Role In Epigenetic Control Research Articles

CSIG-10. DEFINING THE ROLE OF EZHIP ON CHROMOSOMAL INSTABILITY IN EPENDYMOMA

Abstract Diffuse Midline Gliomas (DMGs) are highly aggressive pediatric cancers with no effective treatments. Over 80% of DMGs harbor histone mutations, particularly H3.1 K27M, H3.3 K27M, and H3.3 G34R/V. These mutations impair the function of the Polycomb Repressive Complex 2 (PRC2), leading to reduced levels of H3K27me3. Overexpression of the EZHIP has been identified in ependymomas and DMGs. EZHIP overexpression is marked by widespread H3K27me3 loss and is mutually exclusive with histone mutations. EZHIP overexpression mimics the effects of the H3K27M mutation by reducing H3K27me3 levels. It is believed to inhibit the catalytic activity of EZH2 by binding to its SET domain, similar to the K27M mutant. Expression of EZHIP is tightly regulated and typically absent in most cell types. The transcriptional regulation of EZHIP is poorly understood; however, promoter hypermethylation is likely involved in regulating its expression. We investigate EZHIP’s role in DMG tumorigenesis, focusing on its regulation, impact on histone modifications, and chromosomal stability. Initial analyses have revealed transcription factor binding sites within the EZHIP promoter, including EGR2, SOX2, and ICP4, indicating that various transcription factors and viral infections might directly affect EZHIP expression. Utilizing RNA-Seq, we have identified both shared and unique transcriptomic alterations in DMG cells related to EZHIP expression and H3.3 K27M mutations. To study the role of EZHIP in tumorigenesis, we developed a mouse model expressing EZHIP-HA p2A PDFGB in RCAS-Nestin TVA mice. EZHIP expression induces high-grade tumors and significantly reduces survival. Our ongoing research suggests that EZHIP functions similarly to H3.3 K27M mutations in driving tumor growth, not only by reducing K27me3 but also through chromosomal missegregation. We aim to elucidate the full extent of EZHIP’s role in epigenetic control, histone post-translational modifications, chromosomal accessibility, and instability. Our findings will provide critical insights into the mechanisms underlying DMG tumorigenesis and potential therapeutic targets.

CHHM: a Manually Curated Catalogue of Human Histone Modifications Revealing Hotspot Regions and Unique Distribution Patterns.

Histone modification is one of the key elements in epigenetic control and plays important roles in regulation of biological processes and disease development. Currently, records of human histone modifications with various levels of confidence in evidence are scattered in various knowledgebases and databases. In the present study, a curated catalogue of human histone modifications, CHHM, was obtained by manual retrieval, evidence assessment, and integration of modification records from 10 knowledgebases/databases and 3 complementary articles. CHHM contains 6612 nonredundant modification entries covering 31 types of modifications (including 9 types of emerging modifications) and 2 types of histone-DNA crosslinks, that were identified in 11 H1 variants, 21 H2A variants, 21 H2B variants, 9 H3 variants, and 2 H4 variants. For ease of visualization and accessibility, modification entries are presented with aligned protein sequences in an Excel file. Confidence level in evidence is provided for each entry. Acylation modifications contribute to the highest number of modification entries in CHHM. This supports that cellular metabolic status plays a very important role in epigenetic control. CHHM reveals modification hotspot regions and uneven distribution of the modification entries across the histone families. Such uneven distribution may suggest that a particular histone family is more susceptible to certain types of modifications. CHHM not only serves as an important and user-friendly resource for biomedical and clinical researches involving histone modifications and transcriptional regulation, but also provides new insights for basic researches in the mechanism of human histone modifications and epigenetic control.

Evolutionary trajectory of organelle-derived nuclear DNAs in the Triticum/Aegilops complex species.

Organelle-derived nuclear DNAs, nuclear plastid DNAs (NUPTs), and nuclear mitochondrial DNAs (NUMTs) have been identified in plants. Most, if not all, genes residing in NUPTs/NUMTs (NUPGs/NUMGs) are known to be inactivated and pseudogenized. However, the role of epigenetic control in silencing NUPGs/NUMGs and the dynamic evolution of NUPTs/NUMTs with respect to organismal phylogeny remain barely explored. Based on the available nuclear and organellar genomic resources of wheat (genus Triticum) and goat grass (genus Aegilops) within Triticum/Aegilops complex species, we investigated the evolutionary fates of NUPTs/NUMTs in terms of their epigenetic silencing and their dynamic occurrence rates in the nuclear diploid genomes and allopolyploid subgenomes. NUPTs and NUMTs possessed similar genomic atlas, including (i) predominantly located in intergenic regions and preferential integration to gene regulation regions and (ii) generating sequence variations in the nuclear genome. Unlike nuclear indigenous genes, the alien NUPGs/NUMGs were associated with repressive epigenetic signals, namely high levels of DNA methylation and low levels of active histone modifications. Phylogenomic analyses suggested that the species-specific and gradual accumulation of NUPTs/NUMTs accompanied the speciation processes. Moreover, based on further pan-genomic analyses, we found significant subgenomic asymmetry in the NUPT/NUMT occurrence, which accumulated during allopolyploid wheat evolution. Our findings provide insight into the dynamic evolutionary fates of organelle-derived nuclear DNA in plants.

H4K5 Butyrylation Coexist with Acetylation during Human Spermiogenesis and Are Retained in the Mature Sperm Chromatin.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Distinct power of bone marrow microRNA signatures and tumor suppressor genes for early detection of acute leukemia.

Acute leukemia involving lymphocytic and myeloid cells is cancer with a high mortality rate. Swift and timely diagnosis might be a potential approach to improving patient prognosis and survival. The microRNA (miRNA) signatures are emerging nowadays for their promising diagnostic potential. MiRNA levels from bone marrow can be used as prognostic biomarkers. The current study was designed to evaluate if the microRNAs and tumor suppressor genes (TSGs) profiling of hematopoietic bone marrow could help in acute leukemia early detection. Also, we assessed the DNA methyltransferase 3A (DNMT3A) expression and its possible epigenetic effects on miRNAs plus TSGs expression levels. The expression levels of ten miRNAs and four TSGs involved in acute lymphocytic leukemia (ALL) as well as acute myeloid leukemia (AML) were quantified in 43 and 40 bone marrow samples of ALL and AML patients in comparison with cancer-free subjects via real-time quantitative PCR (RT-qPCR). The receiver-operating-characteristic (ROC) analysis of miRNAs was performed in the study groups. Further, the correlation between the DNMT3A and TSGs was calculated. Significant differences were detected in the bone marrow expression of miRNAs and TSGs (P < 0.05) between acute leukemia patients and healthy group. ROC analysis confirmed the ability of miR-30a, miR-101, miR-132, miR-129,miR-124, and miR-143 to discriminate both ALL and AML patients with an area under the ROC curve of ≥ 0.80 (P < 0.001) and high accuracy. The correlation between DNMT3A and P15/P16 TSGs revealed that DNMT3A plays a vital role in epigenetic control of TSGs expression. Our findings indicated that the downregulation of bone marrow miRNAs and TSGs was accompanied by acute leukemia development. The authors conclude that this study could contribute to introducing useful biomarkers for acute leukemia diagnosis.

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.

In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.

DNMT1 maintains the methylation of miR-152-3p to regulate TMSB10 expression, thereby affecting the biological characteristics of colorectal cancer cells.

ObjectiveDNA methyltransferases (DNMTs) take on a relevant role in epigenetic control of cancer proliferation and cell survival. However, the molecular mechanisms underlying the establishment and maintenance of DNA methylation in human cancer remain to be fully elucidated. This study was to investigate that how DNMT1 affected the biological characteristics of colorectal cancer (CRC) cells via modulating methylation of microRNA (miR)‐152‐3p and thymosin β 10 (TMSB10) expression.MethodsDNMT1, miR‐152‐3p, and TMSB10 expression, and the methylation of miR‐152‐3p in CRC tissues and cells were detected. SW‐480 and HCT‐116 CRC cells were transfected with DNMT1 or miR‐152‐3p‐related sequences or plasmids to explore their characters in biological functions of CRC cells. The binding relationship between DNMT1 and miR‐152‐3p and the targeting relationship between miR‐152‐3p and TMSB10 were analyzed. The tumor growth was also detected in vivo.ResultsUpregulated DNMT1, TMSB10, reduced miR‐152‐3p, and methylated miR‐152‐3p were detected in CRC tissues and cells. Silenced DNMT1 or upregulated miR‐152‐3p reduced TMSB10 expression and suppressed CRC progression and tumor growth. Moreover, elevated DNMT1 could reverse the effect of miR‐152‐3p upregulation on CRC development and tumor growth. DNMT1 maintained methylation of miR‐152‐3p. TMSB10 was the direct target gene of miR‐152‐3p.ConclusionThe study highlights that silenced DNMT1 results in non‐methylated miR‐152‐3p to depress TMSB10 expression, thereby inhibiting CRC development, which provides a new approach for CRC therapy.

A monocyte-keratinocyte-derived co-culture assay accurately identifies efficacies of BET inhibitors as therapeutic candidates for psoriasiform dermatitis

BackgroundBromodomain and extra-terminal (BET) proteins perform key roles in epigenetic control of gene expression that is involved in inflammatory conditions, including psoriasiform dermatitis (PsD). Predicting which (of many potential available BET inhibitors) will be effective in vivo is challenging. ObjectiveWe determine if a novel in vitro assay that includes two critical cell types involved in human psoriasis can predict the therapeutic potential of specific BET inhibitors in vivo. MethodsAn in vitro model consisting of U-937 and HaCaT cell co-culture was created to screen small molecule BET antagonists for inhibition of cutaneous inflammatory genes. Efficacious BET inhibitors were tested in a mouse imiquimod (IMQ)-induced PsD model. ResultsIn the co-culture system, HaCaT cells exhibited a marked increase in the secretion of a characteristic set of proinflammatory and Th17-associated cytokines. Of the ten commercially-available small molecules targeting BET proteins assayed, most compounds exhibited inhibitory functions at 1 μM against inflammatory activation, but responded variably at lower concentrations. OTX015, a typical representative for most of the compounds, barely inhibited the inflammatory reactions at 0.1 μM. By contrast, ABBV075 was effective in concentrations as low as 0.01 μM. While oral administration OTX015 in IMQ-treated mice reduced disease severity, ABBV075 equally decreased the symptoms and molecular and cellular severity markers at one-tenth of the minimal dosing required for OTX015. ConclusionIn vitro screening system combined with an in vivo animal model, can serve as a convenient pre-clinical screening tool for the selection of BET inhibitors (and possibly other drugs) that may have clinical potential in psoriasis therapy.

Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation.

DNA methyltransferases (DNMTs) play a relevant role in epigenetic control of cancer cell survival and proliferation. Since only two DNMT inhibitors (azacitidine and decitabine) have been approved to date for the treatment of hematological malignancies, the development of novel potent and specific inhibitors is urgent. Here we describe the design, synthesis, and biological evaluation of a new series of compounds acting at the same time as DNMTs (mainly DNMT3A) inhibitors and degraders. Tested against leukemic and solid cancer cell lines, 2a–c and 4a–c (the last only for leukemias) displayed up to submicromolar antiproliferative activities. In HCT116 cells, such compounds induced EGFP gene expression in a promoter demethylation assay, confirming their demethylating activity in cells. In the same cell line, 2b and 4c chosen as representative samples induced DNMT1 and -3A protein degradation, suggesting for these compounds a double mechanism of DNMT3A inhibition and DNMT protein degradation.

DNMT3A with Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novoacute myeloid leukemia (AML) and are associated with poor prognosis. Although studies demonstrated survival benefit of induction chemotherapy dose intensification, outcomes remain unsatisfactory in most patients due to advanced age, comorbidities, and hence inability to tolerate treatment. Clinical trials of low-intensity regimens combining cytarabine and cladribine, nucleoside analog chain terminators that stall DNA replication, appear to be safe and effective, and tend to particularly benefit patients with DNMT3Amutations. Consistently, we observe increased sensitivity to cytarabine, fludarabine, and cladribine in multiple cellular systems harboring mutant DNMT3Ain vitro (Figure 1A, B). Differential sensitivity to cytarabine was confirmed in normal and leukemic primary bone marrow cells derived from mice with and without Dnmt3a mutations ex vivo (Figure 1C). Dynamic chromatin organization plays a pivotal role in DNA-associated cellular processes including DNA replication and damage repair. We previously found altered chromatin remodeling in cells expressing mutant DNMT3A after genotoxic stress. Gene expression studies by us and others demonstrated negative enrichment of cell cycle related signatures including G2/M checkpoint adaptation, in cells with DNMT3A mutations. These signatures are implicated in DNA damage response and replication fork integrity and suggest sensitivity to replication stress. To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed wildtype (WT) or R882 mutant (MUT) forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. Analysis of the DNA damage signaling proficiency in response to cytarabine revealed persistent intra-S phase checkpoint activation (phospho-CHK1), accompanied by accumulation of DNA damage visualized by γH2A.X foci and by Comet assay in the DNMT3A(mut) overexpression cells (Figure 1D). This damage was only partially resolved after drug had been removed and cells were allowed to repair the DNA (Figure 1E), and was carried through mitosis, resulting in increased rate of micronucleation.At the same time, DNMT3A mutant cells remained proficient in initiating homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways, evidenced by RAD51 and 53BP-1 foci formation, respectively. These data demonstrate enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, rather than defects in double-strand DNA break repair. In support of this, cells with mutant DNMT3Awere characterized by accentuated replication stress as evidenced by high levels of phospho-RPA, which persisted after drug wash-out (Figure 1F). Consistently, DNMT3A-mutant cells treated with cytarabine were characterized by a higher number and a larger area of PCNA foci. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that while the overall kinetics of replication restart remained unchanged, cells with DNMT3A(mut) showed higher rate of fork collapse and increased reliance on latent replication origins (Figure 1G). Gene expression profiling by RNA-seq identified dysregulation of pathways associated with cell cycle progression, specifically G1/S phase transition, DNA replication, DNA integrity checkpoint, and chromatin. Our studies show cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication and suggest that cytarabine-induced replication fork stalling may further synergize with other agents aimed at DNA damage and replication. Figure 1 Disclosures No relevant conflicts of interest to declare.

SA97 - CHANGES OF OPEN CHROMATIN REGIONS REVEAL STAGE-SPECIFIC TRANSCRIPTIONAL NETWORK DYNAMICS IN HUMAN IPSC-DERIVED NEURONS

Background The application of human induced Pluripotent Stem Cells (iPSCs)-differentiated neurons has served as a promising model for gaining insights into the molecular and cellular mechanisms of genetic risk for mental disorders. Multiple factors determine the fate and trajectory of neuronal cell differentiation, of which transcriptional regulation plays a major role. Chromatin accessibility (openness) to Transcription Factors (TF) strongly influences gene transcription and cell differentiation. However, the dynamic changes of open chromatin and the TF networks during neuronal differentiation from iPSCs are poorly understood. Methods Here, we performed a global mapping of Open Chromatin Regions (OCRs) using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) at different cell stages of glutamatergic neuronal differentiation, examined the correlation of changes of open chromatin and mRNA abundances (assayed by RNA-Seq), and constructed the neuronal stage-specific TF networks through a genome-wide inference of TF-binding footprints in OCRs. Results We found that OCRs were highly dynamic during neuronal differentiation, and more importantly that OCR accessibility at core promoter regions was positively correlated with mRNA abundances at their respective cell stages. Furthermore, we found that the dynamic changes of OCRs during neuronal differentiation were accompanied by the binding events of cell stage-specific TFs. As expected, binding footprints of OCT4 and NANOG, genes that encode pluripotent stem cell-specific markers, are most enriched in iPSCs. Intriguingly, binding footprints of NEUROD1 and NEUROG2, the two TFs that have been reported to rapidly induce high efficient conversion of iPSCs to excitatory neurons, are among those most enriched in the relatively mature neurons. Discussion Further TF network analysis based on the inferred TF binding footprints in OCRs successfully identified core TF networks and their master regulators for different neuronal stages. Interestingly, both NEUROD1 and NEUROG2 are in the same core TF network specific to more mature neurons, suggesting their pivotal role in epigenetic control of neuronal differentiation and maturation.

Cells with DNMT3A Mutations Are More Sensitive to Cytarabine-Induced DNA Damage

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) (20-35%) and are associated with poor prognosis. Although studies demonstrated survival benefit after daunorubicin dose intensification for DNMT3A mutant AML, these patients tend to be older (median age at diagnosis 67 y.o. in DNMT3A-mutated cases compared to 60 y.o. in DNMT3A-WT) and more likely to be unfit for high-dose chemotherapy. In older patients, cytarabine monotherapy, a nucleoside analog chain terminator that stalls DNA replication, may be preferred due to lower toxicity. However, its efficacy in DNMT3A mutant AML has not been evaluated.Previous gene expression studies in large AML cohorts and in a mouse model of Dnmt3a-mutant hematopoiesis demonstrated negative enrichment of the cell cycle related signatures, including G2/M checkpoint and E2F targets. Hence, we hypothesized cell cycle-specific drugs cytarabine and fludarabine may be effective in DNMT3A-mutant context. DNMT3A mutant AML cell lines (MUT) SET-2 and KO-52 were more sensitive to cytarabine and fludarabine than DNMT3A wild-type (WT) cells KU-812 and K-562 in vitro (cytarabine IC50 46.1 and 165.8 μM in MUT SET-2 and KO-52 compared to 465.2 μM and not reached in WT KU-812 and K-562, respectively; fludarabine IC50 1.1 and 1.3 μg/ml in MUT vs 16 μg/ml and not reached in WT, Figure 1A). Higher proportion of cells with mutant DNMT3A were undergoing apoptosis 24 hours after cytarabine exposure (26.5±2.3% and 13.1±2.3% Annexin V+ cells in SET-2 and KO-52 vs 6.9±2.6% and 4.6±2.5% in KU-812 and K-562, respectively, p<0.05). Analysis of the DNA damage signaling revealed increased levels of phospho-CHK1, γH2A.X, and cleaved PARP (stalled replication forks, DNA damage, and apoptosis markers).To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed WT or R882 mutant forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. MUT cells were more sensitive to cytarabine compared to WT (IC50 38 μM vs 213 μM after 48 hours of exposure, Figure 1B), which was accompanied by increased apoptosis (22.3±7.6% in MUT vs 7.2±0.3% in WT Annexin V+ cells, p<0.03). DNMT3A-MUT cells showed persistence of the phospho-CHK1 levels over 12 hours of continuous cytarabine exposure, whereas WT showed resolution of the CHEK1 signaling after initial activation. Consistently, MUT cells accumulated significantly more DNA damage over 36 hours of continuous exposure compared to WT, as evidenced by γH2A.X immunofluorescent staining, and by Comet assay (Figure 1C). Fewer MUT cells were able to complete replication and progress to the G2 phase of the cell cycle (5.3±0.2% in MUT vs 11.2±0.6% in WT, flow-cytometric analysis by DNA content, p<0.01). Notably, both WT and MUT cells were proficient in DNA repair by homologous recombination (HR, tested by RAD51 foci formation visualized by immunofluorescence) and by non-homologous end joining (NHEJ, 53BP1 foci). These data demonstrate that enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, and not to defects in DNA repair. Consistently, even though MUT cells showed more DNA damage by Comet assay after 12 hours of cytarabine treatment, it was largely repaired 60 minutes after drug wash-out, similar to WT, Figure 1D). These observations support continuous infusion of cytarabine as a delivery method of choice in the clinic, rather than bolus administration. Finally, bone marrow cells derived from Dnmt3a-mut conditional knock-in mice showed impaired clonogenic survival in MethoCult when exposed to 25 nM cytarabine ex vivo, compared to Dnmt3a-WT (p<0.003). We are currently investigating gene expression, DNA methylation, and chromatin accessibility profiles in WT and MUT cells; and cytarabine efficacy in a pre-clinical mouse model of Dnmt3a(mut):Flt3(ITD):Npm1(c) leukemia.In conclusion, our studies show cells with DNMT3A mutations may be sensitive to DNA damage induced by clinically relevant nucleoside analogs such as cytarabine. Our data support continuous infusion of cytarabine rather than bolus administration, and establish its mechanistic basis. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication. [Display omitted] DisclosuresLicht:Celgene: Research Funding.

Abstract 4321: Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer

Abstract Epigenetic alteration is a common trait of human cancers. Chemical modifications on DNA and core histone proteins are the major mechanisms for epigenetic regulation. Recently, emerging evidence suggested that reversible chemical modifications on RNA also play a critical role in epigenetic control of gene expression. N6-methyladenosine (m6A) is the most abundant modification found in mammalian mRNA. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. In this study, we investigated the expression of m6A methyltransferases and demethylases in human hepatocellular carcinoma (HCC). We found the major m6A methyltransferase METTL3 was remarkably up-regulated in HCC. High METTL3 expression was associated with poor overall and disease-free survival in HCC patients. Consistent with the METTL3 overexpression, we found that mRNA m6A level was significantly elevated in human HCC. To investigate the roles of METTL3 in human HCC, we employed lentiviral-based shRNA and CRISPR/Cas9 systems to inactivation METTL3 in HCC cell lines. We showed that knockdown and knockout of METTL3 drastically suppressed HCC proliferation and migration in vitro and abolished HCC tumorigenicity and lung metastasis in vivo. In contrast, overexpression of METTL3 by CRISPR/dCas9 SAM system promoted HCC growth. Using RNA-seq and m6A-Seq, we identified tumor suppressor gene SOCS2 as a novel target of METTL3. We detected m6A modification in SOCS2 mRNA and the m6A modification was diminished upon METTL3 knockdown. m6A modification promoted SOCS2 mRNA degradation. We demonstrated that knockdown of METTL3, mutation of m6A modification sites, and treatment of demethylating agent augmented SOCS2 mRNA expression in HCC cells. In addition, knockdown of YTHDF2, an m6A reader protein, also rescued SOCS2 mRNA expression in HCC cells. The above findings together demonstrated that up-regulation of METTL3 lead to m6A modification of SOCS2, which in turn promotes SOCS2 mRNA degradation through YTHDF2 dependent mechanism. Our findings provided a proof-of-concept model to demonstrate the importance of aberrant m6A modification in epigenetic silencing of tumor suppressor genes. Citation Format: Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice H. Tsang, Jialing Shen, Carol L. Cheng, Long-Hin Tsang, Daniel W. Ho, David K. Chiu, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4321.

Epigenetic regulation of the Hedgehog and Wnt pathways in cancer.

The Hedgehog (Hh) and wingless-Int1 (Wnt) pathways are important for tissue patterning in the developing embryo. In adult tissue, both pathways are typically dormant but are activated under certain conditions such as tissue damage. Aberrant activation of these pathways by mutations in key pathway regulators contributes to the genesis and progression of several cancer types. In addition, the impact of epigenetic regulation of the Hh and Wnt pathways on cancer is becoming increasingly clear. In this review, current knowledge on the epigenetic control of Hh and Wnt and the impact on tumor formation will be discussed. First, the role of epigenetic control on ligand production will be discussed, followed by the epigenetic regulation of the extra- and intracellular pathway members. Furthermore, the epigenetic control of pathway target genes will be highlighted. Lastly, an overview of current therapeutic strategies to target aberrant epigenetic control of the Hh and Wnt pathways is provided.

Novel Drugs Targeting the Epigenome

Epigenetic drug discovery has its beginning in the cancer research arena, focusing first on DNA methylation and histone deacetylation. There are currently two DNA methyltransferase (DNMT) inhibitors and four histone deacetylase (HDAC) inhibitors approved by the US Food and Drug Administration (FDA) during the past 13 years. Over the past few years, breakthrough discoveries of chromatin-modifying enzymes and associated mechanisms have exploded, providing new insights into the role of epigenetic control in gene regulation and leading to the discovery of a variety of new and specific drug targets. Among them, epigenetic “reader”—bromodomain and extra-terminal protein (BET), “writers”—disruptor of telomeric silencing 1-like (DOT1L), enhancer of zeste homolog 2 (EZH2), and protein arginine methyltransferase 5 (PRMT5), and “erasers”—lysine-specific histone demethylase 1 (LSD1) as well as isocitrate dehydrogenase (IDH) attract greater attention due to the ongoing clinical trials. This article provides a brief overview of new drugs modulating the above epigenetic targets, including their indication, mechanism of action, and disclosed chemical structures. The trend of epigenetic drug approval in the following few years is expectable, at least partially, from current clinical trials summarized in this review.

A postprandial FGF19-SHP-LSD1 regulatory axismediates epigenetic repression of hepaticautophagy.

Lysosome-mediated autophagy is essential for cellular survival and homeostasis upon nutrient deprivation, but is repressed after feeding. Despite the emerging importance of transcriptional regulation of autophagy by nutrient-sensing factors, the role for epigenetic control is largely unexplored. Here, we show that Small Heterodimer Partner (SHP) mediates postprandial epigenetic repression of hepatic autophagy by recruiting histone demethylase LSD1 in response to a late fed-state hormone, FGF19 (hFGF19, mFGF15). FGF19 treatment or feeding inhibits macroautophagy, including lipophagy, but these effects are blunted in SHP-null mice or LSD1-depleted mice. In addition, feeding-mediated autophagy inhibition is attenuated in FGF15-null mice. Upon FGF19 treatment or feeding, SHP recruits LSD1 to CREB-bound autophagy genes, including Tfeb, resulting in dissociation of CRTC2, LSD1-mediated demethylation of gene-activation histone marks H3K4-me2/3, and subsequent accumulation of repressive histone modifications. Both FXR and SHP inhibit hepatic autophagy interdependently, but while FXR acts early, SHP acts relatively late after feeding, which effectively sustains postprandial inhibition of autophagy. This study demonstrates that the FGF19-SHP-LSD1 axis maintains homeostasis by suppressing unnecessary autophagic breakdown of cellular components, including lipids, under nutrient-rich postprandial conditions.

Detection of 5-carboxylcytosine in Mammalian Cells DNA

5-carboxylcytosine (5caC) is an important derivative of cytosine modification in DNA demethylation pathway. However, the accurate quantification of 5caC is a challenge, especially in mammalian tissue. This plight probably results from the trace amount of 5caC in mammalian and the inadequate sensitivity of the existing technologies. Herein, we report a novel LC-MS/MS method to precisely quantify 5caC in mammalian cells. The 5caC standard reagent was synthesized, and the genomic DNA was hydrolyzed with formic acid and the target compound 5caC was detected by HILIC LC-MS/MS. The results showed 5caC generally exist in mice organs and cancer tissues. The content of 5caC in mice brain was higher than in lungs and liver, and was obviously decreased in colorectal cancer tissues compared with the adjacent tissue. This result suggests that 5caC probably associates with tumorigenesis and plays a dominated role in epigenetic control of neuronal function.

Non-coding RNAs as regulators in epigenetics (Review).

Epigenetics is a discipline that studies heritable changes in gene expression that do not involve altering the DNA sequence. Over the past decade, researchers have shown that epigenetic regulation plays a momentous role in cell growth, differentiation, autoimmune diseases, and cancer. The main epigenetic mechanisms include the well-understood phenomenon of DNA methylation, histone modifications, and regulation by non-coding RNAs, a mode of regulation that has only been identified relatively recently and is an area of intensive ongoing investigation. It is generally known that the majority of human transcripts are not translated but a large number of them nonetheless serve vital functions. Non-coding RNAs are a cluster of RNAs that do not encode functional proteins and were originally considered to merely regulate gene expression at the post-transcriptional level. However, taken together, a wide variety of recent studies have suggested that miRNAs, piRNAs, endogenous siRNAs, and long non-coding RNAs are the most common regulatory RNAs, and, significantly, there is a growing body of evidence that regulatory non-coding RNAs play an important role in epigenetic control. Therefore, these non-coding RNAs (ncRNAs) highlight the prominent role of RNA in the regulation of gene expression. Herein, we summarize recent research developments with the purpose of coming to a better understanding of non-coding RNAs and their mechanisms of action in cells, thus gaining a preliminary understanding that non-coding RNAs feed back into an epigenetic regulatory network.

MicroRNA in sperm from Duroc, Landrace and Yorkshire boars

Sperm contain microRNAs (miRNAs), which may have roles in epigenetic control. Regarding phylogenetic relationships among various swine breeds, Yorkshire and Landrace, are considered phenotypically and genetically very similar, but distinctly different from Duroc. The objective of the present study was to compare abundance of boar sperm miRNAs in these three breeds. Overall, 252 prioritized miRNAs were investigated using real-time PCR; relative expression of miRNAs in sperm was similar in Yorkshire and Landrace boars, but significantly different compared to Duroc. Seventeen miRNAs (hsa-miR-196a-5p, hsa-miR-514a-3p, hsa-miR-938, hsa-miR-372-3p, hsa-miR-558, hsa-miR-579-3p, hsa-miR-595, hsa-miR-648, hsa-miR-524-3p, hsa-miR-512-3p, hsa-miR-429, hsa-miR-639, hsa-miR-551a, hsa-miR-624-5p, hsa-miR-585-3p, hsa-miR-508-3p and hsa-miR-626) were down-regulated (P < 0.05; fold regulation ≤−2) in Yorkshire and Landrace sperm, compared to Duroc sperm. Furthermore, three miRNAs (hsa-miR-9-5p, hsa-miR-150-5p, and hsa-miR-99a-5p) were significantly up-regulated in Yorkshire and Landrace sperm compared to Duroc sperm, However, 240 miRNAs were not significantly different (within + 2 fold) between Yorkshire and Landrace sperm. We concluded that miRNAs in sperm were not significantly different between Yorkshire and Landrace boars, but there were significant differences between those two breeds and Duroc boars. Furthermore, integrated target genes for selected down-regulated miRNAs (identified via an in-silico method) appeared to participate in spermatogenesis and sperm functions.

Dissecting Epigenetic Dysregulation of Primary Antibody Deficiencies.

Primary antibody deficiencies (PADs), the most prevalent inherited primary immunodeficiencies (PIDs), are associated with a wide range of genetic alterations (both monogenic or polygenic) in B cell-specific genes. However, correlations between the genotype and clinical manifestations are not evident in all cases indicating that genetic interactions, environmental and epigenetic factors may have a role in PAD pathogenesis. The recent identification of key defects in DNA methylation in common variable immunodeficiency as well as the multiple evidences on the role of epigenetic control during B cell differentiation, activation and during antibody formation highlight the importance of investing research efforts in dissecting the participation of epigenetic defects in this group of diseases. This review focuses on the role of epigenetic control in B cell biology which can provide clues for the study of potential novel pathogenic defects involved in PADs.