The tumor viruses human T-lymphotropic virus 1 (HTLV-1), hepatitis C virus (HCV), Merkel cell polyomavirus (MCPyV), high-risk human papillomaviruses (HR-HPVs), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV) and hepatitis B virus (HBV) account for approximately 15% of all human cancers. Although the oncoproteins of these tumor viruses display no sequence similarity to one another, they use the same mechanisms to convey cancer hallmarks on the infected cell. Perturbed gene expression is one of the underlying mechanisms to induce cancer hallmarks. Epigenetic processes, including DNA methylation, histone modification and chromatin remodeling, microRNA, long noncoding RNA, and circular RNA affect gene expression without introducing changes in the DNA sequence. Increasing evidence demonstrates that oncoviruses cause epigenetic modifications, which play a pivotal role in carcinogenesis. In this review, recent advances in the role of host cell epigenetic changes in virus-induced cancers are summarized. Viruses are infectious agents that can cause malignant and non-malignant diseases. Approximately 15% of all human cancers have a viral etiology and six human viruses are firmly associated with cancer ,3,4. DesIt is very difficult to study the epigenetic changes in virus-induced cancer cells for several reasons. Tumors are usually not detected in an early stage and tumor cells represent end products rather than initiation products. Moreover, oncoviruses have often a very long incubation time and virus-induced tumors often occur several decades after the original infection ,9,10. ItViruses also employ epigenetic changes to regulate their life cycle. This review focuses predominantly on the role of virus-induced epigenetic modifications of the host cell in carcinogenesis. The reader is referred to excellent reviews that expound how epigenetic changes modulate the viral life cycle replication ,13,14,15DNA methylation occurs at cytosine residues in CpG dinucleotides and is a fundamental mechanism in silencing gene transcription and is catalyzed by a family of DNA methyltransferases (DNMTs). DNMT3A and DNMT3B are responsible for establishing DNA methylation. DNMT3L is catalytically inactive but stimulates the enzymatic activity of DNMT3A/3B. DNMT1 is responsible for maintaining the DNA methylation pattern. Erasing DNA methylation is executed by the demethylating enzymes ten-eleven translocation (TET), activation-induced cytidine deaminase (AICDA) and thymine DNA glycosylase (TDG). Methylation of DNA reduces gene expression, whereas demethylation has the opposite effect. Methylation of DNA can prevent transcription regulatory proteins to bind or allow proteins with high affinity for methylated CpG to bind. There are three families of such proteins: methyl-CpG-binding domain (MBD), ubiquitin-like, containing PHD and RING finger domain (UHRF), and Zinc-finger domain. The MBD family comprises MeCP2, MBD1, MBD2, MBD3, and MBD4. The UHRF family contains UHRF1 and UHRF2, and the last family includes Kaiso, Zinc finger and BTB domain containing 4 (ZBTB4) and ZBTB38 ,17. MeCPAberrant methylation is associated with diseases, including cancer ,23. InduTax gene, suggesting that the Tax protein is not essential for the maintenance of ATL [The retrovirus HTLV-1 infects 10–20 million people worldwide, but only 3–5% of infected individuals will develop adult T-cell leukemia-lymphoma (ATL) 30–50 years after initial infection ,58. HTLVe of ATL . HBZ is e of ATL .The integrated HTLV-1 genome is often hypermethylated. Tax was able to increase the transcriptional activity of HLTLV-1 LTR even when heavily methylated . StimulaKLF4) and early growth response 3 (EGR3) were among the genes that were hypermethylated. Ectopic expression of KLF4 and EGR3 in ATL cell induced apoptosis, indicating that hypermethylated-mediated silencing of these genes enables ATL cell to escape from cell death [MHC-I) genes were also hypermethylated. This may result in altered gene expression and may help ATL cells to evade the immune system [PRDM16), resulting in elevated expression of the protein encoded by the PRSM16 gene, transcription factor MEL1. Overexpression of this protein is associated with leukemogenesis [FOX3P locus was found to be hypomethylated in cells from ATL patients and higher FOX3P protein levels were observed [Methylation analysis of ATL genomes showed prominent CpG hypermethylation and hypomethylation in comparison with controls ,67,68,69ll death . Transcrobserved . Tax wasobserved ,73. Howeobserved .SHP-1) gene is hypomethylated in ATL cells and SHP-1 expression is lost. The authors showed that Tax repressed SHP-1 expression by recruiting HDAC1, but whether demethylation of the promoter depended on Tax was not investigated [NDRG2) is frequently downregulated in ATL. Tax indirectly contributed to repression of this promoter by increasing the expression of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase. Overexpression of EZH2 suppressed transcription of NDRG2 via DNA methylation and trimethylation of histone 3 at lysine 27 (H3K27me3) [The mechanisms by which HTLV-1 enforces DNA methylation are incompletely understood. Although DNMT1 and DNMT3B were upregulated in HTLV-1 transformed T cells, not all cells expressed Tax, suggesting a Tax-independent mechanism . The prostigated . The tum3K27me3) . Both ex3K27me3) . A possi3K27me3) ,77. HCV is a (+) RNA virus belonging to the family Flaviviridae and is one of the leading causes of hepatocellular carcinoma (HCC). The viral genome is translated into a polypeptide of approximately 3000 amino acids that is cleaved by viral-encoded and cellular proteases to generate structural and non-structural proteins . In vitrCDKN2A (cyclin-dependent kinase inhibitor 2A), CDH1 (cadherin 1), SOCS1 , RASSF1A (Ras associated domain family member 1), APC (adenomatous polyposis coli protein), GSTP1 (glutathione S-transferase Pi 1), STAT1 , and PRDM2 (PR/SET domain 2) in HCV-positive HCC has been established. Hampered expression of these genes contributes to cancer by promoting cell proliferation, mobility and invasion, and immune evasion [DNMT1 gene is an E2F1 target gene and the core protein has been shown to phosphorylate pRb, resulting in activation of E2F1-dependent transcription.The methylation landscape of HCV-positive HCC tissues differs from non-tumor controls and a correlation between HCV infection and aberrant methylation of genes such as  evasion ,82,83,84 evasion ,28,29,30 evasion . The DNMPolyomaviridae family [MCPyV is the most recently identified virus to be linked to a human cancer. It is associated with about 80% of Merkel cell carcinoma (MCC), a rare, but aggressive cutaneous malignancy. The MCPyV genome is always integrated in all virus-positive MCCs examined ,85. MCPye family . The vire family . RASSF1A promoter hypermethylation was observed in MCPyV-positive MCC [RASSF1A promoter and 22% of the CDKN2A promoter, whereas the promoters of the tumor suppressor genes fragile histidine triad diadenosine triphosphate (FHIT), tumor promoter p73 (TP73), and protein tyrosine phosphatase receptor type G (PTPRG) had no or infrequent hypermethylation. However, no significant correlation between viral infection and hypermethylation was observed, indicating that MCPyV infection may not induce DNA hypermethylation of these promoters [RASSF2, RASSF5A, RASSF5C and RASSF10 and the TERT gene (encoding telomerase reverse transcriptase) was frequently detected in MCCs compared to normal skin samples, but again no correlation with MCPyV infection was found [RB1 gene (encoding retinoblastoma protein pRb) was hypermethylated in MCCs compared to normal skin samples, but the pattern of hypermethylation of the RB1 promoter was similar in all MCCs independent of the MCPyV status [INK4A-ARF (CDNK2A) locus and DUSP2  gene were found to be frequently hypermethylated in MCC tumors, but the viral status in these tumors was not specified, so that a possible role for MCPyV in hypermethylation cannot be determined [INK4A-ARF methylation was found between virus-positive and virus-negative MCC tumors [PTCH1 gene (encoding the Patched 1) and the gene for Atonal BHLH transcription factor 1 (ATOH1) was detected in both virus-negative and virus-positive MCC cell lines [REST promoter was observed in all tissues and cell lines, indicating that silencing of REST is not caused by hypermethylation and occurred independently of the virus status. The authors speculated that miR-9, which is upregulated in MCCs and targets the 3′ untranslated region of REST mRNA, may prevent REST synthesis [The DNA methylomes of MCPyV-negative and MCPyV-positve MCCs display significant differences in several genes that are associated with cancer. Frequent occurrence of tive MCC . DNA metromoters . Hypermeas found ,90. The V status . MCPyV LV status . This iltermined ,94. In aC tumors . Hypometll lines ,97. MCC ynthesis . The losynthesis . This fiPDCD1) promoter was hypomethylated in 42 out of 69 MCCs tissues and hypomethylation was significantly more frequent in virus-positive tumors. Virus-positive MCC patients with hypomethylated PDCD1 promoter had a better prognosis than those with high PDCD1 methylation [In conclusion, aberrant DNA methylation of cancer-related genes is common in both MCPyV-negative and MCPyV-positive MCCs and does not seem to be provoked by MCPyV infection. Viral-independent modification of host DNA methylation was further confirmed in a study that showed that DNA methylation in MCC tissues was significantly lower as compared to the patients’ chronological age. The accelerated DNA methylation in patients was irrespective of the viral presence . Althoughylation . FurtherHuman papillomaviruses (HPV) are non-enveloped viruses with a circular dsDNA genome of approximately 8000 base-pairs . More thDNMT promoter, E6 releases the repression by appropriating p53 [Methylome analyses of HPV-positive cancers revealed differences in DNA methylation compared to matching normal tissue or HPV-negative tumors and transfection studies have confirmed that the E6 and E7 oncoproteins provoked hypermethylation tumor suppressor genes and hypomethylation of proto-oncogenes ,113,114.ting p53 ,32. Furtting p53 . The mecEBV or HHV4 is an enveloped virus with a dsDNA genome of around 170 kilobase-pairs. More than 90% of the world population have lifelong infection with this virus. EBV is associated with Burkitt’s lymphoma, Hodgkin’s disease, primary effusion lymphoma (PEL), nasopharyngeal carcinoma lymphoma, gastric carcinoma, but also with non-malignant diseases, including infectious mononucleosis ,5,116. EEBV-associated cancers such as gastric cancer, nasopharyngeal carcinoma and Burkitt’s lymphoma are characterized by extensive hypermethylation of the host DNA compared with non-infected tumors and cell culture studies have illustrated that EBV infection induces de novo methylation ,121,122.CDH1 promoter and downregulation of cadherin 1 by augmenting the expression and activity of DNMT1, 3A and 3B [CDH1 gene contributes to cancer progression by increasing proliferation, invasion, and metastasis [RASSF10, which encodes a protein that inhibits cell proliferation, invasion, and migration and induces apoptosis was hypermethylated in EBV-positive gastric cancer compared to EBV-negative gastric cancers. The authors demonstrated that LMP1 promoted DNMT1 expression, which was responsible for hypermethylation of the RASSF10 gene. Overexpression of LMP1 in human gastric adenocarcinoma AGS cells stimulated migration, invasion and cell colony formation and this was counteracted when RASSF10 was co-expressed. Xenograft studies with LMP1 and LMP1 plus RASSF10 cells confirmed that RASSF10 thwarted the LMP1-malignant phenotype. These results suggest that LMP1-mediated methylation and silencing of the RASSF10 gene plays a role in EBV-induced oncogenesis [PTEN) gene through stimulation of DNMT1 in a STAT3-dependent manner [RASSF1A promoter by enhancing DNMT3A expression. This epigenetic modification results in decreased RASSF1A expression, leading to increased cell proliferation [KDM2B) and trigger hypermethylation. KDM2B demethylates histone 3 at lysine 4 (H3K4me3). H3K4me3 is commonly associated with active transcription and demethylation will result in transcriptional silencing [LMP1 induces hypermethylation of the A and 3B . Loss oftastasis . The genogenesis . Other sogenesis ,35. LMP2ogenesis ,40,41. Hogenesis . LMP2A ct manner . EBNA3C,feration . Finallyilencing . Thus, EKSHV or HHV8 is the causative agent of Kaposi sarcoma and associated with the lymphoproliferative disorders, multicentric Castleman’s disease and PEL ,127. No CDNK2A, CDH1 and CDH13 (cadherin 1 and 13), LDHB (lactate dehydrogenase B), HLTF , CCND2 (cyclin D2). The authors showed that KHSV LANA recruited DNMT3A to chromatin, and induced hypermethylation and transcriptional inactivation of these genes [TGFBR2) through inducing hypermethylation of Sp1 binding sites, thereby preventing Sp1 binding. Epigenetic silencing of this promoter contributed to the pathogenesis of KSHV-associated tumors [CpG methylation analysis of the human DNA in KSHV-infected cells and KSHV-associated PELs revealed both hyper- and hypomethylated promoters compared with KSHV-negative lymphoma BJAB cells. Genes encoding proteins involved in cell cycle control, signaling pathways and metastasis were differently methylated in the KSHV-positive cells and tumors compared to control cells ,129. Somse genes ,44,45. Lse genes . Moreoved tumors . Two othd tumors ,49. vIL6d tumors . AnotherIt is estimated that more than 250 million people globally are chronically infected with HBV, and each year around 800,000 patients died from HBV- and HCV-related HCC. Of these, approximately 50% of are caused by HBV . HBV-indCIP1/WAF1 (CDKN1A), p14ARF (CDKN2A) and p14INK4B (CDKN2B), cadherin 1, RASSF1A, the spleen associated tyrosine kinase SYK (SYK), GSTP1, the protein phosphatase 1 regulatory subunit 13B (PP1R13B), the tumor promotor p53 binding protein 2 (TP53BP2), and insulin like growth factor binding protein 3 (IGFBP3) [Comparing the DNA methylation profile of HBV-associated HCC and HBV-negative tumors or healthy adjacent liver tissue, HBV-infected and non-infected cells, and HBx transgenic mouse model and control mice disclosed differentially methylation. Several cellular promoters were hypermethylated in the presence of HBV or HBx, including the promoters of the genes encoding cyclin-dependent protein kinases inhibitors p21(IGFBP3) ,137,138.(IGFBP3) .INK14A, resulting in activation of the cyclin-dependent kinase 4/6-pRb-E2F1 pathway, and ultimately in stimulation of DNMT1 expression [PP1R13B and TP53BP2 promoters [HBV seems to affect DNA methylation by several mechanisms. One study showed that HBx could cause hypomethylation through releasing DNMT3A from promoters . HBx alspression . Moreovepression ,56. Anotpression . HBx waspression . The rearomoters . SimilarHost cell DNA is packed and present in a highly organized structure called chromatin, which is a complex of DNA, histones and other proteins. Chromatin is a dynamic structure that regulates the accessibility of DNA for transcription, replication, DNA repair and recombination. Nucleosomes are the basic units of chromatin and consist of two copies of the canonical histones H2A, H2B, H3 and H4 around which DNA is twisted. The linker histone H1 is interspersed between nucleosomes. Posttranslation modifications (PTMs) of histones will affect the chromatin structure and hence the accessibility of the DNA. The most studied and best understood histone PTMs are acetylation of lysine (K) and methylation of lysine and arginine (R) residues, and phosphorylation of serine (S), threonine (T) and tyrosine (Y) ,140,141.Another mechanism to change the chromatin structure is by chromatin remodelers ,144. ATPPerturbed histone and modifications and remodeling of chromatin are pivotal events in oncogenesis . In the CIP1/WAF1 encoding gene. Expression of this cyclin-dependent kinase inhibitor was upregulated in HTLV-1 infected cells and it was shown that histone H4, but not histone H3 was acetylated [HTLV-1 infection can affect histone acetylation as demonstrated for the p21etylated . Both Taetylated ,148,149.etylated . As not etylated ,201,202.etylated . Proteinetylated . The mecetylated . These fEVC1) and EVC2 genes though stimulating histone H3 acetylation and H3K4me3 [BCL2L11) gene, which encodes the proapoptotic protein BCL2 interacting mediator of cell death (BIM), was decreased in ATL cells compared to HTLV-negative T cell lines and normal PBMC. Ectopic expression of HBZ in T cells inhibited transcription of the BCL2L11 gene. The authors showed that HBZ-mediated repression of BCL2L11 transcription involved inactivation of the transcription factor Forkhead box O3A (FOXO3A), hypermethylation, upregulation of H3K9me2 and H3K27me3, and reduced acetylation of histone H3. HBZ-mediated silencing of BIM expression led to decreased apoptosis and may thus contribute to HTLV-1 induced oncogenesis [Altered histone methylation may also contribute to HTLV-1-induced cancer. The H3K27me3 pattern in ATL cells was different from normal CD4+ T cells, indicating that HTLV-1 reprograms the H3K27me3 profile. H3K27me-silenced genes included genes whose products are involved in control of cell proliferation, cell migration, transcriptional regulation, immune response and cellular metabolism ,204. Fuj H3K4me3 . The EVC H3K4me3 . Mukai a H3K4me3 . Transcrogenesis .Two studies demonstrated that HTLV-1 could induce chromatin remodeling. The integrated HTLV-1 genome bound CCCTC-binding factor (CTCF), a chromatin remodeling protein and regulator of transcription. Recruitment of CTCF by HTLV-1 provirus may spread abnormalities in the chromatin structure of host cells, thereby affecting gene expression . Mass spSFRP1) promoter. The core protein was shown to downregulate SFRP1 expression by an epigenetic mechanism. The core protein increased the levels of DNMT1 and HDAC1 and stimulated their binding to the SFRP1 promoter. This resulted in hypermethylation and reduction in histone H3 acetylation. Silencing of SRFP1 led to deregulated activation of the Wnt signaling pathway and may thus contribute to HCC-induced HCC [HCV can modulate histone acetylation as shown for secreted frizzled related protein 1 (uced HCC . c-MYC), PTEN, CDH1, epidermal growth factor (EGF), CDKN2A, and IGFBP3 [HOX) genes. Kasai et al. reported that the expression of several HOX genes was induced in HCV infected or core protein expressing cells. HCV and core protein stimulated HOX gene expression by impairing histone H2A monoubiquitination via degradation of PRC1 component E3 ligase RNF2 (ring finger protein 2) [HCV infection is associated with changes in histone methylation. Ectopic expression of the entire HCV polypeptide resulted in a significant loss of H4K16ac, H4R3me2, and H4K20me3, and was correlated with the altered expression of genes important in hepatocarcinogenesis such as avian myelocytomatosis viral oncogene homolog (d IGFBP3 . Increasd IGFBP3 . HCV infd IGFBP3 . The autotein 2) . As HOX KDMA1, indicating that sT may affect histone methylation. sT:MYCL:EP400 complex formation was required to transform IMR90 human diploid fibroblasts, suggesting that complex formation is important in the development of MCPyV-positive MCC [The LTs of the murine and SV40 polyomaviruses were found to bind to, and to upregulate the expression and the activity of p300/CBP ,212,213.tive MCC .CIP1/WAFf1 levels. RNA interference-mediated silencing of HDAC2 in HPV18-positive HeLa cells increased expression of the p21CIP1/WAFf1 tumor suppressor and stimulated apoptosis [Several studies have shown that HATs and HDACs can play a role in HR-HPV associated cancers. Expression levels of HDAC1 and HDAC1 were increased in invasive HPV-positive cervical cancers compared normal epithelium and inversely correlated with p21poptosis . It is npoptosis ,166. E7 poptosis . The intpoptosis . TIP60 apoptosis .c-FOS) gene transcription. Hence, E7-mediated upregulation of c-FOS protein levels may contribute to deregulation of cell cycle control [HR-HPV E7 was shown to interact with Mi2β, HDAC1 and HDCA2, which are constituents of the NuRD complex, a CHD chromatin remodeling complex. HPV E7 could through this interaction downregulate expression of proteins involved in immune responses and promote cell growth ,216. Fur control .HOX genes. In accordance with cervical cancer, expression of these genes was upregulated in the E6/E7 transformed fibroblasts and in E7-expressing keratinocytes cells compared to control cells [hTERT promoter activity by increasing H3K4me3 and H3K9ac, which are transcription activation modifications, and decreasing methylation of the transcription repressive modification H3K9me2 [HR-HPV can affect histone methylation by several mechanisms. The PRC2 complex mediates H3K27me3, which is associated with transcriptional repression. Subsequently, PRC1 binds to H3K27me-marked chromatin and further silences gene expression by monoubiquitinating lysine 119 of histone H2A. PRC2 contains the histone methyltransferase EZH2, which catalyzes mono-, di-, and trimethylation of H3 ,218. Perol cells ,170. E6  H3K9me2 . HPV16-p H3K9me2 . E6 coul H3K9me2 .In conclusion, changes in histone acetylation and methylation resulted in dysregulation of cellular gene expression and may contribute to HPV-induced oncogenesis.Increased histone acetylation and increased cellular gene expressed were observed in EBV-transformed lymphoblastoid cell lines compared to control cells . EBNA2 wFCER2 or CD23) promoter [EBV infection was also associated with changes in histone methylation. EBV infection of nasopharyngeal epithelial cells reduced the transcriptional activation mark H3K4me3 and enhanced the suppressive mark H3K27me3 at the promoter regions of several genes, including 16 DNA damage repair genes. The reduced DNA repair ability in EBV-infected nasopharyngeal epithelial cells may play an important role in nasopharyngeal carcinoma . Infectipromoter . It was promoter , suggestpromoter .Taken, together, EBV-induced histone modifications and chromatin remodeling may be a potential cancer driver in EBV-related tumors.KSHV-infected cells displayed changes in the level of H3K27me3 at promoters of genes encoding proteins relevant in KSHV-induced carcinogenesis such as vascular endothelial growth factor (VEGF), p53, and toll-like receptors (TLRs) . SeveralIL-8 and proliferating cell nuclear antigen (PCNA) genes. IL-8 possesses mitogenic, motogenic and angiogenic properties, whereas PCNA is implicated in DNA synthesis. Increased expression of these proteins may represent key steps in neoplastic transformation by HBV [HBx protein of HBV was shown to activate or repress cellular gene expression. This opposite effect depended on whether HBx attracted HATS or HDACs to the promoter. HBx stimulated CRE binding protein (CREB)-dependent transcription by recruiting p300/CBP. Induction of CREB target genes may play a role in the development of HCC associated with HBV infection . HBx alsn by HBV . On the n by HBV . HBx wasn by HBV ,229.c-MYC proto-oncogene [EpCAM) promoter [HBx-caused changes in histone methylation is mediated by different enzymes. HBx stimulated the expression of the histone lysine 9-specific methyltransferase SETDB1, leading to the release of transcriptionally silenced HBV genome . The effoncogene . HBx uproncogene . The KDMoncogene . PLK1-meoncogene . HBx waspromoter . HBx waspromoter . Taken together, these findings emphasize an important role of HBV-induced histone modifications in the development of HCC.MicroRNAs are short, non-coding RNAs that are involved in the regulation of gene expression. Most miRNA genes are transcribed by RNA polymerase II and generate an immature precursor pri-miRNA, which is processed by the RNase III enzymes Drosha and Dicer to produce mature microRNA of 21–23 bases. The mature miRNA is incorporated into the RNA-inducing silencing complex (RISC), which binds to complementary or quasi complementary sequences in the 3′ untranslated region of target mRNAs and induces their degradation or prevents their translation . MicroRNNABP1) gene encoding the ssDNA binding protein HSSB2. Silencing of this DNA repair factor stimulated cell proliferation and genomic instability, indicating that HTLV-1 infection may trigger proliferation and genomic instability by the HBZ/miR-17+miR-21/HSSB2 axis [No HTLV-1-encoded microRNA has been described so far, but HTLV-1 can alter the expression levels of cellular microRNAs. HTLV-1-transformed cells and ATL-derived cell lines had reduced levels of miR-150 and miR-223. STAT1, whose mRNA is a direct target for these miRNAs, was upregulated in HTLV-1-transformed and ATL cells and was required for the proliferation of these cells. MHC-I levels were also increased in these cells and enhanced MHC-I expression helped the tumor cell to avoid immune clearance . STAT1 hSB2 axis .HCV does not seem to encode viral microRNA probably because of its cytoplasmic location, which deprives the virus from nuclear proteins, such as RNA polymerase II and Drosha, required for microRNA biogenesis. However, comparative microRNAome profiling of HBV-associated HCCs and HBV-negative HCCs, and of HepG2 hepatocytes stably transfected and full-length HCV genome and control cells demonstrated that HCV elicited changes in cellular miRNA expression ,262,263.MCPyV encodes a microRNA, referred to as miR-M1, which negatively regulates the expression of LT, a viral protein involved in transcription and replication of the MCPyV genome ,265. ThiComparative microRNAome studies between virus-positive and virus-negative MCC cell lines and tumors have identified several cellular microRNAs whose expression is associated with the MCPyV status (for a recent review see ). These A subset of miRNAs associated with tumor metastasis and MCC-specific survival has been identified. Functionally, overexpression of miR-203 was able to inhibit cell growth, to induce cell cycle arrest, and to regulate survivin expression in MCPyV negative-MCC cells, but not in MCPyV-positive MCC cells. These findings reveal a mechanism for survivin expression regulation in MCC cells and offer insights into the role of miRNAs in MCC tumorigenesis .MCPyV has also been detected in other cancer types, including non-small cell lung cancer . LasithiIn conclusion, the MCPyV-encoded microRNA miR-M1 does not seem to be involved in MCC, but MCPyV infection modifies cellular microRNA expression, which may play a role in tumorigenesis and the tumor microenvironment.MicroRNA prediction algorithms have been used to forecast putative HPV16- and HPV18-encoded miRNAs ,280. By Several studies have shown that HR-HPV positive tumors and cell lines expressing the HR-HPV oncoproteins E5, E6 or E7 have altered cellular microRNAomics compared to control tissue and cells. Upregulation and downregulation of cellular microRNAs have been observed. The microRNAs dysregulated in HPV-positive cervical cancers are involved in cell proliferation, cell survival, angiogenesis, invasion, and migration underscoring their role in HR-HPV pathogenesis (One mechanism by which HPV affected microRNAs expression was by modifying the promoter methylation pattern of the genes encoding microRNAs . For exaThe EBV BHRF1 cluster and the BamHI-A rightward transcript (BART) clusters 1 and 2 encode >40 mature miRs, which can regulate host and viral gene expression. These viral miRs are crucial for EBV-associated tumorigenesis by e.g., inhibiting apoptosis, immune evasion, and cell growth ,248,249.EBV infection also altered the expression of host cell miRNAs. Comparison of the microRNAomes from EBV-positive nasopharyngeal tissue and non-tumor tissue disclosed several cellular miRNAs that were upregulated, but also many were downregulated. One of the cellular miRNAs induced by EBV is miR-155, an oncomir crucial for B cell transformation and proliferation . The micThe mechanisms by which EBV modulate microRNA expression have been less studied but may include changes in DNA methylation and chromatin of the microRNA genes induced by viral proteins as discussed in CIP1/WAF1 expression. KSHV miR-K12-5, miR-K12-9 and miR-K10a/b targeted the pro-apoptotic protein Bcl-2-associated factor 1 (BCLAF1), whereas miR-K12-1, miR-K12-3, and miR-K12-4-3p suppressed caspase 3 expression. These microRNAs allowed the virus to avoid apoptosis. KSHV evaded the innate immune system by miR-K12-5- and miR-K12-9-mediated reduction of myeloid differentiation primary response 88 (MYD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), respectively. Finally, KSHV microRNAs promoted angiogenesis by downregulating the levels of the anti-angiogenic factor thrombospondin, SH3 domain binding glutamate-rich protein (SH3BGR) and CD82 [KSHV produces 25 mature microRNAs and more than 2000 host transcripts that encode proteins associated with KSHV pathogenesis can be directly targeted by these viral microRNAs ,287,288.and CD82 ,288,289.and CD82 . Jarid 2and CD82 . The role of KSHV-provoked dysregulated expression of host cell microRNAs in cancer has been extensively reviewed ,251,252.HBV encodes two viral miRNAs: HBV-miR-2 and HBV-miR-3. HBV-miR-2 may act as an oncomiR because it was found to promote cell growth, migration and invasion by downregulating the expression of the E3 ubiquitin-protein ligase tripartite motif containing 35 (TRIM35) and upregulating protein levels the GTPase RAN. TRIM35 is a proapototic protein and can inhibit the Warburg effect, whereas RAN is involved in nucleocytoplasmic transport, but also in metastasis. HBV-miR-3 enhances cell invasion and proliferation by e.g., silencing PP1A and PTEN ,256. Several studies showed a role for HBx in up- and downregulating the expression of cellular microRNAs, including miR-10, miR-132, miR-143, and miR-193b. This has been the topic of excellent reviews ,291,292.Long non-coding RNAs (lncRNAs) are a heterogeneous group of RNAs that are more than 200 nucleotides long and are not translated into functional proteins. Most lncRNAs are generated by RNA polymerase II and can contain a 5′ cap and 3′ polyA tail. So far, ~18,000 lncRNA genes have been identified in the human genome, but their number is still increasing. The lncRNA genes produce almost 50,000 transcripts, but many remain to be annotated . LncRNAsHTLV-1 produces the antisense mRNA HBZ that is inefficiently polyadenylated and as a result the minor fraction of properly polyadenylated HBZ mRNA is transported to the cytoplasm and translated into the HBZ protein, while the majority of aberrant polyadenylated antisense mRNA is retained in the nucleus and acts as lncRNA. Nuclear HBZ mRNA could bind to the promoters of the cellular genes, including the genes encoding C-C chemokine receptor type 4 (CCR4) and E2F1, and enhanced transcription of these genes, resulting in stimulation of proliferation of HTLV-1-infected cells. HTLV-1 antisense mRNA also promoted expression of survivin . The exaCIP1/WAF1 expression by increasing H3K27me of the CDKN1A promoter [Comparing the levels of cellular lncRNA in ATL cells, HTLV-1-infected cell lines and control cells revealed upregulation of lncRNAs ANRIL (antisense noncoding RNA in the INK4 locus), H19, and SAF (Fas-Antisense) and slight downregulation of HOTAIR (HOX antisense intergenic RNA) and TUSC7 (tumor suppressor candidate 7) by HTLV-1 . The autpromoter . In concSo far, no HCV-encoded lncRNAs have been identified. However, it was shown that the 5′ untranslated region could be processed by the cellular endoribonuclease XRN1, generating subgenomic viral RNAs that are not translated and therefore may act as viral lncRNAs . The funResults from several studies comparing HCV-positive HCC with healthy liver tissue showed that several lncRNAs have significantly different expression levels ,305,329.The existence of putative MCPyV-encoding lncRNAs and the effect of MCPyV infection on host cell lncRNA expression have not been addressed. One study in head and neck squamous cell carcinoma cells demonstrated that miR-375 silenced the expression of the glucose transporter protein solute carrier family 2 member 1  by targeting SCL2A1 mRNA. The head and neck squamous cell carcinoma glycolysis-associated 1 (HNGA1) lnc RNA was upregulated and HNGA1 functioned as a sponge for miR-375. Ectopic expression of lcnRNA HNGA1 in head and neck squamous cell carcinoma cells stimulated cell proliferation and glycolysis, and accelerated tumor growth in xenograft mouse . The expTMPOP2 gene and inhibited its expression. E6 released p53-mediated inhibition of TMPOP2 expression. Interestingly, overexpression of TMPOP2 enhanced E6 and E7 protein levels because TMPOP2 sponged miR-375 and miR-139, which target E6/E7 mRNA [FANC-2. E6 and E7 were found to reduce miR-29a levels, which targets transcription factor YY1 and E7 altered the activity of YY1 [Several lncRNAs previously found to be involved in cancer are also differentially expressed in cervical cancers compared to control samples. RNA interference-mediated silencing of E7 in HPV18-positive HeLa cells or expression of E6 in primary human keratinocytes resulted in altered expression of several lncRNAs compared to control cells ,333,334./E7 mRNA . Moreove/E7 mRNA . E7 and y of YY1 . The exa6 for EBER1 and ~2.5 × 105 for EBER2) in infected cells. Although shorter than classical lncRNAs, EBERs are considered lncRNAs. EBER2 acts as a guide RNA to recruit transcription PAX5 to viral target sites, where the complex suppresses transcription. The role of EBER1 remains poorly understood, but both EBER1 and EBER2 play a role in suppressing the innate immune system, avoiding apoptosis and activating the oncogenic phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway [IFNB1) and CXCL8 genes. BHLF1 lncRNAs promote EBV replication but may also contribute to viral latency [EBV encodes two small non-coding RNAs EBV-encoded RNA1 (EBER1) and EBER2 of 167 and 172 nucleotides long, respectively. Both are RNA polymerase III transcripts and they are present in high copy numbers  interfere with processes such as apoptosis, migration and invasion, proliferation, and DNA repair ,314,315.Approximately 16 potential KSHV lncRNAs have been described, with polyadenylated nuclear RNA (PAN) lncRNA the most important and best-characterized. This lncRNA is involved in viral gene expression, replication, and immune modulation. PAN RNA was shown to bind transcription factor interferon regulatory factor 4 (IRF4) and inhibit transcription of IRF4-responsive genes. Moreover, PAN lncRNA could interact with H3K27-specific demethylases UTX and JMJD3, and with the PRC2 components EZH2 and SUZ12. Hence, PAN lncRNA affects chromatin modification, resulting in altered host gene expression and seems to be required for efficient nuclear export of mRNA ,314,316.ATOM, which encodes angiomotin, a protein that promotes cell migration and angiogenesis. In accordance, overexpression of KIKAT/LINC01061 in SLK cells induced cell migration [Several cellular lncRNAs, including H19, growth arrest specific 5 (GAS5), deleted in lymphocytic leukemia 2 (DLEU2) and MALAT1 are abnormally expressed in KSHV-infected cells and aberrant expression of these lncRNAs has been associated with oncogenic processes such as proliferation, migration, invasion, cell survival and angiogenesis . KSHV caigration . Hence, The expression of several lncRNAs are dysregulated in HBV-associated HCC compared to healthy liver tissue. Some of these lncRNAs are discussed here. For a complete overview, the reader is referred to excellent recent reviews ,322,326.CDKN1B promoter, whose gene encodes cyclin dependent kinase inhibitor p27KIP1, and repressed transcription. Another lncRNA that is upregulated is ANRIL, which binds PRC2 and represses transcription of Krüppel-like factor 2 and sequesters miR-122-5p. Knockdown of ANRIL expression induced apoptosis and suppressed proliferation, invasion and migration of HCC cells in vitro. Integration of HBV adjacent to long interspersed nuclear element 1 (LINE1) resulted in the transcription of a chimeric lncRNA, HBx-LINE1, which can be detected in ~23% of HBV-associated HCC tumors. HBx-LINE1 activated the Wnt signaling pathway by inducing nuclear localization of β-catenin, and stimulated cell proliferation and metastasis in vitro. An HBx-LINE1 transgenic mouse model revealed that the animals were more susceptible to diethylnitrosamine-induced tumor formation than wild-type mice and nuclear localization of β-catenin in hepatocytes of the transgenic animals was observed [Examples of cellular lncRNAs upregulated by HBV include highly expressed in HCC (HEIH), UCA1, HOTAIR, HULC, and LINC00152, which all were shown to interact with EZH2 and repress gene expression, thereby promoting proliferation, migration and invasion, cell survival, and tumor growth. UCA1 recruited EZH2 to the observed ,323,326.Reduced expression of the lncRNAs n346077 and downregulated expression by HBx (DREH) was observed in HBV-positive HCC included. DREH inhibited proliferation and n346077 prevented migration and invasion in vitro ,323,326.The mechanisms by which HBV modulates expression of lncRNAs remains largely unknown. Studies with HBx expression in liver cell lines and with an HBx transgenic mouse model showed that HBx is directly involved in regulating lncRNA expression. HBx increased expression of HULC and HEIH ,320,324.Circular RNAs (circRNAs) are single-stranded, RNAs produced from pre-mRNA by a back splicing mechanism in which the 5′ and 3′ termini are covalently joined. They vary in length from less than a hundred to several thousands of nucleotides ,340. CirThe existence of HTLV-1 encoded circRNA and whether HTLV-1 can induce the production of cellular circRNA have not been investigated. However, after converting its ssRNA virus genome into dsDNA, the dsDNA integrates into the host chromosome and RNA polymerase II-derived viral transcripts are spliced, suggesting that viral circRNA may be produced.It is still unclear whether HCV encodes circRNA. The fact that this virus replicates in the cytoplasm, in the absence of the nuclear splicing machinery, may explain why no HCV circRNA is generated. A recent study examined the cellular circRNA profile in uninfected and HCV-infected liver cells. The authors found 10 circRNAs that were significantly upregulated, whereas 63 had decreased levels in the HCV-positive cells compared to control cells. The authors elaborated on the role of the upregulated circPSD3, which was generated by a backsplicing event between exon 5 and exon 8 from the pleckstrin and Sec 7 domain containing (PSD) transcript and found that depletion of circPSD3 diminished viral infectivity . The mecThe LT and sT encoding region of MCPyV contains an alternative reading frame which encodes the ALTO protein with unknown function . A recenA number of HPV-encoded circRNAs have been identified in HPV positive cervical cancers. Among these viral circRNAs (v-circRNA), is circE7 the most abundant and can be translated into E7. Multiple microRNA binding sites have been identified on circE7, suggesting that it can act as a miRNA sponge. circE7 has been reported also in HPV-positive anal and head and neck cancers ,349. SurTP53 (circ_0000263) or SNAI2 (circ_000284) mRNA. The latter encodes a protein involved in epithelial–mesenchymal transition (EMT) [High throughput RNA sequencing studies of HPV-positive anogenital and oropharyngeal cancers and matched adjacent non-tumor tissues discovered numerous differentially expressed cellular circRNA ,364,365.on (EMT) .EBV encodes more than 30 different v-circRNAs from dissimilar regions of its genome, which are stably expressed in all EBV-associated tumors ,366,367.KSHV produces more than 100 circRNAs, which can be detected in Kaposi’s sarcoma tumors, PEL, and multicentric Castleman’s disease ,354,355.An HBV-encoded circRNA, HBV_circ_1, has been detected in HBV-infected cells and in HBV-associated HCC. HBV_circ_1 is mainly located in the cytoplasm and it was found to bind DXH9, as well as the ribosomal protein P0/P1/P2. Knockdown of DXH9 increased HBV_circ_1 levels which is in agreement with a previous study that described a role of DXH9 in repressing circRNA production . IncreasThere is compelling evidence that cellular circRNAs are involved in the etiology of HBV-associated HCC. By comparing the landscape of circRNA from HBV-positive HCC tissue and control tissue, differentially expressed cellular circRNAs were identified ,370,371.Oncovirus infection has a substantial impact on the host’s epigenetic landscape, which plays a crucial role in virus-driven oncogenesis. Reversing or preventing tumor virus induced epigenetic changes may therefore be a strategy for treating virus-associated tumors As mentioned in In vitro and in vivo studies have demonstrated that HDAC inhibitors, such as the FDA approved drugs vorinostat, belinostat and panobinostat, could be a promising therapy for HPV-positive cancers . A phaseAnti-microRNAs have been designed to target specific microRNAs. The anti-miR-122 (Miravirsen) is used for treatment of HCV infections , and bloHow to exploit lncRNAs and circRNAs for therapeutic purposes in virus-associated cancers remains in its infancy. A recent study showed that a peptide that blocks the interaction between lncRNA HOTAIR and EZH2 decreased invasion of cancer cells in vitro and reduced tumor formation in ovarian tumor xenograft . This ma6-methyladenosine RNA methylation adds another layer of complexity to epigenetic changes and has been shown to play a role in cancer [6-methyladenosine modified and can have an effect on the viral life cycle and pathogenicity, as was shown for HCV and HBV [6-methyladenosine modification of circRNAs is not uncommon and plays a role in their regulation and function [6-methyladenosine RNA methylation of viral and host cell mRNA. These epitransciptomic changes promoted EBV infection in vitro [6-methyladenosine RNA methylation contributes to cancer remains unknown, but it would not be a surprise. Viruses keep amazing scientists with their creativity.All known human tumor viruses show great diversity in their structure and genome sequence. Their oncoproteins have no similarity, yet these viruses use the same mechanisms to induce cancer. They convey the hallmarks of cancer on the host cell. One way to obtain this is by altering gene expression in the infected cell and their viral proteins may do so by functioning as transcriptional regulators, by regulating the activity of transcriptional activators and repressors, or by inducing mutations in the host genome. During recent years, it has become clear that tumor viruses also apply epigenetic mechanisms to alter cellular gene expression. Again, all human tumor viruses seem to apply the same strategies . They can cancer . Viral g and HBV . N6-methfunction . Two recin vitro ,392. Onc


Changes in transcription factor occupancy due to changes in chromatin accessibility have been implicated in congenital heart disease. However, the factors controlling this process and their role in changing gene expression during development or disease remain unclear. In this review, we focus on recent advances in the understanding of epigenetic factors controlling cardiac morphogenesis and their role in diseases.Epigenetic control of gene expression during cardiac development and disease has been a topic of intense research in recent years. Advances in experimental methods to study DNA accessibility, transcription factor occupancy, and chromatin conformation capture technologies have helped identify regions of chromatin structure that play a role in regulating access of transcription factors to the promoter elements of genes, thereby modulating expression. These chromatin structures facilitate enhancer contacts across large genomic distances and function to insulate genes from  Nkx2-5, Gata4, and Tbx5 coalesce at midline and form a linear tube that circulates blood in the developing embryo , an enzyme that catalyzes transfer of methyl groups to CpG motifs, is deleted within the endocardium using Nfatc1Cre . Has3 is an extracellular matrix remodeling enzyme and, in the context of heart development, is required for endothelial-to-mesenchymal transition and valve formation, therefore suggesting a link between the HAS3 methylation status and regulation during cardiac development , which partially relieves repression of Tnni1  shows that loss of Tet3 alone or Tet1, Tet2, and Tet3 triple knockout leads to improper adoption of cardiac mesodermal fate at the expense of neural cell fate as determined by qRT-PCR analysis of neuronal markers Sox1 and Foxg1 and CM markers Nkx2-5, Myh6, Myh7, and Tnnt2.DNA methylation is an early epigenomic change, occurring during DNA replication to mark the daughter strand . Methylafatc1Cre , qRT-PCRelopment . Knockdoassembly . RNA-seqnd Tnnt2 . The imppression . MethylCof Tnni1 . CpG metelopment . Work byNPPA and NPPB, with methylation status validation by MassARRAY  profiles of cardiac-specific TFs  showed that only 16% of regions with TF occupancy overlapped with H3K27ac chromatin marks within fetal cardiac tissues . AnalysiGata4, Nkx2-5, and Mef2c to activate the transcription of these cardiac TFs (Ezh2) of PRC2, which establishes the chromatin mark H3K27me3, using CreNkx2-5 (fl/fl Nkx2-5CreEzh2) causes perinatal lethality with only 2% of pups surviving to postnatal day 20  that acetylates H3 on lysine 27 (H3K27ac) and binds within the promoter regions of a number of critical heart genes such as diac TFs . Indeed,diac TFs . Conditil day 20 . In thisactivity .Baf60c is conditionally knocked out from CMs using Myh6-Cre, showed mis-regulation of structural CM proteins, not TFs involved in cardiac development, possibly mediated by the direct interaction between myocardin (MYOCD) and BAF60C  tag with an eGFP cassette at the 3′ end of the CTCF coding sequence 12.5 and myocardial thinning  and chromatin structure (4C-seq) at the Iroquois (Irx) gene cluster and adjoining genes, including a mitochondrial subunit gene Ndufs6. Mitochondria in fl/fl;Nkx2-5CreCtcf E11.5 CMs appear swollen and disorganized (α-Myosin Heavy chain tamoxifen-inducible Cre recombinase (MerCreMer) mouse to generate CTCF conditional knockout (CTCF-CKO) in adult CMs (CTCF-CKO mice shows little change (<2%) in TAD boundaries and A/B compartmentalization as compared to controls. However, Fit Hi-C, a method to analyze genome-wide chromosomal contacts that are statistically significant, revealed that interactions and accessibility at a large number of enhancer regions are changed in CTCF-CKO mice: 4,037 increase/decrease in contact compared to 1,013 unchanged. The genes in the surrounding chromosomal regions are enriched for cardiac pathology pathways , which is transcribed from the human HOXC locus to trans regulate transcriptional silencing of the HOXD locus in a tissue-dependent manner by direct interaction with the histone methyltransferase PRC2  are greater than 200 nucleotides (nt) in length and function by binding DNA, other RNAs, or RNA binding proteins or can contain their own transcriptional start site to make micropeptides (<100 amino acids) . There aase PRC2 . Furtherase PRC2 , includiilencing , which cilencing . The bioilencing .i) of CTCF protein in endogenous mESC lines (i deleted cell lines show modest gene expression changes (∼500 genes mis-regulated with an average fold change of 2.7) and no change in A/B compartmentalization similar to what was seen by Saldana-Meyer et al. Chromatin conformation capture experiments find that almost half of all CTCF loops are lost in RBRi deletion mutants, suggesting that there are two kinds of CTCF-mediated loops: RBRi dependent and RBRi independent. Even with modest gene expression changes in RBRi deletion mutants, some TAD formation requires RNA binding with CTCF , are alternatively spliced lncRNA transcripts that lie within the myosin gene locus  single-stranded non-coding RNA molecules known as microRNAs (miRNAs) have also been implicated in epigenomic regulation . The rolGATA4/6, MYH6/7, MYH7, ACTN2, HAND2, TBX1/5, and TDGF1, within the NIPBL haploinsufficient samples as compared to control patient samples  , causes congenital heart defects and is linked to ficiency . Tbx1 isy trunk) . TBX1 rey trunk) . The imp embryos .Children with Down syndrome  present with a higher-than-normal incidence rate (>50%) of ventricular septal and atrial septal defects . Transcrde novo, resulting in the generation of a loss-of-function protein  . Nonsens protein . Seventy protein . ChIP-qPin vitro . Furtherignaling .Other than congenital cardiac disease, postnatal epigenetic changes are also correlated with gene expression changes leading to cardiovascular aging, a complex process characterized by decreased heart function and ventricular and atrial remodeling . These eExtensive work in recent years has uncovered some of the molecular mechanisms which control chromatin regulation and disease conditions arising from epigenetic mis-regulation. The conserved nature of chromatin domains and the regulatory mechanisms controlling their establishment and maintenance would suggest that changes to chromatin landscape would lead to dramatic changes in gene expression. However, for the most part, this has not been the case, suggesting that in an epigenetic context, overlapping layers of modifications regulate transcription. Although our understanding of chromatin biology has vastly advanced in recent years with chromatin conformation capture technologies, much remains unclear about what the functional role is of these domains in regulating enhancer accessibility or enabling/repressing transcription. In adult disease conditions, epigenetic changes play a more complex role with a large variability of phenotypes between patients, further confounding the analysis of causative factors. Alterations to epigenetic factors that correlate with disease suggest that these aberrant proteins undergo changes in subunit function and lack biological redundancies. Interestingly, cardiomyopathy phenotypes in human patients appear to be sensitive to these epigenetic aberrations, suggesting specificity in both tissue and type of epigenetic factor expressed during heart development. Refining our understanding of the epigenetic mechanisms at play in cardiac development is required to parse out these phenotypes.Both authors conceptualized, wrote, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27‐fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence‐independent control by adopting it for elongation control, achieving 8‐fold repression and 4‐fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis‐regulatory logic on CRISPRi‐targeted native genes.Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in  A regulatory control mechanism is developed based on the competitive binding of dCas9 to DNA by two guide RNAs. One represses a target gene (CRISPRi) and the second displaces the repressing dCas9 without interfering with the gene’s transcription. Integration can occur at individual promoters through the arrangement of DNA operators to which regulatory proteins bind and either recruit or interfere with transcriptional machinery  to overlapping positions that either recruit or block RNAP  directs dCas9 to the first position and a second derepressing sgRNA (sgD) directs dCas9 to a mutually exclusive second position that does not impact transcription Fig . We findet al, et al, E. coli constitutive promoter can be repressed by targeting the repressing sgRNA (sgR) to overlap the −10 or −35 σ70‐binding sites  and sgD (Vanillic acid‐inducible PVan) encoded on a p15A plasmid. The targeted constitutive promoter (PC1) was built based on the −35 to +1 core of PGate08 . From the same plasmid, dCas9 was expressed using the aTc‐inducible PTet promoter. A concentration of 1.25 nM aTc was used for all experiments and leads to approximately 500 dCas9 molecules per cell during exponential growth  and induced with aTc and either IPTG or Vanillic acid (Van) for 5.5 h . Fluorescence was then measured using flow cytometry. First, we compared the repression obtained by targeting the −10 (sgRP1) or −35 (sgRP2) positions of the promoter  of RFP fluorescence and ymax/ymin are the maximum/minimum measured values. Rx is the strength of the promoter driving the expression of sgR, measured as au of RFP fluorescence and Rκ is rescaled to be in the same units. This assumes that the binding of sgR to dCas9 to form RS is in the linear (unsaturated) regime. Equation P1 and sgRP2 induction curves ‐inducible promoter and sgDP5 from a Van‐inducible promoter  binding to the promoter to form DP,DK is the association constant between DS and P. The production of mRNA transcripts m from the promoter is described bymβ/Rα and mβ/Dα are the maximum transcription rates when either RS or DS are bound to the promoter. Solving for steady‐state yieldsOur model was then expanded to include derepression. Equation C2,As described for Equation P2  constant. The fit is shown as the heatmap coloration in Fig Dκ for sgD is similar to Rκ indicating that sgRP2 and sgDP5 have similar apparent binding strengths. Additionally, the observed αD of 1 is consistent with sgDP5 having little repressive effect on its own.This equation was then fit to the 2‐dimensional response of the promoter to the expression of sgR and sgD Fig . This fiet al . Starting with uninduced cells in exponential phase, we diluted cells into repressing conditions . Consistent with previous work, RFP fluorescence reduced at the rate of cellular division . This led to 47‐fold derepression within 2 h.We monitored the dynamics of repression and derepression for the promoter repression/derepression sgRNA pair sgRsion Fig . After 6et al, et al, et al, dCas9 can block transcription when it is directed to bind internally to a gene by physically interfering with the progression of RNAP Fig . Howeveret al, et al, O1) . sgRO1 was placed under the control of a Chol‐inducible PBetI promoter. It has been observed that elongation repression can be weaker with stronger promoters, presumably due to dislodgement of dCas9 by RNAP  to drive the transcription of the rfp reporter. When the promoter is strong (1 mM IPTG), we observed an 8‐fold repression of RFP upon the maximum induction of sgRO1 (10 mM Chol)  Fig . This lohol) Fig . While w Fig . Thured Fig . Represssion Fig . PreviouO2) and down‐stream (sgDO1) of the sgRO1 position  with the strongest induction of the rfp reporter (1 mM IPTG). Maximum expression of sgDO2 (100 µM Van) showed 4‐fold derepression . For each level of PTac induction, the relative expression during derepression was calculated as the ratio of the RFP fluorescence from the PTac‐only plasmid to the RFP fluorescence from the circuit plasmid with maximal sgRO1 and sgDO2 induction (10 mM Chol and 100 µM Van). From this experiment, we observed that derepression is invariant over a wider range of transcription rates than that observed for repression.The dependence of derepression on the PIPTG Fig . Similaret al, et al, et al, Van / PBetI). From this, we observed that the promoter repression/derepression circuit effectively responded to over 2 orders of magnitude of sgD/sgR ratios with a 50‐fold dynamic range  overrides the regulation imposed by sgR (A) Figs  and 3G. P2 and sgDP5) is capable of responding to transcriptional input ratios over two orders of magnitude  calculation, which if connected to a cooperative switch‐like output could act as a single neuron in a neural network  or other regulatory motifs  was used for all routine cloning. All genetic circuit measurements were done using E. coli K‐12 MG1655 * [F‐ λ‐ ilvG‐ rfb‐50 rph‐1 Δ(araCBAD) Δ(LacI)]  with the GC content set to 50%. Non‐linear fitting was completed with the Python scipy.optimize.curve_fit function.The random 100‐bp sequence within PAll growth was performed in 96‐well V‐bottom plates  at 1,000 rpm in a microplate shaker . The day before, individual colonies were inoculated into 150 µl EZ Rich media and Kan for overnight (16 h) growth at 37°C. The next day, cultures were diluted 200‐fold by adding 0.75 µl of overnight culture into 150 µl of EZ Rich media and Kan. After 2‐h growth, cultures were diluted 1,000‐fold into inducing conditions by adding 6 µL of culture into 198 µl fresh media and Kan and then 5 µl of that dilution into media with inducers and Kan. To induce dCas9, aTc was added to a final concentration of 1.25 nM in all experiments. Growth was performed for 5.5 h, after which samples were prepared for flow cytometry.https://github.com/cytoflow/cytoflow) was used for analysis, including gating. The geometric mean fluorescence is calculated for all cytometry distributions.Fluorescence characterization was performed using a BD LSR Fortessa flow cytometer with the HTS attachment . Samples were prepared by aliquoting 40 μl of cell culture into 160 μl of PBS containing 200 µg/ml Kan. All samples were run in standard mode at a flow rate of 2 μl/s. RFP fluorescence measurements were made using the green (561 nm) laser, and all data were derived from the PE‐Texas Red‐A channel (PMT voltage of 700 V). The FSC and SSC voltages were 650 and 270 V, respectively. At least 10,000 events were collected for each sample, and the Cytoflow Python package (Tac) reported in RFP fluorescence (au). This approach has been described previously  to the activities of the inducible promoter , and then resuspended in 300 µl of PBS and 2 mg/ml Kan and analyzed by cytometry. To transfer into de‐repressing conditions, 28 ml of culture was washed twice by centrifugation at 12°C at 4,300 g for 5 min. Then, the cultures were diluted to OD600 = 0.035 in 40 ml of media with 100 µM Van, 10 mM Choline, and 1.25 nM aTc.All growth was performed at 37°C and 350 rpm in an Innova44  with a 1‐inch throw. The day before the experiment, a starter culture was initiated by inoculating a colony into 2 ml EZ‐rich media with Kan in a 15‐ml test tube. The next day, this culture was diluted to an ODDAA and CAV conceived the study and designed the experiments. DAA performed the experiments and analyzed the data. DAA and CAV wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.


Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves.The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC  TFs (AcNAC1–4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis.A detailed molecular study in kiwifruit (AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.Our results indicate that in kiwifruit the NAC TFs AcNAC2–4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of The online version contains supplementary material available at 10.1186/s12870-021-03154-8. The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by stress signals such as flooding, wounding, drought, and pathogen attack –3, as weS-adenosylmethionine (SAM) by SAM-synthase. SAM is converted to 1-aminocyclopropane carboxylic acid (ACC) and 5′-methylthioadenosine (MTA) by the enzyme ACC synthase (ACS) . For the binding assay, ~ 2.5 μg of recombinant NAC protein was mixed with 0.9 pmol of double-stranded 3′-biotinylated DNA probe  for > 30 min and incubated with 1:2000 Streptactin-HRP  for 1 h in blocking buffer and washed according manufacturer’s instructions . Imaging was conducted with ECL Select substrate  using a ChemiDoc MP imager (Bio-Rad).The DNA binding domain  was blunt cloned in frame behind the Maltose Binding Protein purification tag (MBP) in the vector pMAL-c2x  using the Additional file 1: Table S1 qRT-PCR results for ACO, ACS and NAC TF genes in A. chinensis ‘Hort16A’ fruit and leaves. Table S2 Putative NAC transcription factors identified in the A. chinensis ‘Red5’ genome. Table S3 Primer sequences for qRT-PCR, EMSA and cloning. Table S4 Reference gene validation metrics using GeNorm and BestKeeper. Fig. S1 Amino acid alignment of the C-terminus of ACS proteins from Arabidopsis, tomato and kiwifruit. Fig. S2 Electrophoretic mobility shift assays (EMSA) of AcACS1 promoter fragments (27 bp) with recombinant NAC1–4 proteins. Bottom blot: Original image of Fig. Fig. S3 Potential transcription factor binding sites in the AcACS1, AcACS2 and AcNAC1–4 promoters


While it is already difficult to identify cis-REs in the epigenetically marked sites, interrogating cis-REs residing within the unmarked sites is even more challenging. Here, we report adapting Reel-seq, an in vitro high-throughput (HTP) technique, to fine-map cis-REs at high resolution over a large region of the human genome in a systematic and continuous manner. Using Reel-seq, as a proof-of-principle, we identified 408 candidate cis-REs by mapping a 58 kb core region on the aging-related CDKN2A/B locus that harbors INK4ap16. By coupling Reel-seq with FREP-MS, a proteomics analysis technique, we characterized two cis-REs, one in an epigenetically marked site and the other in an epigenetically unmarked site. These elements are shown to regulate the INK4ap16 expression over an ∼100 kb distance by recruiting the poly(A) binding protein PABPC1 and the transcription factor FOXC2. Downregulation of either PABPC1 or FOXC2 in human endothelial cells (ECs) can induce the INK4ap16-dependent cellular senescence. Thus, we confirmed the utility of Reel-seq and FREP-MS analyses for the systematic identification of cis-REs at high resolution over a large region of the human genome.Growing evidence suggests that functional  In addition, transcription can also be regulated on the epigenetic level with heritable alterations that are not due to changes in the DNA sequence of the underlying cis-REs. Rather, epigenetic modifications, such as DNase hypersensitive sites, DNA methylation and histone modification, alter DNA accessibility by opening chromatin structure, thereby regulating gene transcription by making the underlying cis-REs accessible to regulatory proteins  locus (ANRIL)  . In partclerosis . This cocis-REs over a large region of the human genome at high resolution in a cell type-specific fashion. We used INK4ap16 as a model system to evaluate this approach. By using Reel-seq, we screened the aging-related CDKN2A/B locus using the nuclear extract (NE) isolated from human primary endothelial cells (ECs) and identified 408 candidate cis-REs within the 58 kb core region. As a proof-of-principle, we performed an in-depth analysis with FREP-MS, a proteomics analysis technique  were purchased from Lonza. ECs were free of mycoplasma and cultured in basal medium EGM-2 (Lonza) supplemented with 10% fetal bovine serum. All cells were cultured at 37°C in 5% COAll primers used in this study were purchased from IDT and are listed in cis-REs, two Reel-seq libraries were created with the construct sequence shown in To identify Epigenetically marked sites are defined as regions that contain any of the following markers: H3K27ac, H3K4me1, H3K4me3, known transcription factor (TF)-binding sites, DNase I hypersensitivity sites, predicted DNase I hotspots or enhancers as predicted from chromatin modifications to defined DNA elements based on UCSC Genome Browser as well as the ENCODE database. We designated elements that do not contain any of these marks as an unmarked site.cis-RE with two negative controls and two samples. In brief, ∼10 μg of FREP construct DNA . After incubating the DNA with NE isolated from human ECs at RT for 30 min, the DNA-protein complex was resolved on a 6% TBE native gel for mobility shifting. The data represent three independent biological replicates (n = 3).EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. For the probe, a 35 bp DNA fragment containing a Relilla luciferase (Promega). The firefly luciferase reporter activity was normalizing to the Relilla luciferase activity using the Dual-Glo® Luciferase Reporter Assay System (Promega). All experiments were performed according to the manufacturer's protocol. The data represent six independent biological replicates (n = 6).Luciferase reporter assay was performed in 293T cells using the reporter vector, pGL3-Promoter vector . The 35 bp S1606 and S961 fragments shown in CRISPR/Cas9 gene editing was performed using the LentiCRISPR v2 vector system  with the gRNA sequences listed in n = 3).ChIP was performed as described previously . Brieflyn = 3).DNA-pulldown western blot was performed as previously described . In brie® III Reverse Transcriptase (Invitrogen) according to manufacturers’ protocol. For qPCR, 50 ng cDNA was mixed with 0.5 μmol/l of each primer pair in a 20 μl total reaction using the Power SYBR Green PCR Master Mix (Applied Biosystems) or using the TaqMan Universal PCR Master Mix (Applied Biosystems). PCR amplification was carried out using an initial incubation at 94°C for 10 min, followed by 40 cycles of amplification step , with the StepOne real-time PCR system. Data were analyzed using the ΔΔCT method with GAPDH as an endogenous reference. All the primers used are listed in ARFp14 (Cat#: Hs99999189_m1); INK4bp15 (Cat#: Hs00793225_m1); INK4ap16 (Cat#: Hs02902543_mH); ANRIL (Cat#: Hs04259472_m1) and GAPDH (Cat#: Hs02786624_g1). Data represent the combination of three independent samples (n = 3).Total RNA was isolated with the RNeasy Mini kit (Qiagen). 1 μg of RNA was digested with 1 U DNase I (Invitrogen) at 37°C for 30 min, followed by 65°C for 10 min after adding 1 μl 50 mM EDTA. cDNA was synthesized with SuperScriptn = 3).Whole cell lysates were prepared using RIPA buffer (Sigma). Cytosolic proteins and nuclear proteins were isolated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer's instructions. Proteins were resolved on SDS-PAGE gels and transferred to PVDF membranes. Proteins were detected with gene-specific antibodies. All antibodies were purchased and used as listed in n = 3).The SA-β-Galactosidase Staining Kit  was used to stain senescent ECs. The staining was visualized using an RVL-100-G microscope . Images were analyzed using ImageJ software . The data represent three independent biological replicates (n = 3).Cells were plated on glass coverslips and fixed in 4% paraformaldehyde. For γ-H2AX staining, cell membranes were solubilized in PBS containing 5% FBS and 0.5% Triton X-100. Cells were first incubated with γ-H2AX antibodies in the solubilizing buffer for 1 h and immunofluorescence was detected with Alexa Fluor 488-conjugated secondary antibody. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) . The staining was visualized using an RVL-100-G microscope . Images were analyzed using ImageJ software . The data represent three independent biological replicates . The targeted sequences are listed in For siRNA transient knockdown in human ECs, siRNAs for human FOXC2 and PABPC1 were purchased from Thermo Fisher (Cat #: 4427037 with ID: s194415 for FOXC2 and ID: s25664 for PABPC1) and knockdown was performed according to the manufacturer's protocol. For cis-REs with the local epigenetic states, we investigated the overlaps of candidate cis-REs and epigenetic annotations reported in the Segway encyclopedia . If a candidate cis-RE overlapped multiple Segway encyclopedia elements, we reported the average CAASs of all overlapping encyclopedia elements.To assess the potential association of candidate clopedia . Since tcis-REs on the 58 kb core region at the CDKN2A/B locus.The UCSC genome browser  was usedThe Layered H3K4Me1 and Layered H3K27Ac tracks show where modification of histone proteins is. We used the tracks of H3K4me1 and H3K27ac markers for all cell types listed in ENCODE.We used the track of DNase I Hypersensitivity on all cell types listed in ENCODE.This track set contains regulatory elements, gene transcription starting sites, interactions between regulatory elements and genes, and clustered interactions.cis-regulatory elements in the human genome.This track displays the ENCODE Registry of candidate This track displays literature-curated regulatory regions, transcription factor binding sites, and regulatory polymorphisms.P-values were calculated using Student's t test with two tails.For normally distributed data, all data were represented as standard error of mean (SEM). cis-REs at high resolution over a large region of the human genome. For this, we re-designed the synthetic DNA library as shown in Figure cis-REs within a large region of the human genome in an HTP fashion.Previously, we introduced Reel-seq to identify functional SNPs (fSNPs) that are associated with diseases based on GWAS analysis in a high-throughput (HTP) fashion . Reel-secis-REs at high resolution in an HTP manner, we chose to screen the 58 kb core region on the CDKN2A/B locus. A genomic view of the CDKN2A/B locus is diagramed in Figure ARFp14, INK4bp15, INK4ap16 and ANRIL located at this locus and the relative position of this 58 kb region located within the 3′ end of ANRIL. We are particularly interested in this 58 kb core region because GWAS have identified a strong association of this 58 kb region with different types of cardiovascular diseases including coronary artery disease (CAD), and all these diseases are recognized as age-related diseases since their incidence dramatically increases as a function of age . Using this method, we identified a total of 1439 fragments in Library 1 and 1313 fragments in Library 2. Next, we calculated the P-value using a Student's t test on those fragments with a Slope <0 in round 1, 4, 7 and 10. Using this approach, we identified a total of 209 fragments in Library 1 and 199 fragments in Library 2 with a P-value < 0.05 in round 1, 4, 7 and 10 and a slope <0. We classified these 408 DNA fragments as candidate cis-REs. Among these 408 DNA fragments, there are 88 fragments that are overlapped between the two libraries. Based on a Pearson's Chi-squared test, this observed number of overlaps was statistically significant , and the expected number of overlapping fragments was 25. The detailed distribution of these 408 candidate cis-REs in the two libraries is present in the content of the 58 kb genomic region as shown in Figure To identify in sillico analysis on the 58 kb core region at the CDKN2A/B locus using ENCODE database with all the cell types, including human endothelial cells, and conditions. In total, we identified 46 candidate regulatory regions including 45 predicted enhancers and one predicted promoter region and the average length of these regulatory regions is 271 bp . The position of these 10 candidate cis-REs is presented in cis-REs showed clear and unique EMSA bands    Figure , left, a) Figure , right. ) Figure . For S96) Figure . To deted Figure . In contL Figure , upper. cis-REs are fundamentally involved in gene expression regulation, their functionality relies on recruiting specific regulatory proteins including transcription factors to modulate gene expression. In order to determine the regulatory protein(s) that specifically bind to the cis-REs on S1606 and S961, we applied FREP-MS, a proteomics tool recently developed in our lab . None of these proteins were previously characterized as transcriptional regulators. To demonstrate that these four proteins are also regulators of cellular senescence via modulating the 6 Figure . While a6 Figure , similar6 Figure . Next, wn Figure . Howeverse cells . Except l Figure  and γ-H2l Figure  staining1 Figure .cis-REs are located in the non-coding regions of the human genome. Their identification is challenging, especially if they reside within epigenetically unmarked sites. One strategy to identify these elements is to develop a technique that can fine-map these cis-REs base-by-base, tiling a large region of the human genome in a systematic and continuous manner. However, considering the size of the human genome, such techniques have to be extremely simple in design and easy to perform. We believe that Reel-seq provides such a straightforward technique. We also believe that by coupling Reel-seq with FREP-MS, we will now be able to decipher the mechanisms of gene transcription regulation at a resolution that was not previously possible.cis-REs regulating the INK4ap16 expression. Two such identified cis-REs were further characterized to regulate INK4ap16-dependent cellular senescence by recruiting PABPC1 and FOXC2. FOXC2 is a transcription factor that belongs to the forkhead box family and participates in embryonic development  based on our knowledge that a typical transcription factor occupies 6–12 nucleotides  in terms of their impact on regulating gene expression. Nonetheless, the strategy outlined here presents a simple, scalable approach to identify cis-REs contained within large genome regions of epigenetically marked or unmarked sites.We also recognize the disadvantage of using Reel-seq and FREP-MS. Unlike most  as MPRA ,16 and Mleotides . TherefoAll data are available in the supplementary files.gkab890_Supplemental_FilesClick here for additional data file.