A distal enhancer of GATA3 regulates Th2 differentiation and allergic inflammation.

Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation.

An integrative transcriptional logic model of hepatic insulin resistance.

Abnormalities of lipid/lipoprotein and glucose metabolism are hallmarks of hepatic insulin resistance in type 2 diabetes. The former antedate the latter, but the latter become progressively refractory to treatment and contribute to therapeutic failures. It's unclear whether the two processes share a common pathogenesis and what underlies their progressive nature. In this study, we investigated the hypothesis that genes in the lipid/lipoprotein pathway and those in the glucose metabolic pathway are governed by different transcriptional regulatory logics that affect their response to physiologic (fasting/refeeding) as well as pathophysiologic cues (insulin resistance and hyperglycemia). To this end, we obtained genomic and transcriptomic maps of the key insulin-regulated transcription factor, FoxO1, and integrated them with those of CREB, PPAR-α, and glucocorticoid receptor. We found that glucose metabolic genes are primarily regulated by promoter and intergenic enhancers in a fasting-dependent manner, while lipid genes are regulated through fasting-dependent intron enhancers and fasting-independent enhancerless introns. Glucose genes also showed a remarkable transcriptional resiliency (i.e., the ability to compensate following constitutive FoxO1 ablation through an enrichment of active marks at shared PPAR-α/FoxO1 regulatory elements). Unexpectedly, insulin resistance and hyperglycemia were associated with a "spreading" of FoxO1 binding to enhancers and the emergence of unique target sites. We surmise that this unusual pattern correlates with the progressively intractable nature of hepatic insulin resistance. This transcriptional logic provides an integrated model to interpret the combined lipid and glucose abnormalities of type 2 diabetes.

Comprehensive molecular phenotyping of ARID1A-deficient gastric cancer reveals pervasive epigenomic reprogramming and therapeutic opportunities.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. DESIGN: Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analysed to examine tumour microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell and epigenomic level, and targeted by pharmacological inhibition. RESULTS: We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven proinflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. CONCLUSION: Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes.

Tumorigenic activation around HPV integrated sites in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP-seq, RNA-seq and Hi-C and 4C-seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI-SCC-090 through target enrichment sequencing, 4C-seq revealed 0.5 to 40 Mb of HPV-interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super-enhancers, the remaining non-amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI-SCC-090-specific super-enhancer formation around the ITPR3 promoter and in the 80-kb-downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM-SCC-47 and UM-SCC-104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC.

The Link of mRNA and rRNA Transcription by PUF60/FIR through TFIIH/P62 as a Novel Therapeutic Target for Cancer.

The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.

Transcriptional dysregulation by aberrant enhancer activation and rewiring in cancer.

Cell identity is controlled by regulatory elements, such as promoters, enhancers, and insulators, within the genome. These regulatory elements interact in the nucleus and form tissue-specific chromatin structures. Dysregulation of these elements and their interactions can lead to loss of cell identity and promote the development of diseases such as cancer. Tumor cells acquire aberrantly activated enhancers at oncogenic driver genes through various mechanisms. Small genomic changes such as mutations, insertions, and amplifications can form aberrant enhancers. Genomic rearrangements at the chromosomal level, including translocations and inversions, are also often observed in cancers. These rearrangements can result in repositioning of enhancers to locations near tumor-type-specific oncogenes. Chromatin structural changes caused by genomic or epigenomic changes lead to mis-interaction between enhancers and proto-oncogenes, ultimately contributing to tumorigenesis through activation of oncogenic signals. Additional epigenomic mechanisms can also cause aberrant enhancer activation, including those associated with overexpression of oncogenic transcription factors and the mutation of transcriptional cofactors. Exogenous viral DNA can also lead to enhancer aberrations. Here, we review the mechanisms underlying aberrant oncogene activation through enhancer activation and rewiring, both of which are caused by genomic or epigenomic alterations in non-coding regions.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.

Identification and characterization of the binding sequences and target genes of p53 lacking the 1st transactivation domain.

The tumor suppressor gene p53 encodes a transcriptional activator that has two transactivation domains (TAD) located in its amino terminus. These two TAD can transactivate genes independently, and at least one TAD is required for p53 transactivation function. The 1st TAD (a.a. 1-40) is essential for the induction of numerous classical p53 target genes, while the second TAD (a.a. 41-61) suffices for tumor suppression, although its precise molecular function remains unclear. In this study, we comprehensively identified the sites to which p53 lacking the 1st TAD (Δ1stTAD-p53) binds, as well as its potential target genes. We found that the binding sequences for Δ1stTAD-p53 are divergent and include not only the canonical p53 consensus binding sequences but also sequences similar to those recognized by a number of other known transcription factors. We identified and analyzed the functions of three Δ1stTAD-p53 target genes, PTP4A1, PLK2 and RPS27L. All three genes were induced by both full-length p53 and Δ1stTAD-p53, and were dependent on the transactivation activity of the 2nd TAD. We also found that two of these, PTP4A1 and PLK2, are endoplasmic reticulum (ER) stress-inducible genes. We found that upon ER stress, PTP4A1 suppresses apoptosis while PLK2 induces apoptosis. These results reveal a novel Δ1stTAD-p53 downstream pathway that is dependent on the transcription activation activity of the 2nd TAD.

Epstein-Barr virus-positive gastric cancer involves enhancer activation through activating transcription factor 3.

Epstein-Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing (-seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3- enhancers in EBV-infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein-Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation-seq analysis on ATF3 binding sites and RNA-seq analysis on ATF3 knocked-down MKN7_EB revealed 96 genes targeted by ATF3-activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV-positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV-infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV-positive GC.

PHLDA1, another PHLDA family protein that inhibits Akt.

The PHLDA family (pleckstrin homology-like domain family) of genes consists of 3 members: PHLDA1, 2, and 3. Both PHLDA3 and PHLDA2 are phosphatidylinositol (PIP) binding proteins and function as repressors of Akt. They have tumor suppressive functions, mainly through Akt inhibition. Several reports suggest that PHLDA1 also has a tumor suppressive function; however, the precise molecular functions of PHLDA1 remain to be elucidated. Through a comprehensive screen for p53 target genes, we identified PHLDA1 as a novel p53 target, and we show that PHLDA1 has the ability to repress Akt in a manner similar to that of PHLDA3 and PHLDA2. PHLDA1 has a so-called split PH domain in which the PH domain is divided into an N-terminal (ß sheets 1-3) and a C-terminal (ß sheets 4-7 and an α-helix) portions. We show that the PH domain of PHLDA1 is responsible for its localization to the plasma membrane and binding to phosphatidylinositol. We also show that the function of the PH domain is essential for Akt repression. In addition, PHLDA1 expression analysis suggests that PHLDA1 has a tumor suppressive function in breast and ovarian cancers.

Ethyl ß-d-glucoside: a novel chemoattractant of Ralstonia solanacearum isolated from tomato root exudates by a bioassay-guided fractionation.

A chemoattractant of Ralstonia solanacearum isolated from the activated charcoal-adsorbed fraction of tomato root exudates was identified as ethyl ß-d-glucopyranoside by instrumental analyses and comparison with synthetic preparations. Ethyl ß-D-glucopyranoside showed unambiguous activity at above 1 µmol/disc. Its stereoisomers and D-glucose were inactive.

Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells.

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

Chromatin activation with H3K36me2 and compartment shift in metastatic castration-resistant prostate cancer.

Epigenetic modifiers are upregulated during the process of prostate cancer, acquiring resistance to castration therapy and becoming lethal metastatic castration-resistant prostate cancer (CRPC). However, the relationship between regulation of histone modifications and chromatin structure in CRPC has yet not fully been validated. Here, we reanalyzed publicly available clinical transcriptome and clinical outcome data and identified NSD2, a histone methyltransferase that catalyzes H3K36me2, as an epigenetic modifier that was upregulated in CRPC and whose increased expression in prostate cancer correlated with higher recurrence rate. We performed ChIP-seq, RNA-seq, and Hi-C to conduct comprehensive epigenomic and transcriptomic analyses to identify epigenetic reprogramming in CRPC. In regions where H3K36me2 was increased, H3K27me3 was decreased, and the compartment was shifted from inactive to active. In these regions, 68 aberrantly activated genes were identified as candidate downstream genes of NSD2 in CRPC. Among these genes, we identified KIF18A as critical for CRPC growth. Under NSD2 upregulation in CRPC, epigenetic alteration with H3K36me2-gain and H3K27me3-loss occurs accompanying with an inactive-to-active compartment shift, suggesting that histone modification and chromatin structure cooperatively change prostate carcinogenesis.

Enhancer infestation drives tumorigenic activation of inactive B compartment in Epstein-Barr virus-positive nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignant epithelial tumor endemic to Southern China and Southeast Asia. While previous studies have revealed a low frequency of gene mutations in NPC, its epigenomic aberrations are not fully elucidated apart from DNA hypermethylation. Epigenomic rewiring and enhancer dysregulation, such as enhancer hijacking due to genomic structural changes or extrachromosomal DNA, drive cancer progression. METHODS: We conducted Hi-C, 4C-seq, ChIP-seq, and RNA-seq analyses to comprehensively elucidate the epigenome and interactome of NPC using C666-1 EBV(+)-NPC cell lines, NP69T immortalized nasopharyngeal epithelial cells, clinical NPC biopsy samples, and in vitro EBV infection in HK1 and NPC-TW01 EBV(-) cell lines. FINDINGS: In C666-1, the EBV genome significantly interacted with inactive B compartments of host cells; the significant association of EBV-interacting regions (EBVIRs) with B compartment was confirmed using clinical NPC and in vitro EBV infection model. EBVIRs in C666-1 showed significantly higher levels of active histone modifications compared with NP69T. Aberrant activation of EBVIRs after EBV infection was validated using in vitro EBV infection models. Within the EBVIR-overlapping topologically associating domains, 14 H3K4me3(+) genes were significantly upregulated in C666-1. Target genes of EBVIRs including PLA2G4A, PTGS2 and CITED2, interacted with the enhancers activated in EBVIRs and were highly expressed in NPC, and their knockdown significantly reduced cell proliferation. INTERPRETATION: The EBV genome contributes to NPC tumorigenesis through "enhancer infestation" by interacting with the inactive B compartments of the host genome and aberrantly activating enhancers. FUNDING: The funds are listed in the Acknowledgements section.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted "cell migration" and "extracellular matrix" programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis. SIGNIFICANCE: Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7 upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencing of gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPR interference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

Hi-C Analysis to Identify Genome-Wide Chromatin Structural Aberration in Cancer.

Hi-C is a method that analyzes genome-wide chromatin structure using next-generation sequencer. Chromatin structure is crucial for regulating transcription or replication, and Hi-C has revealed the hierarchical chromatin structures, such as loop, domain , and compartment structures. Aberrant alteration of these structures causes disease, and a number of structural aberrations in cancer cells have been reported recently. Besides, Hi-C can identify chromosome rearrangements that frequently occurred in cancer. Therefore, Hi-C is a powerful technique to analyze epigenomic and genomic aberrations in tumorigenesis. Here we will introduce the basic protocol of Hi-C in experimental and analytical aspects.

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term "Myc active pathway" according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed "MYC_TARGETS_V2" with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as "TNFA_SIGNALING_VIA_NFKB", "APOPTOSIS", and "TGF_BETA_SIGNALING" were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.