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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.

The number and variety of identified histone post-translational modifications (PTMs) are continually increasing. However, the specific consequences of each histone PTM remain largely unclear, primarily due to the lack of methods for selectively and rapidly introducing a desired histone PTM in living cells without genetic engineering. Here, we report the development of a cell-permeable histone acetylation catalyst, BAHA-LANA-PEG-CPP44, which selectively enters leukemia cells, binds to chromatin, and acetylates H2BK120 of endogenous histones in a short reaction time. Time-course analyses of this in-cell catalytic reaction revealed that H2BK120 acetylation attenuates the chromatin binding of negative elongation factor E (NELFE), an onco-transcription factor. This H2BK120 acetylation-mediated removal of NELFE from chromatin reshapes transcription, slows leukemia cell viability, and reduces their tumorigenic potential in mice. Therefore, this histone acetylation catalyst provides a unique tool for elucidating the time-resolved consequences of histone PTMs and may offer a modality for cancer chemotherapy. Here, the authors develop a cell-permeable chemical catalyst that acetylates histone H2BK120 in leukemia cells, attenuates NELFE chromatin binding, reprograms transcription, and reduces the tumorigenic potential of leukemia cells in mice. [ABSTRACT FROM AUTHOR]

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)‐specific methyltransferase that is associated with both various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double‐strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that it is not further mobilized by DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose‐dependent effects of NSD2 on homologous recombination (HR), canonical‐non‐homologous end joining (c‐NHEJ), and non‐canonical‐NHEJ (non‐c‐NHEJ). Endogenous NSD2 has a role in repressing non‐c‐NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c‐NHEJ repair and suppresses HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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 (β sheets 1‐3) and a C‐terminal (β 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. Through a comprehensive screening for p53 target genes, we identified pleckstrin homology‐like domain family, member 1 (PHLDA1) as a novel p53 target, and we show that PHLDA1 has the ability to repress Akt in a manner similar to that of other PHLDA family proteins. We show that the PH domain of PHLDA1 is responsible for its localization to the plasma membrane, and PHLDA1 has the ability to bind to phosphatidylinositols and repress Akt. In addition, PHLDA1 expression analysis suggests that PHLDA1 has a tumor suppressive function in breast and ovarian cancers. [ABSTRACT FROM AUTHOR]

Tomato root exudates were analyzed using a bioassay to detect the chemoattractant for Ralstonia solanacearum. An activated charcoal-adsorbed fraction of root exudates from tomato cultivar Oogata-fukuju had chemoattractant activity for R. solanacearum strain MAFF 730138. The active component, purified using a Sep-Pak C18 cartridge, an activated charcoal column, diol-modified silica gel, and NH-modified silica gel, is a new hydrophobic attractant. The final purified fraction produced a single peak in a diol-modified silica gel HPLC analysis. [ABSTRACT FROM AUTHOR]

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 Ch IP-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. [ABSTRACT FROM AUTHOR]

Selective activation of p53 target genes in response to various cellular stresses is a critical step in determining the ability to induce cell-cycle arrest or apoptosis. Here we report the identification of the microRNA miR-22 as a p53 target gene that selectively determines the induction of p53-dependent apoptosis by repressing p21. Combinatorial analyses of the AGO2 immunocomplex and gene expression profiles identified p21 as a direct target of miR-22. Induction of p21 was inhibited by miR-22 after exposure to the genotoxic agent Adriamycin (doxorubicin; Bedford Laboratories), sensitizing cells to p53-dependent apoptosis. Interestingly, the activation of miR-22 depended on the intensity of the stresses that induced cells to undergo apoptosis in the presence of p21 suppression. Our findings define an intrinsic molecular switch that determines p53-dependent cellular fate through post-transcriptional regulation of p21. Cancer Res; 71(13); 4628-39. ©2011 AACR. [ABSTRACT FROM AUTHOR]

Recent analyses revealed the essential function of chromatin structure in maintaining and regulating genomic information. Advancements in microscopy, nuclear structure observation techniques, and the development of methods utilizing next-generation sequencers (NGSs) have significantly progressed these discoveries. Methods utilizing NGS enable genome-wide analysis, which is challenging with microscopy, and have elucidated concepts of important chromatin structures such as a loop structure, a domain structure called topologically associating domains (TADs), and compartments. In this chapter, I introduce chromatin interaction techniques using NGS and outline the principles and features of each method., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2024 The Authors; Published by the American Association for Cancer Research.)

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., Competing Interests: Competing interests: PT has stock and other ownership interests in Tempus Healthcare, previous research funding from Kyowa Hakko Kirin and Thermo Fisher Scientific, and patents/other intellectual property through the Agency for Science and Technology Research, Singapore (all outside the submitted work). RS has received honoraria from Bristol-Myers Squibb, Lilly, Roche, Taiho, Astra Zeneca, DKSH and MSD; has advisory activity with Bristol-Myers Squibb, Merck, Eisai, Bayer, Taiho, Novartis, MSD and AstraZeneca; received research funding from MSD and Paxman Coolers; and has received travel grants from AstraZeneca, Eisai, Roche and Taiho Pharmaceutical., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mutations in the DNA mismatch repair gene MSH2 are causative of microsatellite instability (MSI) in multiple cancers. Here, we discovered that besides its well-established role in DNA repair, MSH2 exerts a novel epigenomic function in gastric cancer. Unbiased CRISPR-based mass spectrometry combined with genome-wide CRISPR functional screening revealed that in early-stage gastric cancer MSH2 genomic binding is not randomly distributed but rather is associated specifically with tumor-associated super-enhancers controlling the expression of cell adhesion genes. At these loci, MSH2 genomic binding was required for chromatin rewiring, de novo enhancer-promoter interactions, maintenance of histone acetylation levels, and regulation of cell adhesion pathway expression. The chromatin function of MSH2 was independent of its DNA repair catalytic activity but required MSH6, another DNA repair gene, and recruitment to gene loci by the SWI/SNF chromatin remodeler SMARCA4/BRG1. Loss of MSH2 in advanced gastric cancers was accompanied by deficient cell adhesion pathway expression, epithelial-mesenchymal transition, and enhanced tumorigenesis in vitro and in vivo. However, MSH2-deficient gastric cancers also displayed addiction to BAZ1B, a bromodomain-containing family member, and consequent synthetic lethality to bromodomain and extraterminal motif (BET) inhibition. Our results reveal a role for MSH2 in gastric cancer epigenomic regulation and identify BET inhibition as a potential therapy in MSH2-deficient gastric malignancies., Significance: DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer., (©2022 American Association for Cancer Research.)

The chemotactic activity of the pathogen of bacterial wilt disease, Ralstonia solanacearum, was tested against 30 aromatic acids and plant hormones infused on filter discs in bioassays on agar plates. 4-Hydroxycinnamic acid ( p-coumaric acid) and 4-hydroxybenzoic acid were strong chemoattractants, 3,4-dihydroxybenzoic acid (protocatechuic acid) and jasmonic acid were weak attractants, and 2-hydroxybenzoic acid (salicylic acid) showed both attracting and repelling activity depending on dose. Examination of the dose dependency revealed that the ED 50 for 4-hydroxycinnamic acid and 4-hydroxybenzoic acid was 0.08 and 0.39 μmol/disc, respectively. 2-Hydroxybenzoic acid showed chemoattractant activity at 0.33 μmol/disc but chemorepellent activity at 3.3 μmol/disc, and bacterial random motility was activated at 1.0 μmol/disc and bacterial activity was suppressed at 33 μmol/disc. Although water-soluble attractants including amino acids and organic acids have been previously investigated, this is the first report of hydroxylated aromatic acids (HAAs) as chemoattractants of R. solanacearum.

Epigenome regulates gene expression to determine cell fate, and accumulation of epigenomic aberrations leads to diseases, including cancer. NCD38 inhibits lysine-specific demethylase-1 (LSD1), a histone demethylase targeting H3K4me1 and H3K4me2, but not H3K4me3. In this study, we conjugated NCD38 with a potent small molecule called pyrrole (Py) imidazole (Im) polyamide, to analyze whether targets of the inhibitor could be regulated in a sequence-specific manner. We synthesized two conjugates using β-Ala (β) as a linker, i.e., NCD38-β-β-Py-Py-Py-Py (NCD38-β 2 P 4 ) recognizing WWWWWW sequence, and NCD38-β-β-Py-Im-Py-Py (NCD38-β 2 PIPP) recognizing WWCGWW sequence. When RKO cells were treated with NCD38, H3K4me2 levels increased in 103 regions with significant activation of nearby genes ( P = 0.03), whereas H3K4me3 levels were not obviously increased. H3K27ac levels were also increased in 458 regions with significant activation of nearby genes ( P = 3 × 10 -10 ), and these activated regions frequently included GC-rich sequences, but less frequently included AT-rich sequences ( P < 1 × 10 -15 ) or WWCGWW sequences ( P = 2 × 10 -13 ). When treated with NCD38-β 2 P 4 , 234 regions showed increased H3K27ac levels with significant activation of nearby genes ( P = 2 × 10 -11 ), including significantly fewer GC-rich sequences ( P < 1 × 10 -15 ) and significantly more AT-rich sequences ( P < 1 × 10 -15 ) compared with NCD38 treatment. When treated with NCD38-β 2 PIPP, 82 regions showed increased H3K27ac levels, including significantly fewer GC-rich sequences ( P = 1 × 10 -11 ) and fewer AT-rich sequences ( P = 0.005), but significantly more WWCGWW sequences ( P = 0.0001) compared with NCD38 treatment. These indicated that target regions of epigenomic inhibitors could be modified in a sequence-specific manner and that conjugation of Py-Im polyamides may be useful for this purpose., Competing Interests: CONFLICTS OF INTEREST All authors have no conflicts of interest to disclose.

Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infection-mediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site., Competing Interests: CONFLICTS OF INTEREST All authors have no potential conflicts of interest to disclose.

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCF(Fbxo22)-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCF(Fbxo22) ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCF(Fbxo22)-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22(-/-) mice are almost half the size of Fbxo22(+/-) mice owing to the accumulation of p53. These results indicate that SCF(Fbxo22)-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.

The genetically amenable organism Drosophila melanogaster has been estimated to have 14,076 protein coding genes in the genome, according to the flybase release note R5.13 (http://flybase.bio.indiana.edu/static&#95;pages/docs/release&#95;notes.html). Recent application of RNA interference (RNAi) to the study of developmental biology in Drosophila has enabled us to carry out a systematic investigation of genes affecting various specific phenotypes. In order to search for genes supporting cell survival, we conducted an immunohistochemical examination in which the RNAi of 2,497 genes was independently induced within the dorsal compartment of the wing imaginal disc. Under these conditions, the activities of a stress-activated protein kinase JNK (c-Jun N-terminal kinase) and apoptosis-executing factor Caspase-3 were monitored. Approximately half of the genes displayed a strong JNK or Caspase-3 activation when their RNAi was induced. Most of the JNK activation accompanied Caspase-3 activation, while the opposite did not hold true. Interestingly, the area activating Caspase-3 was more broadly seen than that activating JNK, suggesting that JNK is crucial for induction of non-autonomous apoptosis in many cases. Furthermore, the RNAi of essential factors commonly regulating transcription and translation showed a severe and cell-autonomous apoptosis but also elicited another apoptosis at an adjacent area in a non-autonomous way. We also found that the frequency of apoptosis varies depending on the tissues.