethylating activity, 5AC and DAC induced widely different histone PTMs and considering them interchangeable should be carefully evaluated. The mechanism of these histone PTM changes is not clear and may involve modulation of the activity or the expression of the enzymes inducing histone PTMs.

Abstract PR-008: Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification

Abstract Aberrant epigenetic regulation is a hallmark of pancreatic cancer. Since changes in histone methylation are potentially reversible, they present an attractive target for therapeutic intervention. Kdm6a and Kdm6b belong to a family of histone demethylases specific for lysine 27 of histone 3 and have been implicated in multiple types of cancer, including pancreatic ductal adenocarcinoma (PDA). We have gathered data from the extensive investigation of a mouse model of PDA in which either Kdm6b or both Kdm6a and Kdm6b have been deleted with concomitant activation of oncogenic KrasG12D restricted to adult acinar cells. While prior work had demonstrated an important role for Kdm6a in PDA formation, our data now show that the closely related Kdm6b is implicated in PDA tumorigenesis as well. Partial or complete loss of Kdm6b results in accelerated acinar-to-ductal metaplasia (ADM) and formation of pancreatic intraepithelial neoplasia (PanIN) with increased inflammation and fibrosis. Accelerated ADM formation was also observed ex vivo upon culturing acinar cells in the absence of oncogenic Kras, indicating that Kdm6b activity is critical for the maintenance of acinar identity and suppresses the early stages of pancreatic neoplasia development. However, only mice heterozygous for Kdm6b continue to exhibit a reduced overall median survival compared to KrasG12D alone, while complete loss of Kdm6b results in a similar overall median survival compared to KrasG12D. This suggests a requirement for oncogenic Kdm6b function during later progression of PanIN to invasive cancer. Conversely, concomitant loss of Kdm6a and Kdm6b protects against ADM and PanIN formation in vivo and ex vivo and double mutants display a trend towards an increased overall median survival compared to mice carrying only the KrasG12D mutation. Strikingly, the resulting tumors of Kdm6a/Kdm6b deficient mice exhibit features of an extremely rare subtype of sarcomatoid pancreatic cancer found in human patients. Taken together, this suggests that Kdm6 demethylase function is required during the initiation and progression of PDA. Furthermore, targeting Kdm6a/Kdm6b with specific inhibitors at distinct stages might provide opportunities for novel treatment avenues for pancreatic cancer. Citation Format: Laura Leonhardt, Lucia Y. Li, David I. Berrios, Sudipta Ashe, Audrey M. Hendley, Grace E. Kim, Matthias Hebrok. Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-008.

Methionine affects the expression of pluripotency genes and protein levels associated with methionine metabolism in adult, fetal, and cancer stem cells.

Intracellular and extracellular regulatory factors promote the potency and self-renewal property of stem cells. Methionine is fundamental for protein synthesis and regulation of methylation reactions. Specifically, methionine metabolism in embryonic and fetal development processes regulates gene expression profile/epigenetic identity of stem cells to achieve pluripotencyand cellular functions. We aimed to reveal the differences in methionine metabolism of bone marrow (BM)-mesenchymal stem cells (MSCs), umbilical cord blood (UCB)-MSCs, and cancer stem cells (CSCs), which reflect different metabolic profiles and developmental stages of stem cells. UCB-MSC, BM-MSCs, and breast CSCs were treated with different doses (0, 10, 25, 50, and 100 µM) of l-methionine. Cell surface marker and cell cycle assessment were performed by flow cytometry. Changes in gene expressions (OCT3/4, NANOG, DMNT1, DNMT3A, and DNMT3B, MAT2A, and MAT2B) with methionine supplementation were examined by quantitative real-time polymerase chain reaction and the changes in histone methylation (H3K4me3, H3K27me3) levels were demonstrated by western blot analysis. S-adenosylmethionine//S-adenosylhomocysteine (SAM/SAH) levels were evaluated by enzyme-linked immunosorbent assay. Cells that were exposed to different concentrations of l-methionine, were mostly arrested in the G0/G1 phase for each stem cell group. It was evaluated that BM-MSCs increased all gene expressions in the culture medium-containing 100 µM methionine, in addition to SAM/SAH levels. On the other hand, UCB-MSCs were found to increase OCT3/4, NANOG, and DNMT1 gene expressions and decrease MAT2A and MAT2B expressions in the culture medium containing 10 µM methionine. Moreover, an increase was observed in the He3K4me3 methylation profile. In addition, OCT3/4,NANOG, DNMT1, and MAT2B gene expressions in CSCs increased starting from the addition of 25 µM methionine. An increase was determined in H3K4me3 protein expression at 50 and 100 µM methionine-supplemented culture condition. This study demonstrates that methionine plays a critical role in metabolism and epigenetic regulation in different stem cell groups.

Dynamic changes of histone methylation in mammalian oocytes and early embryos.

Histone methylation is a key epigenetic mechanism and plays a major role in regulating gene expression during oocyte maturation and early embryogenesis. This mechanism can be briefly defined as the process by which methyl groups are transferred to lysine and arginine residues of histone tails extending from nucleosomes. While methylation of the lysine residues is catalyzed by histone lysine methyltransferases (KMTs), protein arginine methyltransferases (PRMTs) add methyl groups to the arginine residues. When necessary, the added methyl groups can be reversibly removed by histone demethylases (HDMs) by a process called histone demethylation. The spatiotemporal regulation of methylation and demethylation in histones contributes to modulating the expression of genes required for proper oocyte maturation and early embryonic development. In this review, we comprehensively evaluate and discuss the functional importance of dynamic histone methylation in mammalian oocytes and early embryos, regulated by KMTs, PRMTs, and HDMs.