Sessile plants possess an assembly of signaling pathways that perceive and transmit environmental signals, ultimately resulting in transcriptional reprogramming. Histone is a key feature of chromatin structure. Numerous histone-modifying proteins act under different environmental stress conditions to help modulate gene expression. DNA methylation and histone modification are crucial for genome reprogramming for tissue-specific gene expression and global gene silencing. Different classes of chromatin remodelers including SWI/SNF, ISWI, INO80, and CHD are reported to act upon chromatin in different organisms, under diverse stresses, to convert chromatin from a transcriptionally inactive to a transcriptionally active state. The architecture of chromatin at a given promoter is crucial for determining the transcriptional readout. Further, the connection between somatic memory and chromatin modifications may suggest a mechanistic basis for a stress memory. Studies have suggested that there is a functional connection between changes in nuclear organization and stress conditions. In this review, we discuss the role of chromatin architecture in different stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory. Plants utilize highly evolved mechanisms to improve their growth and development to face various biotic and abiotic stresses, in part due to their sessile nature. The plasticity of plants allows them to adapt and survive through these environmental challenges . ChromatIn all eukaryotes, chromatin is packed into nucleosomes; the histone family of proteins makes up a large portion of the chromatin protein component. A nucleosome is a repetitive unit composed of 147 bp of DNA coiled in 1.67 left-handed turns around a histone octamer comprised of pairs of H2A, H2B, H3, and H4 histones . HistoneWhen highly condensed, the chromatin architecture prevents access by transcription factors, polymerases, and other nuclear proteins to DNA. Some modifications due to stress signals take place in the chromatin structure, which enables DNA to become accessible. These chromatin remodeling includes shifting or removing histones, introducing histone variants, or posttranslationally modifying existing histones .via ATP hydrolysis, and the other utilizes specialized enzymes that methylate DNA or modify histone residues through the addition of covalent modifications (There are two different strategies among many processes involving two different enzymatic mechanisms to accomplish chromatin organization: One operates through chromatin remodelers that change DNA-histone interactions ications .The SWITCHING DEFECTIVE2/SUCROSE NON-FERMENTING2 (SWI2/SNF2) family of chromatin remodeling complexes (CRCs), part of a large superfamily of helicases and translocases, use the energy obtained from ATP hydrolysis to gain access to DNA sequences . The SWISaccharomyces cerevisiae  and At3g06010 (CHR12), and two affirmed proteins, At2g28290 (SPLAYED or SYD) and At2g46020 (BRM), as the nearest homologs of yeast and human SWI/SNF ATPase subunits. BRM and SYD (2193 and 3574 amino acids) represent huge proteins, while CHR12 and CHR23 (1132 and 1054 amino acids) are altogether more modest. AT-hook motifs are present in the C-terminal regions of BRM and SYD, whereas there is no such distinctive C-terminal domain in the CHR12 and CHR23. Decrease in DNA methylation 1 (DDM1) encodes a SWI2/SNF2-like protein, showing that chromatin remodeling is a crucial process for maintenance of DNA methylation ; small losses can also be seen in genes , and successive DNA transposition have additionally been noticed and may represent a considerable amount of phenotypic variability , SANT , and Leucine Zipper space with yeast SWI3 and its orthologs in mouse (Srg3), Drosophila (Moira), and human (BAF170 and BAF155).The SWI/SNF subfamily remodelers comprise 8–14 subunits initially purified from revisiae . A C-terhylation . In Arabomic DNA . DDM1 isomic DNA . In Arabin genes . Arabidohromatin . A genomiability . There aArabidopsis, only BSH (At3g17590) shows significant similarity to SNF5 (in yeast), which plays a key role in the organization and functioning of SWI1/SNF1 complexes. The Arabidopsis genome encodes two exceptionally comparable homologs of yeast SWP73: At3g01890 (named AtSWP73A) and At5g14170 (named AtSWP73B), which show 83.7% arrangement personality to one another. SWP73 has a functional role in transcriptional activation. The SWI2/SNF2-type ATPase domain belongs to the helicase and NTP-driven nucleic acid translocase superfamily 2 (SF2). This SF2 facilitates interaction with different targeting domains and functional modules, which activates remodeling activities in chromatin structure and thus helps in transcription regulation and DNA repair  subfamily remodelers comprise of two to four subunits initially purified from d motifs . In mostXenopus laevis. They vary in their structure due to the diversity in their chromodomains. They can act as transcriptional activators or repressors depending on CHD (The chromodomain helicase DNA-binding (CHD) subfamily remodelers comprise of 1–10 subunits first purified from g on CHD . In loweS. cerevisiae is characterized by the presence of a split ATPase subunit with a long insertion found in the middle of the ATPase domain, which binds with the helicase-related (AAA-ATPase) Rvb1/2 proteins and one ARP protein. It is involved in transcription activation and DNA-double-strand break (DSB) repair  necessary for the binding Arps and actin components is also present in the motor subunits of INO80 protein. The involvement of IN080 complexes in DNA repair is suggested by the presence of RuvB-like helicases.The inositol requiring 80 (INO80) subfamily initially purified from ) repair . Higher Arabidopsis, during stress, RESTRICTED TO NUCLEOLUS 1 (REN1) was found to be incorporated with nucleoli and helps in pollen development  CRCs and (ii) chromatin-modifying enzymes, to overcome various biotic and abiotic stresses . In Arabelopment . STRESS elopment , whereaselopment .Arabidopsis, HISTONE DEACETYLASE 6 (HDA6) is upregulated by cold stress and positively regulates freezing tolerance  and HDACs catalyzing histone acetylation and deacetylation show a role in cold responses . In Arabolerance . HDACs aolerance . Accordiolerance .Arabidopsis, salinity tolerance is determined by expression levels of DEK3 (a DEK domain-containing protein), which acts in association with DNA topoisomerase , there was no change observed in a large portion of chromatin. Chromatin accessibility was increased in case of extreme temperatures, while the result for chromatin accessibility did not change much in case of drought and salt stresses  subfamily is highly conserved and comprises widespread domain architectures, which enables it to link with epigenetic regulation and signaling-dependent chromatin remodeling , show de-repression of DNA-methylated genes and TEs. Enhanced interaction of pericentromeric regions and the genome, decondensation of pericentromeric heterochromatin, and transcriptional defects that are mainly focused on loci residing in pericentromeric regions are noticed in atmorc1 and atmorc6 mutants. In eukaryotes, MORC ATPases are proposed to be the conserved regulators of gene silencing , while SlMORC1 or NbMORC1 expression represses it , identified as a hereditary screen to recognize components associated with the TCV resistance signaling pathway  is represented by the resistance protein HRT (HR to TCV) and its related avirulence factor, the viral coat protein. Plants not having HRT fail to build up an HR after TCV infection permits systemic viral spread and results in the death of the plant. CRT1 physically interact with HRT and 10 other R proteins; these R proteins are mainly inactive. CRT1 possesses two close and four distant homologs; silencing of the two closest homologs, CRH1 (CRT1 homolog 1) and CRH2, compromised TCV resistance to a far extent in comparison to crt1. crt1-1 mutation and silencing of CRT1 family members compromise cell death triggered by the R proteins. Reduced resistance to avirulent Pseudomonas syringae (Pst) and Hyaloperonospora arabidopsidis was observed in double knockout (dKO) in the Col-0 background, crt1-2 crh1-1, which lacks CRT1 and its closest homolog. The knockout of CRT1 gene results in severe susceptibility to both virulent and avirulent H. arabidopsidis. These results show that CRT1 is a very crucial factor in multiple levels of plant immunity (Hsp90), rearranging DNA structure (gyrase or topoisomerases), signal transduction (histidine kinase), or DNA mismatch repair  localized in the nucleus, formed protein complexes, and acted as H2A/H2B chaperones. These protein complexes help in the regulation of chromatin organization in epigenetic inheritance, as they specifically bind to histones H2A and H2B  GHKL ATPases  superfamily. MORC protein was initially isolated from mouse, which is important for meiotic nuclear division . Thereaf elegans , and difbidopsis , tobaccomodeling . The rolbidopsis . MORCs iilencing . The MOResses it . Arabido pathway . Arabidod repair  It has bimmunity . GHKL ATir MutL; . In Arab and H2B . NAP1 isc stress . NRP proc stress  geneticaof H2A.Z .A. thaliana  is involved in biotic stress signaling and resistance towards pathogen (SlyWRKY75 gene is induced in response to biotic stress (Evolutionary conserved SnRK1 kinases (Snf1-RELATED KINASE1) govern metabolic adaptation during low extended darkness by controlling C/S1-bZIP signaling in thaliana . Plants thaliana . SnRK1s bidopsis . Calcine2-4 CAF1 mutant. The maternal participation to the phenotype severity is more than the paternal contribution when early and late generation fas2-4 plants were crossed. It shows that the preferred maternal transmission uncovers a more prominent reprogramming of epigenetic data in the male in comparison to female germline. Epigenetic mechanisms underlie the progressive developmental phenotype aggravation in CAF1 mutants in Arabidopsis  in SAM and SCARECROW (SCR) in RAM  in RAM . This shertility . FAS5, wactivity .Arabidopsis, MSI1 is having an important function in polycomb repressive complexes (PRC2) due to which msi mutants are lethal to the embryo  on DNA or histones . MethylaArabidopsis and rice that, upon stress, histone variants are also differentially expressed like histone chaperons  originating from TTS by BRM does not depend on the presence of linked sense promoters, which suggests that 3'-bound BRM utilizes antisense promoters to maintain sense expression of those genes. It has been observed that the antisense transcripts arising therefrom and the TTS regions of genes have been implicated in environmental signals sensing in many systems, including cold sensing by the FLC 3' region and sulfur sensing by the 3' untranslated region (UTR) of SULTR2; 1 in plants, or yeast, the requirement for the 3' region of KCS1 for phosphate sensing. This suggests that a large fraction of the 3' SWI/SNF targets are stress-related genes  of protein-coding genes. ed genes .In vitro somatic embryogenesis induced in response to external signals is an example of plant developmental plasticity developed by the chromatin-regulating molecular machinery  and ASCORBATE PEROXIDASE 2. The expression pattern of these genes found to be strong in the case of inducible HS when comparing with nonmemory genes (like HSP70 and HSP101). HSFA2 was reported to be the most strongly heat-induced HSF, as it is required specifically for HS memory. The first reported HS memory-associated gene, specifically involved in HS memory is HSA32. It was studied that HSA32 is required for HSP101 protein stability, which suggests a similar role to chaperons. ROF1, which is the peptidyl-prolyl-isomerase and member of the FK506-binding protein family, is also seen to be specifically required for HS memory by directly interacting with HSP90.1, which further interacts with HSFA2  is silenced during vernalization by the polycomb repressive complex 2 (PRC2), which accumulates H3K27me3 at target loci  and REPRESSOR OF SILENCING 1  pathway, which uses them as guides and target the DNA methylation machinery to homologous sequences  or the theory of “inheritance of acquired characteristics.” According to Lamarck, alterations in phenotypic traits are a result of the environment and are associated with evolution. Lamarckism says that simple organisms tend to evolve into more complex ones by an adaptive force. The environment creates needs to which organisms respond by utilizing features, which are then emphasized or weakened through use and disuse; this generates characteristics that an individual organism acquires and then are pass on to its offspring. Plants have elaborate mechanisms to deal with different environmental conditions. When the memory effect is present only in the first stress-free generation, it is called intergenerational memory, while if the memory is traceable in a minimum of two stress-free generations, it is termed transgenerational memory . Transgebidopsis . Accordibidopsis . The mat mutants . DME enc mutants . The rol 1 ROS1; . DME dem 1 ROS1; . It has  1 ROS1; . Due to  1 ROS1; . These sequences . It is hequences . As imprequences . The rolequences . DNA metequences . These vSchizosaccharomyces pombe, a role for RNAi in TGS was observed where it is necessary for the formation of constitutive heterochromatin at pericentromeric. The release of the passenger strand from Ago1 and dsRNA requires catalytic activity and thus is necessary for the pairing of bases between loaded siRNA and their targets. These interactions provide the RNA-induced transcriptional silencing complex (RITSC) a critical place, integrating transcription and chromatin modification, which creates a positive loop between siRNA generation, RITSC localization, and H3K9 methylation. In S. pombe, the coupling of transcription, production of siRNA, and silencing indicates that TGS occurs in cis; however, in plants, it has been seen that it can also occur in trans. In A. thaliana microRNA (miRNA)-directed siRNA biogenesis is a mechanism that particularly targets transposon transcripts and triggers epigenetic reactivation during reprogramming of the germ line  following mitosis and meiosis to ensure epigenetic inheritance . Double-erm line . In Arabh siRNAs . AnalysiA. thaliana, flowering is initiated when H3K27me3 established at the floral repressor FLOWERING LOCUS C (FLC), which is a result of H3.1 deposition during DNA replication. At FLC, H3K27me3-mediated silencing finally reset in the future generation to ensure transcriptional reactivation in the early embryo  histone demethylases counteracts the activity of PRC2. There are three closely related JMJ H3K27 demethylases reported were EARLY FLOWERING 6 (ELF6), RELATIVE OF ELF6 (REF6), and JUMONJI 13 (JMJ13), and all are expressed in the sperm. As PRC2 is absent in the sperm, the H3K27me3 demethylation by JMJ proteins is supposed to occur globally, whereas in somatic tissues, H3K27 demethylases occupy the border of H3K27me3 domains in presence of PRC2  by interacting with HD2C  genes  and removes acetyl groups of histone H3 and H4 from the cognate regions, thus represses their expression specifically when ABA is present. A reduction in ABA sensitivity is observed in HDA15-deficient mutants, thus are affected by drought stress. Various transcription factors from bZIP, MYC, NAC, and MYB families then get activated and initiate downstream ABA responses. HDA15 represses ROP genes and acts as a positive regulator of ABA signaling by repressing . HDA15 activity also affects the expression of some ABA-responsive genes. The expression of PKL (SWI/SNF type chromatin remodeling factor) is induced by seed imbibition in Arabidopsis, and it mediates the repression of embryonic traits during germination. Seed germination is mediated by induction of ABI3 and ABI5 transcription factors expression in response to induced expression of PKL. This discussion suggests that there is a change in expression or activity of HDACs due to ABA accumulation, which in turn regulates growth under stress  gene, which remains until progression to flowering. Due to prolonged cold, COOLAIR, which is a set of long noncoding RNA (lncRNA)-transcribed antisense from FLC in A. thaliana, gets induced, which is a characteristic of polycomb silencing. As discussed earlier in the review, the polycomb group (PcG) proteins are responsible for gene silencing in higher eukaryotes. PcG regulates many genes and several developmental processes. It has been found during cold conditions that the expression of FLC gets reduced when COOLAIR gets associated with the FLC locus. The synchronized replacement of H3K36 methylation with H3K27me3 gets disturbed at the FLC nucleation site when COOLAIR is removed during cold stress -mediated H3K27 deacetylation is necessary . PsSNF5 interacts with Arabidopsis SWI3-like proteins (SWI3A and SWI3B), which further interacts with FCA  experiments and more recent Hi-C analysis, revealing their role in the spatial organization of chromosomes  was discovered for heat-stress-intolerant mutants; in excessive heat stress, it is required for chromocenter decondensation upon heat stress . A study conducted in yeast shows that transcriptional repression of ∼540 genes occurs at a faster rate if, during carbon source shifts, the genes have been previously repressed .Various biochemical changes take place in chromatin structure to maintain gene activity: Some of these modifications have the capacity to be stably transmitted through cell division stages, which suggest that modifications in the chromatin state could help in coping with different biotic and abiotic stresses . FurtherWhat are the kinetics of changes in histone modifications and transcripts following the stress signal perception?Which transcription factor interact with which coactivator or corepressors under a given stress situation and cell type?What is the role of cell type in determining transcriptional regulation through its chromatin status?What is the exact composition of native chromatin modifying complexes in different tissues, developmental stages, and stress situations?Can we design epigenetic switches to regulate agronomically important traits under stress conditions?Can we exploit the strength of epigenome modification in horticultural crops since their breeding is difficult? Can grafting change methylation and acetylation state in horticultural crops?SB and SM: writing and reviewing. MB and GL: reviewing and editing. RM: conceptualizing, reviewing, writing, and editing. All authors contributed to the article and approved the submitted version.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.


GDNF is a dual promoter gene, and the promoter II with two enhancers and two silencers plays a major role in transcription initiation. We had previously reported that histone hyperacetylation and DNA hypermethylation in GDNF promoter II region result in high transcription of GDNF in GBM cells, but the mechanism remains unclear. In this study, we investigated whether these modifications synergistically regulate high GDNF transcription in GBM.Glial cell line-derived neurotrophic factor (GDNF) is highly expressed in glioblastoma (GBM) and blocking its expression can inhibit the initiation and development of GBM. GDNF transcription and promoter II activity. CREB regulated GDNF transcription via the cyclic AMP response elements (CREs) in enhancer II and silencer II of GDNF promoter II. However, the two CREs played opposite regulatory roles. Interestingly, hypermethylation of CRE in silencer II occurred in GBM tissues and cells which led to decreased and increased phosphorylated CREB (pCREB) binding to silencer II and enhancer II, respectively. Moreover, pCREB recruited CREB binding protein (CBP) with histone acetylase activity to the CRE of GDNF enhancer II, thereby increasing histone H3 acetylation and RNA polymerase II recruitment there and at the transcription start site (TSS), and promoted GDNF high transcription in U251 cells. The results indicated that high GDNF transcription was attributable to DNA hypermethylation in CRE of GDNF silencer II increasing pCREB binding to CRE in enhancer II, which enhanced CBP recruitment, histone H3 acetylation, and RNA polymerase II recruitment there and at the TSS.Cyclic AMP response element binding protein (CREB) expression and phosphorylation at S133 were significantly increased in human GBM tissues and GBM cell lines (U251 and U343). In U251 GBM cells, high expressed CREB significantly enhanced GDNF promoter II enhanced GDNF high transcription, providing a new perspective for GBM treatment.Our results demonstrate that pCREB-induced crosstalk between DNA methylation and histone acetylation at the  GDNF gene was initially cloned from the rat B49 glial cell line [GDNF is a single-copy gene with two promoters. Promoter I is located upstream of exon IV. Promoter II is located upstream of exon I and contains two enhancers, two silencers, and various transcription factor binding sites; it plays a major role in transcription initiation [GDNF expression in primary gliomas and multiple GBM cell lines, which was positively correlated with pathological grade [GDNF or its binding receptor, GFRα1, effectively inhibits GBM progression [GDNF expression in GBM cells is mainly caused by its high transcription rather than gene mutations [GDNF promoter II exist in GBM tissue and cells, both of which are involved in regulating high GDNF transcription [Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor (TGF-β) superfamily. The ell line . In humaitiation , 3. Numeitiation , particiitiation –9. GDNF al grade . Subsequal grade , 12. Knogression , 14. Higgression . In addigression . Abnormautations . Moreovecription , 19. HowGDNF expression was synergistically regulated by DNA methylation and various histone modifications in different brain regions of mice [C/EBPα) in bone marrow stromal cells as a cofactor for DNA methylation and histone acetylation in the C/EBPα promoter [GDNF promoter II [GDNF transcription through the abnormal binding of transcription factors. The cyclic AMP response element binding protein (CREB) is a methylation-sensitive nuclear transcription factor involved in tumor cell immortalization and transformation. It binds to the cyclic AMP response element (CRE) in the promoter as a homodimer or heterodimer and activates gene expression by binding to the KID-interacting domain (KIX) in the CREB binding protein (CBP) via its own kinase-inducible domain (KID) [GDNF promoter II in C6 GBM cells was significantly reduced by a CBP inhibitor, curcumin [GDNF promoter II, and abnormal methylation of promoter II affects the binding of CREB to promoter II in GBM tissue [GDNF expression in C6 cells [GDNF promoter II. The abnormal DNA methylation in GDNF promoter II, especially hypermethylation in silencer II, results in selective CREB binding to CRE in enhancer II and increases the acetylation of histone H3 and RNA Pol II recruitment in GDNF promoter II by recruiting CBP, thus increasing GDNF transcription.Uchida et al. recently reported that  of mice . Zhao etpromoter . In addimoter II , 22. Thuin (KID) , 24. Duein (KID) –27, direin (KID) . Our precurcumin , 22, 29,M tissue . A recenM tissue . MoreoveC6 cells . We hypoGDNF transcription by the CRE in the two cis-acting elements of the GDNF promoter II and the methylation status and CREB binding of the two CREs were assessed by site-directed mutagenesis, CRISPR/Cas9, bisulfite sequencing PCR (BSP), and chromatin immunoprecipitation (ChIP)-PCR; effects of S133 phosphorylated CREB (pCREB) on GDNF transcription and recruitment of CBP were determined by point mutation, co-immunoprecipitation (co-IP), and re-ChIP-PCR; effects of CBP on histone H3 acetylation and RNA polymerase II recruitment in the CRE in enhancer II and the TSS of GDNF were examined by RNAi and ChIP-PCR. Our results reveal the molecular mechanisms of synergistic upregulation of GDNF by DNA methylation and histone acetylation in GBM cells, providing a new target for GBM treatment.To test the above hypothesis, expression of CREB and the effect of CREB on GDNF expression were measured by real-time polymerase chain reaction (PCR), RNA interference (RNAi), and a dual-luciferase reporter system; regulation of GDNF promoter II, and the binding sites located at − 984/− 977 nt and − 311/− 304 nt, respectively  database, which revealed significantly higher CREB mRNA expression in GBM tissue than in normal brain (NB) tissue (P < 0.01)  tissues were determined by real-time PCR and western blot, respectively. The results showed that CREB mRNA and protein expression were very significantly higher (P < 0.01) in GBM tissue, whereas the increase was not significant (P > 0.05) in low-grade glioma tissue compared with NB tissue   and protein overexpression (OE) of CREB were performed in U251 GBM cells. CREB mRNA expression was very significantly decreased after lentivirus CREB-KDs infection. The lowest expression was observed in the CREB-KD1 group, which was 61% lower than that of the NC group (P < 0.01)   (GDNF mRNA and protein expression in U251 cells were very significantly decreased by CREB knockdown (P < 0.01)  (GDNF transcription in GBM cells.To investigate the relationship between highly expressed CREB and  < 0.01) . On this < 0.01) , suggestGDNF transcription by affecting promoter II activity, the effect of CREB knockdown and protein overexpression on GDNF promoter II activity in U251 cells was analyzed using a dual-luciferase reporter system. GDNF promoter II activity was significantly decreased by CREB knockdown and increased by CREB overexpression (both P < 0.01)  were overexpressed in U251 cells ; whereas KCREB overexpression did not have a significant effect (P > 0.05)  . In contrast, promoter II activity was significantly increased by the deletion or mutation of CRE in silencer II (P < 0.01) and significantly decreased by that in enhancer II (P < 0.01), indicating that both mutation and deletion can block CREB binding to a specific CRE , but it did not change significantly for that containing mutations of both CREs (P > 0.05). Compared with the EGFP-NC group with each type of promoter II, CREB overexpression only significantly increased promoter activity for the wild-type GDNF promoter II and that containing CRE mutation in silencer II only (P < 0.01). Compared with wild-type GDNF promoter II overexpressing CREB, promoter activity with CREB overexpression was significantly decreased for GDNF promoter II containing CRE mutation in enhancer II only (P < 0.01) and increased for that containing CRE mutation in silencer II only (P < 0.01). These findings indicate that the binding of CREB to CREs in the two cis-acting elements play opposing role in the regulation of GDNF transcription, and CRE in enhancer II may play a stronger role  and sgRNA-CRE-S (P < 0.01), respectively . CREB specifically bound to CRE in GDNF enhancer II and silencer II in vitro, and hypermethylation of silencer II reduced CREB binding   , whereas no significant change was observed in enhancer II , whereas no significant change was observed in enhancer II . CREB overexpression significantly increased the activity of the unmethylated promoter II and hypermethylated silencer II (P < 0.01). Moreover, promoter II with hypermethylated silencer II had significantly higher activity than unmethylated promoter II  CREBs  and significantly decreased by CR133 overexpression ; whereas the binding of pCREB to CRE in silencer II was significantly decreased (P < 0.01), and CBP recruitment was decreased but not significantly  is capable of recruiting CBP with histone acetylase activity to promote gene transcription [ is capabGDNF enhancer II by CREB, CBP binding to GDNF enhancer II in U251 cells infected with NC or CREB-KD1 virus was detected by ChIP-PCR. CREB knockdown significantly reduced CBP binding to enhancer II  for 12, 24, or 36 h to inhibit pCREB binding to CBP. GDNF transcription was then measured by real-time PCR. The results showed that GDNF transcription was significantly and dose-dependently decreased by KG-501 treatment compared to the solvent control group (dimethyl sulfoxide)  . Real-time PCR revealed that CBP knockdown significantly decreased GDNF transcription in U251 cells (P < 0.01)  Fig. g, which 01) Fig. h. To clales Fig. i, j. ChIion Fig. k and RNAion Fig. l in the 01) Fig. m.GDNF transcription in GBM cells, we examined expression of the proto-oncogene CREB in different grades of glioma tissues and GBM cell lines. CREB expression was abnormally increased in GBM tissue and cell lines, which is consistent with previously reported results [GDNF transcription in C6 GBM cells [GDNF transcription in GBM cells. Our results confirmed this hypothesis and revealed that GDNF expression and transcription were significantly decreased or increased by knockdown or enhancement of CREB expression, respectively. Moreover, overexpression of mutant KCREB did not significantly alter GDNF transcription, and overexperession of wild-type CREB significantly increased the binding of CREB to CRE in enhancer II of the GDNF promoter II, indicating that CREB regulation of GDNF transcription is dependent on its binding to promoter II.To investigate the mechanism of crosstalk between DNA methylation and histone acetylation in regulating high  results , 34. DruBM cells , suggestGDNF promoter II [GDNF transcription through different CREs, the effect of CRE deletion in different cis-acting elements on GDNF transcription was assessed using a dual-luciferase reporter system and CRISPR/Cas9. GDNF transcription was significantly increased and decreased by CRE deletion in silencer II and enhancer II, respectively. The CRE in enhancer II may play a larger role in regulating GDNF transcription. CREB binding to different CREs in GDNF promoter II was assessed by EMSA and ChIP-PCR, which revealed that CREB bound to CREs in both enhancer II and silencer II. The binding in enhancer II was significantly higher in U251 and U343 cells than in NHA cells, which was associated with CRE hypermethylation in silencer II. Moreover, hypermethylation of silencer II significantly enhanced the effect of CREB overexpression in promoting GDNF transcription. High methylation of silencer II decreases CREB binding to CRE in silencer II and increases that in enhancer II, thereby promoting GDNF transcription. However, why does increased binding of CREB to CRE in enhancer II increase GDNF transcription? Is it related to histone H3 hyperacetylation in GDNF promoter II? These questions need to be addressed in future studies.CREB binds to DNA sequences via the CRE motif . Bioinfomoter II . To clarGDNF transcription. Takebayashi and Hisaoka groups showed that drug-induced phosphorylation of CREB at Ser133 increased GDNF production through extracellular signal-regulated kinase (ERK) signal pathway in rat C6 glioma cells [GDNF transcription in mouse astrocytes in vivo [GDNF transcription in GBM cells. Valeria et al. found that CREB phosphorylation was only significantly increased in astrocytoma; no significant changes were seen in oligodendroglioma [GDNF expression in astrocytoma but no significant change in oligodendroglioma (data not shown). It suggests that pCREB may only be involved in regulating high GDNF transcription in astroglioma cells. Phosphorylated CREB is able to enter the nucleus and recruit coactivators by forming homodimers or heterodimers [GDNF transcription by recruiting CBP to the CRE in GDNF enhancer II. To test this hypothesis, the binding of pCREB and CBP in the nuclei of GBM cells was confirmed by immunofluorescence and co-IP. A significant increase in pCREB-mediated CBP recruitment in the CRE of GDNF enhancer II was detected by re-ChIP-PCR. Furthermore, RNAi knockdown of CREB significantly reduced the binding of CBP to CRE in GDNF enhancer II, indicating that high recruitment of CBP in the CRE of enhancer II is achieved by pCREB. Finally, U251 cells were treated with different concentrations of KG-501, a small-molecule inhibitor that inhibits pCREB-CBP complex formation by directly targeting the KIX domain of CBP, thereby inhibiting CREB-mediated gene transcriptional activation [GDNF transcription. Collectively, the results suggest that pCREB promotes GDNF transcription in GBM cells by increasing CBP recruitment in the CRE of enhancer II.CREB is activated by Ser133 phosphorylation through oncogenic signaling pathways . Our stuma cells –40. In a in vivo . These rroglioma . TCGA darodimers . It has tivation , 44. TreGDNF promoter II in C6 cells, which could be significantly reduced by a CBP inhibitor, curcumin [GDNF promoter II. To test this hypothesis, ChIP-PCR was performed to measure CBP binding and histone H3 acetylation in the CRE of GDNF enhancer II in GBM and NB tissues and in U251 and NHA cells. The results showed that both parameters were significantly increased in GBM tissue and cells. Histone H3 acetylation in the CRE of GDNF enhancer II was significantly decreased in U251 cells by CBP knockdown, indicating that CBP is involved in the hyperacetylation of histone H3 in GDNF enhancer II. Numerous studies have shown that histone H3 hyperacetylation can open the promoter chromatin, thereby facilitating the binding of regulatory factors [GDNF enhancer II, and then promotes the binding of CBP and enhancer II through the two mechanisms indicated above, thereby forming a positive feedback pathway to maintain hyperacetylation of histone H3 in the CRE of enhancer II in GBM cells. Moreover, CBP has been shown to promote the recruitment and release of promoter-proximal RNA polymerase II by increasing histone acetylation in the first nucleosome, thereby promoting the expression of eukaryotic genes [GDNF promoter II in C6 cells promotes the increase in RNA polymerase II recruitment at the TSS [GDNF enhancer II may act on the TSS via a loop structure, thereby increasing histone H3 acetylation and RNA polymerase II recruitment at this site. This hypothesis is confirmed by the findings that CBP binding, histone H3 acetylation, and RNA polymerase II recruitment at the TSS of GDNF were significantly increased in GBM tissue and cells, and that histone H3 acetylation and RNA polymerase II recruitment at this site were significantly decreased by CBP knockdown.CBP recruited by pCREB has histone acetylase activity and can efficiently acetylate histones (H3 and H4) in mammalian cells , 46. We curcumin , 22, 29. factors . In addi factors . CBP pho factors . Thus, iic genes . Our preGDNF transcription in GBM cells as a coupling factor for DNA methylation and histone acetylation. The molecular mechanisms are as follows: (1) CREB is highly expressed in GBM cells and is activated by S133 phosphorylation; (2) hypermethylation of GDNF silencer II, especially in its CRE, inhibits pCREB binding to silencer II, which results in an increase in its selective binding to CRE in enhancer II as a homodimer; (3) CBP has histone acetylase activity, is highly expressed in GBM cells, binds to pCREB, and is recruited to the CRE of GDNF enhancer II; (4) CBP recruited by pCREB significantly increases histone H3 acetylation in the CRE of GDNF enhancer II, which facilitates CBP binding; and CBP also acts on the TSS of GDNF via a DNA loop structure to increase histone H3 acetylation and RNA polymerase II recruitment at this site, thereby increasing GDNF transcription  tissue samples were obtained from 6 patients with acute brain trauma who underwent intracranial decompression, and 12 human glioma tissue samples  were acquired from the affiliated hospitals of Xuzhou University. Biopsied glioma tissues derived from patients who had not yet undergone cancer therapy were randomly sampled. Specimens with pathological grades I-II and IV were assigned to the low- and high-grade glioma  groups, respectively.Human U251 and U343 GBM cell lines were obtained from ATCC between years 2015 and 2018 and cultured as described previously .http://www.oncomine.org) was used to identify changes in gene expression in GBM [The Oncomine cancer microarray database (n in GBM . “Brain −ΔΔCT). The primer sequences for the target genes and internal reference gene are shown in RNA extraction and real-time PCR were performed as previously described . GlyceraRadioimmunoprecipitation assay lysis buffer was used to isolate the total protein. Samples from each group were subjected to western blotting as described previously . Detaile4 cells/well in a 24-well plate . After 24 h, cells were infected by 1 μL concentrated lentivirus in the presence of polybrene (8 μg/mL).Full-length human wild-type CREB ORF (NM_134442.3) and mutant CREB ORF (CREB-133 and K-CREB) fragments were amplified from the CREB dominant-negative vector set  by PCR technology. CREB133 contains a serine-to-alanine mutation corresponding to amino acid 133 that blocks protein phosphorylation. KCREB contains mutations in its DNA-binding domain, preventing it from binding to CRE. Three types of CREB ORF were subcloned into lentiviral vector pLenti6.3-MCS-IRES-EGFP/V5 DEST. Three pairs of miRNA oligos targeting the human CREB mRNA  were synTM assay was performed to check the lentivirus-sgRNA’s cleavage by using a knockout and mutation detection kit  according to the manufacturer’s instructions. Briefly, genomic DNA was extracted from U251 cells with stably transfected Cas9 after 72 h of infection with sgRNA lentiviruses using a QIAamp DNA Mini and Blood Mini Handbook kit . The products were amplified by two pairs of PCR primers , the following sequences were obtained by chemical synthesis with a KpnI restriction site introduced into the 5'-end and a HindIII restriction site into the 3'-end: wild-type GDNF promoter II (− 1300/+ 149) sequence, CRE-WT; mutant or deletion sequences: mtCRE-E or △CRE-E, with CRE mutant or deletion in enhancer II; mtCRE-S or △CRE-S, with CRE mutant or deletion in silencer II; mtCRE-ES or △CRE-ES, with CRE mutant or deletion in both enhancer II and silencer II, respectively. The sequences were digested with KpnI and HindIII and ligated into the pGL3-basic vector . The mutant sequences of the constructs are listed in According to the human GDNF promoter activity assays were performed as previously described [escribed . RelativGCACGTCACGGAGT G-3', − 997 to − 970 nt; wild-type CREB-S: 5'-CGCGCCGGTTGACGTGGTGTCTCGTTCG-3', − 321 to − 293 nt, underline denotes CREs; methyl-CREB-S: 5'-CGCGCCGGTTG ACGTGGTGTCTCGTTCG-3', − 321 to − 293 nt, all the cytosines in the CpG of the probe were methylated) containing the consecutive putative CREB-binding site was synthesized with and without a biotin label. We mixed 10 μg of cell nuclear extracts and a 100-fold molar excess of unlabeled competing probes  and incubated at room temperature for 20 min followed by the addition of 15 μL of labeled probes (0.1 μmol) for 20 min. For the supershift assay, samples were incubated with 1 μL of an anti-CREB monoclonal antibody  for an additional 20 min at room temperature. Protein/DNA complexes were then separated by non-denaturing polyacrylamide gel electrophoresis on 6% gels and transferred to nitrocellulose membranes (Millipore). Protein/DNA complexes were analyzed using streptavidin–horseradish peroxidase and developed with an enhanced chemiluminescence system .Electrophoretic mobility shift assay was performed with the non-radioactive EMSA kit  according to the user manual. The double-stranded DNA probes  were converted to big-wig files using bedGraphToBigWig and visualized in the Integrative Genomics Viewer software. The results for ChIP-PCR and re-ChIP-PCR were calculated as %Input: %Input = 2(CtInput−CtChIP) × Input dilution factor × 100. Antibody information and primer sequences are shown in ChIP-PCR assays were performed as previously described . For re-http://quma.cdb.riken.jp/.BSP was performed as previously described , 18. TwoGDNF-promoter II-mt containing the mutant GDNF promoter II was extracted. In this plasmid, a methylation-insensitive single restriction site (5'-MluI-silencer II-XhoI-3') was introduced into both ends of silencer II in GDNF promoter II (− 1300/+ 149) by point mutation, which did not affect promoter activity [GDNF-promoter II-mt plasmid was digested with the restriction enzymes MluI and XhoI. The vector backbone and corresponding silencer II fragment were recovered by agarose gel electrophoresis. Part of the recovered silencer II fragment (3 μg) was methylated using CpG methyltransferase  according to the manufacturer’s instructions, and then ligated back into the corresponding luciferase vector backbone with T4 ligase (methylated fragment: vector backbone = 3:1). The remaining unmethylated silencer II fragment was also ligated back into the luciferase vector backbone as a control. Ligated components were separated by gel electrophoresis. Circular plasmid DNA with a high migration rate was recovered. After BSP confirmed > 90% DNA methylation of silencer II in GDNF promoter II treated with M.SssI, the circular plasmid DNA was transfected into U251 cells. Promoter activity was measured using a dual-luciferase reporter system.This assay was performed according to the method previously reported by our group . Brieflyactivity . Next, 1The U251 cells were grown on coverslips in a 24-well plate, fixed with 4% paraformaldehyde (w/v) for 40 min, washed for 5 min with PBS three times and permeabilized with 0.5% (w/v) Triton X-100 in PBS for 15 min. The cells were then blocked for 30 min in PBS containing 10% bovine serum albumin followed by overnight incubation with the primary antibodies against pCREB and CBP diluted in PBS containing 10% FBS. After series of washings with PBS, cells were incubated for 2 h with secondary antibodies  conjugated with fluorescein isothiocyanate or tetra methyl rhodamine isothiocyanate in dark moist environment. After several additional washing steps, the coverslips were mounted with Hydromount containing DAPI to stain the nuclei . The localization of pCREB and CBP protein was examined using fluorescence confocal microscope (Leica Microsystems).Equal amounts of nuclear protein extracts prepared from U251 cells were incubated with the pCREB antibody, CBP antibody, or normal IgG  overnight at 4 °C. Then, the agarose-conjugated protein-A/G beads (SCBT) were added into the immunocomplex and the mixture was incubated at 4 °C for another 12 h. After extensive washing with ice-cold WB/IP lysis buffer with protease inhibitors, the beads were mixed with SDS loading buffer and boiled. The proteins in the supernatant were separated by SDS-PAGE and transferred to NC membranes. Blots were incubated with blocking reagent (TBST solution containing 5% non-fat milk), primary and secondary antibodies diluted in TBST, and then developed with enhanced chemiluminescence (ECL) + plusTM Western blotting system kit (Amersham). X-ray film was exposed to the membrane and then developed.t tests were used to identify significant differences in mean values between two groups. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare the mean values of multiple groups. P < 0.05 was considered statistically significant for all tests.All data were processed with SPSS 25.0 . Data are expressed as mean ± standard deviation (SD). Independent sample Additional file 1:. Supplementary TablesAdditional file 2:. Supplementary Figures


Telomeres are highly conserved tandem nucleotide repeats that include proximal double-stranded and distal single-stranded regions that in complex with shelterin proteins afford protection at chromosomal ends to maintain genomic integrity. Due to the inherent limitations of DNA replication and telomerase suppression in most somatic cells, telomeres undergo age-dependent incremental attrition. Short or dysfunctional telomeres are recognized as DNA double-stranded breaks, triggering cells to undergo replicative senescence. Telomere shortening, therefore, acts as a counting mechanism that drives replicative senescence by limiting the mitotic potential of cells. Telomere length, a complex hereditary trait, is associated with aging and age-related diseases. Epidemiological data, in general, support an association with varying magnitudes between constitutive telomere length and several disorders, including cancers. Telomere attrition is also influenced by oxidative damage and replicative stress caused by genetic, epigenetic, and environmental factors. Several single nucleotide polymorphisms at different loci, identified through genome-wide association studies, influence inter-individual variation in telomere length. In addition to genetic factors, environmental factors also influence telomere length during growth and development. Telomeres hold potential as biomarkers that reflect the genetic predisposition together with the impact of environmental conditions and as targets for anti-cancer therapies. Tetrahymena thermophile, the protozoan telomeres contain 20–70 hexameric TTGGGG tandem repeats [Mus musculus that extend up to 150 kb [Telomeres are conserved tandem repeats at chromosomal ends that differ in length in diverse species ,2,3,4,5. repeats . The tel repeats . In plan repeats ,10. In vo 150 kb ,12. Teloo 150 kb ,14. Teloo 150 kb .Single-stranded 50–300 nucleotide guanine rich telomeric G-tail folds back into the duplex DNA to form a t-loop B that reTelomeres are, in general, associated with three types of proteins that include nucleosomes, shelterin complex, and chromosomal transcription factors ,25,26.The telomeres, organized within tightly packed histone octamer composed nucleosomes , are staBesides the routine post-translational modifications, histone proteins function in telomere capping, telomere transcription, homologous recombination at telomeres, cellular differentiation, and nuclear reprogramming ,34. The Shelterin complex comprises of six protein subunits . TelomerTRF1 and TRF2 that exist as homodimers bind to the double-stranded DNA, and POT1 binds to the single-stranded 3′ G-overhang ,40. TRF1POT1, that binds to single-stranded DNA with high specificity, contains two N-terminal oligonucleotide/oligosaccharide binding (OB) folds . The firRAP1 does not bind directly to the DNA, but rather forms a complex with TRF2 and its Myb domain binds to the primary domain of TRF2 for suppressing telomeric homologous recombination ,55. The Several protein complexes, apart from the shelterin, contribute to telomere regulation and maintenance ,57. ThosSome of the proteins associated with telomeres are also involved in the DDR mechanism . RecQ-faWASH at 9p, 2p, Xq/Yq, 1p, 15q, and 16p; immunoglobulin heavy chain genes at 14q; and olfactory receptor genes at 1p, 6p, 8p, 11p, 15q, 19p, and 3q [Subtelomeres are transcriptionally active chromatin regions A between, and 3q . The sub, and 3q ,84,85. TIncomplete replication at chromosomal ends by DNA polymerase results in progressive shortening of telomeres with each successive cell division and is termed as the “end replication problem” . During Normal human cells in a culture stop dividing after 40 to 60 passages, a phenomenon first observed by Leonard Hayflick and eponymously called the Hayflick limit ,92. IncoThe ribonucleic protein, telomerase, counteracts the replication-related telomere attrition. Telomerase is upregulated in tumors from over 90% of cancers; in 10% to 15% of tumors, telomeres are elongated through a homologous recombination-based alternative lengthening of telomeres (ALT) .Telomerase consists of a catalytic subunit, telomerase reverse transcriptase (TERT), and an RNA component (TERC), which acts as a template for the extension of telomeric nucleotide repeats ,98,99. ATelomerase activity remains tightly controlled at multiple levels- from transcriptional regulation of components for biogenesis to recruitment to the telomeres ,112. TheTERT and TERC amplification, genomic rearrangement of TERT, somatic mutations within the TERT promoter, and epigenetic modifications through TERT promoter methylation [Telomerase reactivation occurs in tumors via multiple genetic and epigenetic mechanisms that include hylation ,118.TERT gene at chromosome 5p15.33 and the TERC gene at chromosome 3q26.3  transcription factors [TERT promoter mutations that create identical binding sites for ETS/TCF transcription factors include that detected at −57 bp, originally discovered as the causal germline mutation in a melanoma pedigree, and at −124/−125 bp and −138/−139 bp as CC > TT tandem mutations that occur mainly in skin cancers [de novo E-twenty-six (ETS) binding sites created by the TERT promoter mutations  [TERT hypermethylated oncological region (THOR) [TERT promoter  . Conventn (THOR) . THOR ispromoter A. Hypermtivation ,147,148.tivation ,150.TERT promoter occurs in cancers that harbor TERT promoter mutations [TERT promoter mutations compared to those without mutations [TERT promoter mutations, the methylation was shown to be allele-specific, and H3K27me3 and H3K9me3 histone marks of inactivation promote the methylation [TERT promoter in vitro [Reduced methylation in the utations ,151,152.utations . In the hylation . The binhylation ,153. Thein vitro .ATRX) and the death domain-associated protein (DAXX) have been associated with ALT-positive tumors [ATRX, together with DAXX, function as a chromatin remodeling complex that facilitates the deposition of histone variant H3.3 at the telomeres [ATRX and DAXX due to mutations leads to a repressed heterochromatin state that activates recombination and initiation of ALT [ATRX loss compromises the cell cycle regulation of TERRA and leads to the persistent association of replication protein A (RPA) with telomeres, resulting in a recombinant nucleoprotein structure [Cancer cells that maintain their telomeres by ALT  are chare tumors . ATRX, telomeres . The losn of ALT ,161. ATRtructure . ALT is tructure ,163.Epidemiological studies have shown telomere length as a complex heritable trait with estimated heritability derived from twin studies from 36% to 82% compared to 34% to 50% from familial studies ,165,166.The variability in telomere length in parental gametes is directly expressed in the offspring zygotes, but a correlation between offspring and paternal telomere length or offspring and maternal telomere length is not clear ,169. In Telomere length inter-individual variation arises early in life due to an interplay between genetic and environmental factors ,176. SevA number of telomere length associated genetic loci associated have been identified through linkage analysis and GWAS ,185,186.OBFC1 and chemokine C-X-C motif receptor 4 (CXCR4) [TERC and OBFC1 loci, several GWAS have identified additional variants associated with telomere length in loci containing TERT, CTC1, NAF1, and RTEL1 [Twelve GWAS  on telom (CXCR4) . Followind RTEL1 ,183,186.TERC), 5p15.33 (TERT) and 10q24.3 (OBFC1), and at chromosome 3p14.1 with the PXK gene [Through a large-scale GWAS based on 26,089 healthy controls from breast, ovarian, and prostate cancer, four loci with telomere length associated SNPs were identified, including 3q26.2  gene, leads to the reduced gene expression by affecting the binding of CTCF and Rad21. DCAF4 forms a complex with DDB1 and CUL4 that is involved in nucleotide excision repair [DCAF4 indirectly influences telomerase activity and telomere length through its interaction with DDB1. DDB1 functions as a binding partner for the transcription factor E2F1, a member of the E2F family of transcription factors that regulate cell proliferation and telomerase activity [DCAF4 on telomere regulation remains unclear [BICD1), which functions in vacuolar traffic and regulates telomere length via telomerase and Ku-protein pathways [TERC expression [The SNP rs2535913, located in intron 8  of the DDB1 and CUL4-associated factor 4  genes, has been reported to contain several independent cancer susceptibility loci [TERT expression, which is located in-between the 5′end of TERT and 3′ end of CLPTM1L, with active histone modification marks and multiple transcription factor binding sites [ZNF148) preferentially binds to the minor allele of the variant that mediates increased TERT expression [TERC gene was shown to alter the secondary structure of TERC mRNA, with the minor allele associating with an increase in the gene expression and telomere length [In addition, SNPs at chromosome 5p15.33 and 3q26.2, not associated with telomere length, were shown to affect ity loci ,206,207.ng sites . Transcrpression . In a ste length .The functional studies provide a framework for a genetic approach to investigate the causal role of telomere length in age-associated diseases ,185,195.Several other factors that influence telomere length include oxidative stress, inflammation, lifestyle factors, physiological stress, and exposure to carcinogens ,211,212.Other lifestyle factors like smoking, obesity, and lack of exercise increase the rate of telomere shortening. In a meta-analysis based on 84 studies, it was reported that smokers had shorter telomeres than non-smokers . VariousThe association between telomere length and risk of cancers has been reported in several epidemiological studies ,228,229.In a Mendelian randomization study on 22 primary cancers involving 4,20,081 cases and 10,93,105 controls, genetically increased telomere length was shown to be associated with increased risk of nine cancers, which included glioma, serous low-malignant potential ovarian cancer, lung adenocarcinoma, neuroblastoma, bladder cancer, melanoma, testicular germ-cell cancer, kidney and endometrial cancer . Those fTERT-dependent anticancer immunotherapy, and chemical inhibition of telomerase are the most commonly studied anti-cancer treatment strategies [Telomeres and telomerase-based therapies are emerging as prospective cancer treatment strategies . Telomerrategies ,252. Telrategies ,253. Trerategies ,257,258.rategies ,254,259.rategies .BRAF-mutant melanoma cells [TERT-based therapeutic vaccination have limited anti-proliferation efficiency, the focus has shifted to personalized interventions specifically for patients with TERT promoter mutations and TERT genomic rearrangements, in combination with immune-checkpoint inhibitors [Telomere dysfunction mediated through telomerase substrate precursor, 6-thio-2′-deoxyguanosine (6-thio-dG), impairs cell viability and tumor growth . 6-thio-ma cells ,261. Thema cells ,263,264.ma cells . G-quadrma cells . The usema cells ,266. Althibitors . Individhibitors . Inhibithibitors ,269.Telomeres, the dynamic structures at chromosomal ends, are crucial for genomic integrity, and through age-dependent attrition, act as tumor suppressors . Telomer


We will also underline the cellular transcription factors and epigenetic modifications that regulate the gene expression of these viruses.Polyomavirus infection is widespread in the human population. This family of viruses normally maintains latent infection within the host cell but can cause a range of human pathologies, especially in immunocompromised individuals. Among several known pathogenic human polyomaviruses, JC polyomavirus (JCPyV) has the potential to cause the demyelinating disease progressive multifocal leukoencephalopathy (PML); BK polyomavirus (BKPyV) can cause nephropathy in kidney transplant recipients, and Merkel cell polyomavirus (MCPyV) is associated with a highly aggressive form of skin cancer, Merkel cell carcinoma (MCC). While the mechanisms by which these viruses give rise to the relevant diseases are not well understood, it is clear that the control of gene expression in each polyomavirus plays an important role in determining the infectious tropism of the virus as well as their potential to promote disease progression. In this review, we discuss the mechanisms governing the transcriptional regulation of these pathogenic human polyomaviruses in addition to the best-studied simian vacuolating virus 40 (SV40). We highlight the roles of viral  Polyomaviruses are a family of nonenveloped small circular dsDNA viruses that infect a variety of host species, including humans and other primates. The human polyomaviruses JCPyV, BKPyV, and MCPyV are known to widely infect the human population, but in rare cases, cause severe diseases . In immucis-regulatory elements as well as the origin of replication [Members of the polyomaviridae family share the same general genomic structure  ,10,11. Pcis-acting regulatory elements such as promoters, enhancers, and silencers is an important factor contributing to the virus’ ability to complete its life cycle in the host cell [cis-acting elements in the NCRR  tandem repeat motifs, now recognized as the enhancer element, within the late proximal region of the SV40 NCRR are essential for early viral transcription [Several significant studies have been devoted to dissecting the cription   also significantly increases the infectious capacity of the virus, suggesting that the activity of enhancer elements have a profound impact on viral infectivity. As discussed by the authors, similar effects may be seen if enhancer recombination occurs during a co-infection in an in vivo immunocompromised setting, making organ transplant recipients an intriguing population to investigate further . HoweverThe temporal control of polyomavirus transcription is tightly regulated not only by viral genomic elements but also by the proteins encoded by the viral genome. The LT protein in particular is involved in the transcriptional control of several primate polyomaviruses. In SV40, LT’s viral transcriptional function is primarily to inhibit early transcription in favor of the late transcription ,52,53,54Not only do JCPyV proteins contribute to temporal control of virus transcription, but they also restrict its tropism. Both early and late JCPyV gene expression is restricted only to glial cells. However, expression of either JCPyV or SV40 LT stimulates late gene reporter expression in both glial and non-glial cells, demonstrating that as a viral transcription factor LT has the potential to expand JCPyV tropism . StudiesThe specific roles of MCPyV and BKPyV proteins in viral transcription have not been fully elucidated, although existing evidence suggests that autoregulation of gene expression still occurs. MCPyV LT, for example, induces early viral gene expression in an NCRR reporter system . In addiInterestingly, proteins encoded by other viruses such as HIV also regulate polyomavirus gene expression. JCPyV, BKPyV, and MCPyV are more prone to cause disease in HIV/AIDS patients ,3,10. BePolyomavirus-encoded miRNAs negatively auto-regulate viral gene expression to control the infectious life cycle. Sullivan and colleagues discovered the specific roles of these miRNAs in each virus, which appear to be evolutionarily conserved ,66,67. Tcis-regulatory elements present within the NCRR and act as activators or repressors of viral gene expression. The presence or absence of these transcription factors within the host cell therefore determines whether expression of the viral genes can occur, and whether an infection can be successfully established within a specific cell type. This has been demonstrated by studies of JCPyV and MCPyV, which revealed that cell type-specific factors are crucial for the viruses to establish infection in their respective host cell [Host transcription factors can bind to the ost cell ,11,70. Early experiments established that JCPyV tropism is restricted in glial cells at the level of T antigen expression—the viral promoter and enhancer are only active in glial cells, but viral replication can occur in multiple nonpermissive cell types as long as the SV40 or JCPyV T antigen is present . ExpressA similarly restricted tropism for MCPyV was revealed in 2016 by Liu and colleagues . After eSeveral NCRR-binding transcription factors have been identified for SV40, JCPyV, and BKPyV. By engaging the viral NCRRs, these transcription factors have demonstrated the potential to up- or down-regulate viral gene expression in different viruses. Sp1, identified by Dynan and Tjian in 1983, was among the first factors found to regulate SV40 transcription by binding to the 21-bp tandem repeat region of the SV40 promoter . A totalSeveral cellular factors that regulate JCPyV transcription have been discovered since the 1990s; the readers are referred to a review containing a visual overview of the binding sites of these transcription factors in the viral NCRR . Tst-1, Certain cell type-specific JCPyV transcription factors are crucial in determining the host cell permissiveness to infection. Tst-1, the key transcription factor controlling JCPyV transcription, is specifically expressed in myelinating glial cells and therefore is likely a major determinant of JCPyV tropism . AdditioResearch into the regulation of BKPyV gene expression has revealed complex relationships among the transcription factors that bind to the viral NCRR; an overview of the binding sites for these factors is included in the following reference . Early wIt should also be noted that many of the SV40 transcription factors identified in early studies by the Tjian lab were found in HeLa cell extracts. However, the SV40 enhancer is active in both HeLa and lymphoid B cells. A comparison of extracted proteins from these two cell types revealed that some of the SV40 enhancer-binding proteins are HeLa- or B lymphoid-specific , revealiA number of well-known transcription factors such as NF-κB, Sp1, Ets1, and NF-1 have been predicted to bind to the MCPyV NCRR in silico , though The small, circular genomes of polyomaviruses are packaged together with cellular histones into a viral episome ,102,103.Largely due to the efforts of the Milavetz lab, the epigenetic regulation of SV40 is, thus far, the most studied among the polyomaviruses. The readers are referred to a recent review by Balakrishnan and Milavetz for findings on the epigenetic regulation of polyomaviruses made by their lab and others . HistoneThe epigenetic regulation of other primate polyomaviruses is less well studied, but early evidence suggest that similar mechanisms may control viral transcription. For example, treatment of JCPyV-infected cells with an HDAC inhibitor significantly induces viral transcription . In factThe epigenetic regulation of MCPyV infection remains largely unknown. However, histones are packaged inside the MCPyV virions . After MThe SV40, JCPyV, and BKPyV genomes have significantly fewer methylatable CpGs in their genomes than is predicted for their genome size , suggestStudies examining the promoters of cellular genes have found that Sp1 binding sites protect nearby CpG islands from de novo methylation ,117. TheDecades of research on SV40 have established it as a model system to study eukaryotic transcription. Research on SV40 and other polyomaviruses has also uncovered viral genomic elements and host cell-specific transcription factors that function together to regulate the viral gene expression. In addition, polyomaviruses have been found to hijack cellular signaling pathways such as the MAPK/ERK signaling cascade ,119,120 JCPyV-associated PML is among several debilitating neurological complications that commonly affect HIV/AIDS patients ,29, whercis-acting viral DNA elements, the host cellular factors, as well as the epigenetic modifications that regulate MCPyV transcription [Compared to SV40, JCPyV, and BKPyV, the transcriptional regulation of MCPyV is much less understood, though it is clear that transcription of viral genes plays a critical role in both maintaining persistent viral infection and defining its host tropism and oncogenic potential. MCPyV DNA is clonally integrated into the host genome in at least 80% of MCCs ,132. In cription ,13,136.


Genome integrity is essential to maintain cellular function and viability. Consequently, genome instability is frequently associated with dysfunction in cells and associated with plant, animal, and human diseases. One consequence of relaxed genome maintenance that may be less appreciated is an increased potential for rapid adaptation to changing environments in all organisms. Here, we discuss evidence for the control and function of facultative heterochromatin, which is delineated by methylation of histone H3 lysine 27 (H3K27me) in many fungi. Aside from its relatively well understood role in transcriptional repression, accumulating evidence suggests that H3K27 methylation has an important role in controlling the balance between maintenance and generation of novelty in fungal genomes. We present a working model for a minimal repressive network mediated by H3K27 methylation in fungi and outline challenges for future research. Even though the first description of different chromatin types was cytological , transcrNeurospora crassa [N. crassa, the plant pathogens Dothistroma septosporum, several Fusarium species, Magnaporthe oryzae, Verticillium dahliae, Zymoseptoria tritici, the plant endosymbiont Epichloë festucae, and the facultative animal pathogen, Cryptococcus neoformans. Compared to the breadth of information amassed in plants and animals , review effects on development and pathogenicity in the absence of fungal PRC2, and discuss how the interplay of various histone marks affects genome plasticity and stability in fungi. We propose a testable working model predicting the minimal components of a reversible H3K27me3-mediated gene silencing system in fungi, touch on ongoing forward genetic screens that promise to discover novel components of the system, and present an outlook describing several key challenges in the area of facultative heterochromatin in fungi for the immediate future.Drosophila development [Drosophila Pc mutant, first isolated in 1947 by P. Lewis, received its name because it altered the number of sex combs, a row of thick bristles, on the legs of male flies (reviewed in [Neurospora [Polycomb group (PcG) proteins were discovered by their impact on elopment , and it elopment ,17). Theelopment . The domiewed in ). Usualliewed in . Biochemiewed in ,23. Subsiewed in ,25 and aiewed in ,23. Inteurospora ; notablyurospora ,23. Chaetomium thermophilum, helped define functional regions of E(z)/EZH2 and Esc/EED homologs, and revealed structural similarity between fungal and animal PRC2s [C. neoformans [Although not easily discerned by overall sequence comparisons, homologs of core PRC2 components found in fungi have well-conserved domain architectures when compared to their plant and animal counterparts ,10,26 T. Crystalal PRC2s ,29,30,31al PRC2s ,33. The oformans . Neurospora [Fusarium [Cryptococcus [Neurospora PRC2 was characterized by deletion of homologous genes for all three core subunits , which abrogated all H3K27me3, while deletion of the gene encoding an associated subunit (NPF/CAC-3/MSL1) resulted in lower H3K27me3 levels in many interstitial regions and complete absence of H3K27me3 from subtelomeric regions [Fusarium graminearum, deletion of kmt6/EZH2 resulted in complete loss of H3K27me3 [eed or suz12, while deletion of msl1/cac3 had no effect on H3K27me3 levels . Loss of the C. neoformans PRC2 core components (Ezh2 and Eed1) or the non-canonical subunit, Bnd1, removed all H3K27me3, while loss of Msl1 or the novel accessory component Ccc1 resulted in regional reduction and relocalization of H3K27me3 [Neurospora, Cryptococcus msl1 mutants showed only depletion of H3K27me3 in subtelomeric regions; ccc1 mutants showed a more pronounced reduction of H3K27me3 in these regions.Reverse genetics studies delineated the PRC2 of fungi in the ascomycetes, urospora  and FusaFusarium , and thetococcus . Neurosp regions . In FusaH3K27me3 , and theH3K27me3 . This waZymoseptoria tritici [Studies in these three models, and more recently the wheat pathogen,  tritici ,37, defiDNA replication requires disassembly of nucleosomes and eviction of DNA- and chromatin-interacting proteins. While strand-specific mechanisms of histone recycling have been uncovered (reviewed in ), it is Drosophila [N. crassa showed that telomeric repeats can recruit H3K27me3 to naïve chromosome segments and spread H3K27me3 across dozens of kilobases [de novo PRC2 activity, likely conferred by sequence-specific binding, DNA structure, or an “epigenetic” assembly of RNA and protein factors, and maintenance activity work together to propagate H3K27me3-mediated repression in a manner dependent on the organism and the locus.Contrasting evidence suggests that recycled H3K27me3 is not necessarily sufficient or required for long-term propagation of transcriptional repression. For example, in animals, lncRNAs have been suggested to target PRC2 to specific regions ,48, someosophila ,49,50 anosophila ,53,54,55ilobases . Furtherilobases ,35,55. FN. crassa identified a novel fungal-specific protein, PRC2 ACCESSORY SUBUNIT (PAS) [Fungi are excellently suited for genetic analyses, and studies in at least three laboratories are under way to determine what other components are part of the H3K27 methylation network. Two recent papers describe new components of the fungal H3K27 methylation network. A forward genetics selection with IT (PAS) . PAS proIT (PAS) ,57. Whildefective in silencing (dis), which exhibit phenotypes that are near-identical to those observed with kmt6 deletion strains. While our analyses are still incomplete and ongoing, one of these proteins, DIS2, resembles a protein also found recently in N. crassa, called EFFECTOR of POLYCOMB REPRESSION-1 (EPR-1) [N. crassa EPR-1 protein may act in a similar fashion. All epr-1 mutants display gene expression changes that are similar to strains lacking PRC2 components, but surprisingly H3K27 methylation appeared unaffected [Studies in our lab have identified several mutants, called  (EPR-1) . EPR-1 h (EPR-1) . In fact (EPR-1)  and EBS  (EPR-1) , have be (EPR-1) . The N. affected . By cytoaffected .Our working model predicts additional factors that are expected to be necessary to complete a feedback loop that establishes and maintains H3K27 methylation until environmental or internal conditions require transcriptional activation . Apart fN. crassa H2A.Z and the CRF, SWR-1, similarly promote H3K27 methylation, although H2A.Z and H3K27 methylation were not found to colocalize [ATP-dependent CRFs catalyze nucleosome sliding, disassembly, and exchange, which ultimately influences transcription via nucleosome positioning or DNA accessibility ,66. Intelocalize .Drosophila) and NURF55, a homolog of Msl1, among other components [Recent evidence from animal studies also suggests that CRFs have various roles in controlling PRC2 activity . Themponents ,80. In cmponents ,82,83,84mponents ,76. Takemponents , and altFusarium, where development and differentiation are severely disturbed by the expression or overexpression of ~2,500 genes following loss of PRC2 [N. crassa, 700–900 genes are enriched with H3K27me3, and ~150 genes are derepressed in PRC2 mutants [C. neoformans, ~75 transcripts increase in expression by more than threefold, and 71% of these are within 40 kb of telomeric regions [Z. tritici, ~1,300 genes are enriched for H3K27me3, but only 98 genes in H3K27me3 regions and 124 genes in total were upregulated in absence of H3K27me3 [Generally, transcriptional activity is lower or absent in chromatin enriched with H3K27me3 . Transcr of PRC2 . In N. c mutants ,35, but  regions . In Z. tH3K27me3 . While dHow is H3K27me3-dependent transcriptional repression mediated? After discovery of the molecular activity of PRC2, a hierarchical model of catalysis and spreading of H3K27me3 by PRC2 and subsequent binding of PRC1 by virtue of its Pc subunit, which reads H3K27me3, was proposed . It is nin vitro, a recent study showed interactions with the BAH-domain containing H3K27me3 readers, SHL and EBS, in a BAH–EMF1 Complex to read H3K27me3 and induce transcriptional silencing [While many fungi have genes for the core PRC2 subunits, no fungal genome examined so far encodes known critical components of the canonical PRC1. There is no evidence for clear homologs of many PRC1 subunits, including KDM2B and JARID2, in fungal genomes. One catalytic activity of PRC1 is ubiquitination of H2A , which is thought to result in transcriptional repression  resulted in severe defects in both linear growth and development. Colony morphology is severely disturbed, presumably because various pathways involved in signaling and asexual growth are expressed at improper times. Sexual development is completely abrogated in kmt6 mutants under normal conditions in this homothallic species [kmt6 mutants are female sterile. Overexpression of numerous secondary metabolite clusters in kmt6 mutants may contribute to defects in development and pathogenicity [F. fujikuroi, attempts to delete kmt6 have been unsuccessful [kmt6 by introduction of constructs to generate siRNA from double-stranded RNA revealed severe defects in growth and sporulation [Fusarium species but not others.Transcriptional regulation of important genes is considered the proximal cause for the requirement of PRC2-mediated H3K27 methylation for normal development in the plants and animals that have been studied. While the number of fungi, including plant pathogens, with experimental evidence for the presence of H3K27 methylation is rapidly increasing, the functional characterization of facultative heterochromatin is lagging behind. Loss of H3K27 methylation can slow growth and development in some fungi, most notably in the genus mt6/EZH2 , eed, or species ; in outcgenicity . In the ccessful . This surulation . FurtherFusarium, deletion of kmt6/EZH2 resulted in only minor defects in development. In the wheat pathogen Z. tritici, strains lacking H3K27me3 were indistinguishable from wild type in growth and stress responses  except for a slight increase in melanization at elevated temperatures; pathogenicity on wheat was only mildly affected [kmt6 in the blast pathogen, Magnaporthe oryzae, abolished all H3K27me3, reduced asexual sporulation, and resulted in severe defects in pathogenicity on wheat and barley [Epichloë festucae is a beneficial endosymbiont of perennial ryegrass (Lolium perenne). Deletion of ezhB/EZH2 abolished all H3K27me3 [eas and ltm, respectively, and only expressed in planta [In vitro suppression of eas and ltm genes was influenced by presence of heterochromatic marks, i.e., H3K9me3 or H3K27me3 [eas and ltm were reduced in planta when compared to in vitro growth. Deletion of the H3K9-methyltransferase-encoding gene, clrD, or ezhB increased expression from both clusters in vitro, and double deletion resulted in additive effects [in vitro metabolite production was not observed in any of the mutants [eas cluster, including genes that were not derepressed in vitro, and increased production of the ergot alkaloid, ergovaline [clrD abolished colonization of ryegrass [Dothistroma septosporum, is regulated by H3K27me3 [D. septosporum, the impact of kmt6 deletion on pathogenesis has not been determined [In plant pathogens other than affected . Deletiod barley . EpichloH3K27me3 . This fuH3K27me3 . Genes un planta ,107. In H3K27me3 . H3K9me3 effects . However mutants . Loss ofgovaline , suggestryegrass , demonstH3K27me3 . During termined . N. crassa or C. neoformans in the absence of H3K27me3 under normal laboratory growth conditions, but loss of accessory components, e.g., NPF/CAC-3/MSL1 in Neurospora and Msl1 or Ccc1 in Cryptococcus, resulted in minor growth reduction, presumably because these subunits are also found in several other complexes involved in chromatin regulation [Candida albicans and Aspergillus fumigatus, lack core PRC2 components and no H3K27 methylation has been detected [C. neoformans and Fusarium oxysporum are the only animal pathogens among the fungi in which H3K27me3 has been demonstrated [Cryptococcus, Msl1 is required for multiple virulence-associated phenotypes, including thermotolerance, cell wall stability, and melanin production, as well as full virulence in a murine infection model [MSL1 deletion, reduced survival in murine macrophages, and decreased virulence in a murine infection model [Fusarium. Furthermore, functional studies in pathogenic or symbiotic fungi have generally focused on specific and regional effects, so direct evidence for genome-wide chromatin changes during host infection remains scant.Overt phenotypes are not observed in gulation ,35. The detected . So far,nstrated ,109, eveon model . Loss ofon model . These rGenome integrity is essential for the maintenance of cellular function and viability. Genome instability is a phenomenon that is frequently observed in malfunctioning cells associated with disease in many eukaryotes but has also been reported as advantageous for rapidly adapting organisms ,112. ThiPathogenic fungi are prominent examples of organisms with a high degree of genetic diversity . StudiesPopulation genetic studies demonstrated that DNA sequences subject to rearrangements and instability are not randomly distributed within the genome, but rather specific regions show higher variability than others. Sequences located close to chromosomal ends, the subtelomeric regions, and sequences harboring active or mutated transposable elements (TEs) show a high degree of diversity, even between isolates of the same species ,121,122.N. crassa, a mutant karyopherin (DIM-3) involved in the assembly of constitutive heterochromatin changed average chromosome structure [set-7/kmt6 deletion mutant resulted in the movement of subtelomeric regions to the interior of the nucleus and occasional disruption of centrochromatin, measured by the appearance of several CenH3 foci, rather than the usual single focus [E. festucae contributes to symbiosis with ryegrass [in planta were overrepresented near these regions. For example, the eas gene cluster , which attracts H3K9me3, and, in turn, allows cytosine DNA methylation [C. neoformans. Mutants of ezh2/kmt6 and the novel ccc1 subunit were tested for conditional relocalization of H3K27me3 to constitutive heterochromatin; H3K27me3 moved into regions of constitutive heterochromatin in ccc1 mutants [N. crassa, the presence of H3K9me2/3 is not necessary for H3K27me3 relocalization, suggesting additional factors involved in this process. Recent reviews proposed models  and H3K27me2/3 distribution. H3K27me3 enrichment is reduced at normal target genes upon loss of 5mC, and relocalizes to loci with normally high 5mC levels , includise, MET1 . One of se, MET1 . In N. cK27me2/3 .C. neoformans PRC2, Ccc1, is similar to proteins found in HDAC complexes and associates with the class II HDAC, Hda1 (Clr3); deletion of Hda1 results in reduced growth as observed for Ccc1 and de-repression of PRC2-silenced genes in subtelomeric regions [So far, little is known about interactions of histone lysine acetylation (KAT) or deacetylation complexes, either HDACs or sirtuins, with PRC2. One of the two novel subunits of  regions ,110. WhiFrom this glimpse into connections between different chromatin marks, it is clear that our understanding of interactions between chromatin modifications and the function of H3K27 methylation is still rudimentary, and that it will take comparative studies in several organisms to uncover all existing connections.N. crassa appeared deleterious rather than compensatory [Population genetic analyses as well as recent experimental work indicated that H3K27me3 is connected to sequence diversity and instability. An intriguing puzzle to solve in the near future is how H3K27me3 can confer instability on a molecular level, and why this mechanism evolved over evolutionary timescales. While functional studies on the molecular mechanism are still lacking, a recent study on early land plants suggested that H3K27me3 together with H3K9me3 might represent an ancient form of genome defense mechanism against transposons . Regionsensatory .Z. tritici isolates, several H3K27me3-marked, isolate-specific regions were identified . These were often flanked by H3K9me3-enriched regions and contained genes that are most similar to transposons or their “cargo”  that result in the formation of a variety of subcomplexes [N. crassa and C. neoformans but again only under standard laboratory growth conditions. Based on the number of putative DNA-binding “transcription factors”  and the increase in percentage of the genome enriched with H3K27me3 , we propose that there is an increased number of PRC2 adapter proteins that interact with SUZ12 to recruit PRC2 activity to its target regions in some fungi, e.g., F. graminearum. This large number of repressor complexes would be balanced by activator complexes that are coupled to HAT co-activator complexes.omplexes . In planomplexes . These vHow does H3K27me3 and facultative heterochromatin control fungal development? In plants and animals, H3K27me3 is involved in differentiation and development but no comprehensive time course studies across a complete life cycle have been carried out in fungi. This is an important knowledge gap that must be filled in the near future.What happens to H3K27me3-enriched regions in pathogenic fungi in response to the host environment? We are largely naïve on this issue, as successful and routine in planta ChIP-seq is still extremely difficult. H3K27me3 covers more than 30% of the F. graminearum genome during growth in vitro, including regions enriched with pathogenesis-associated genes [in vitro are derepressed in planta [F. oxysporum and Verticillium dahliae [ed genes . Many ofn planta ,164; a s dahliae ,165). ThHow are chromatin domains organized in three-dimensional space within the nucleus? Existing Hi-C studies revealed that facultative heterochromatin has major impacts on nuclear organization, and we are currently only at the beginning of these studies. Hi-C investigations in double and triple mutants that are defective for several chromatin marks at the same time are underway.How do we reconcile genome metastability, increase of point mutations, chromosome loss, and transcriptional effects at the evolutionary scale? Comparing existing evidence from all fungi demonstrates that transcriptional regulation is not the only role of PRC2. Using several strains of the same species and several species of the same genus is now possible in all four genera discussed here. Genome sequencing, resulting in complete genome assemblies, ChIP-seq, Hi-C, and RNA-seq, are now routine and no longer prohibitively expensive. We predict that numerous “lab evolution” studies will help to resolve the current uncertainties. To this end, we also have begun to investigate the mechanistic connections between spontaneous and induced DNA lesions, fixed mutations, replication, and H3K27me3 targeting. The surprising observation in Z. tritici suggesting that H3K27me3 generates a “metastable” genome makes this species an ideal model to uncouple transcriptional and genome stability effects in future studies.


E. coli genome. However, some targets are off-limits, such as regions immediately outside the Mu ends (cis-immunity) as well as the entire ~ 37 kb genome of Mu (Mu genome immunity). Paradoxically, MuB is responsible for cis-immunity and is also implicated in Mu genome immunity, but via different mechanisms. This study was undertaken to dissect the role of MuB in target choice in vivo.The target capture protein MuB is responsible for the high efficiency of phage Mu transposition within the E. coli genome, in the presence and absence of MuB. The data reveal that Mu’s ability to sample the entire genome during a single hop in a clonal population is independent of MuB, and that MuB is responsible for cis-immunity, plays a minor role in Mu genome immunity, and facilitates insertions into transcriptionally active regions. Unexpectedly, transposition patterns in the absence of MuB have helped extend the boundaries of the insular Ter segment of the E. coli genome.We tracked Mu transposition from six different starting locations on the The results in this study demonstrate unambiguously the operation of two distinct mechanisms of Mu target immunity, only one of which is wholly dependent on MuB. The study also reveals several interesting and hitherto unknown aspects of Mu target choice in vivo, particularly the role of MuB in facilitating the capture of promoter and translation start site targets, likely by displacing macromolecular complexes engaged in gene expression. So also, MuB facilitates transposition into the restricted Ter region of the genome. E. coli, making it the most efficient transposable element (TE) described to date ).3 family, Tn7, and bacteriophage Mu, display transposition immunity [cis-immunity), and it is thought that this form of self-recognition must also provide protection against self-integration (TE genome immunity)  and ΔMuB prophages placed at six different locations around the E. coli genome, we show that cis-immunity depends on MuB, while Mu genome immunity is only mildly breached in its absence. The data also reveal a previously unappreciated role for MuB in facilitating Mu insertion into transcriptionally and translationally active regions. An unanticipated outcome of this study is the finding that the Ter segment of the E. coli genome, which is more isolated from the rest of the genome, is larger than previously estimated.The present study investigates the role of MuB in the three diverse functions discussed above - target capture, E. coli [We recently exploited the DNA-DNA contact mechanism of phage Mu transposition to directly measure in vivo interactions between genomic loci in E. coli . Thirty-While MuB is essential for target capture in vitro , transpoE. coli genome shown in Fig. The color-coded map of the Why does such a defined Ter segment emerge only in MuB-deficient prophages? Given that the ΔMuB prophage in Ter had no trouble sampling within Ter, but that the other ΔMuB prophages did have difficulty inserting here, we suggest that the answer lies in the existence of some special feature at the Ter boundaries that isolates Ter. MatP, which binds to specific matS sequences distributed within Ter , has beelac operon, which is expected to be only partially transcribed under our growth conditions. The figure also includes the flagellar master regulator gene flhD which has multiple promoters [dnaJ which has no promoter and is exclusively co-transcribed with dnaK [Two prior microarray data sets have shown a negative correlation between transcription and Mu transposition , 38, altromoters , and dnaith dnaK . For alllacZY. Interestingly, two different WT Mu insertion patterns were observed within the lac operon, whose lacZ and lacY genes are repressed by the activity of the lacI repressor, which is expected to be transcribed [. The number of Mu insertions in lacI were roughly half those in lacZY, with a strong suppression of insertions around the TSS and + 1 nt region of lacI for WT. This observation is in agreement with the previous findings of a negative correlation between transcription and transposition.WT Mu had significant difficulty inserting near the + 1 nt of all active genes, in a region that extends up to 50–200 bp upstream, typically including promoter regions (TSS) , and 50–nscribed . The numflhDC operon that control flagellar gene transcription in Salmonella, only two (P1 and P5) were seen to be functional [dnaJ is 2 kb away, as dnaJ is always co-transcribed with dnaK, with a small 370 nt RNA candidate tpke11 between the two genes [flhD, with reduced insertion around the TSS, while ΔMuB prophages show in addition a secondary exclusion zone upstream from the + 1 nt that encompasses both P1 and P5 promoter regions. Even though TSS is absent in dnaJ, WT Mu shows an insertion exclusion zone around + 1 nt of this gene. ΔMuB prophages show an exclusion zone upstream of dnaJ not seen in WT, around the position of tpke11, while revealing an unusually permissive region upstream of dnaK. The latter permissive region in both WT and ΔMuB corresponds to the 377 bp intergenic region between yaaI and the dnaKJ operon promoter. While this set insertion patterns overall is consistent with the negative correlation between transcription and transposition, particularly around the TSS and + 1 nt for WT, the insertion patterns in dnaJ reveal that the + 1 nt region presents a transposition barrier independent of the promoter region, and is likely reflective of the translation activity of the mRNA near this genomic site given that transcription and translation are coupled in bacteria. As nascent mRNA is being translated, the ribosomes could slow down the RNA polymerase enough to provide steric protection to the genomic DNA from Mu transposition.Of six potential promoters in the nctional . These two genes , 43. WT E. coli has 7 ribosomal RNA operons that are highly transcribed [rrnA, uniform across the entire operon, and independent of MuB. rrnE and rrnH receive more insertions in the 23S compared to the 16S region, and are responsive to MuB. rrnG shows a large increase in sampling only at the 5′ end of the 16S region (note that rrnG is on the negative strand). There seems to be an equal level of Mu insertion between rrnB, rrnC, and rrnD. In the 5.3 kb window encompassing the entirety of each of these operons, over 85% of the window is comprised of coding sequences. WT transpositions into the coding sequences make up between 20 and 50% of all reported transpositions, rather than the 85% if it was entirely random. MuB mutants typically faired much worse, ranging from 5 to 10% of observed transpositions in the same area. Thus in the majority of cases, there is a significant reduction of Mu transposition into the ‘coding’ regions of the rRNA operons in the absence of MuB.To examine Mu insertion patterns in genes that are transcribed but not translated, we looked at both ribosomal RNA operons and tRNA genes. nscribed . We obserrn operons should reflect this as well. Accordingly, rrnA is the least transcriptionally active. While early experiments showed little difference in expression levels between the operons in minimal media , more rcription , have deactivity . Our resnE Fig. S. RegardlE. coli genome [ileY and selC (marked with red asterisks), is unmistakable , to distinguish them from late stage transposition (LST) where data were collected after multiple rounds of transposition. For the LST condition, we let the experiment run for 2 h, which allowed WT to complete its lytic cycle (in ~ 50 min) and ΔMuB prophages to accumulate 5 to 10 copies of Mu on average per cell as predicted by genome abundance, assuming an even distribution of Mu copy number among the population. All six prophage strains were used for EST experiments, and one WT plus all six ΔMuB prophages for LST experiments.cis-immunity phenomenon  are shown in Fig. The previously described ratchet-model suggests that intrinsically clustered MuA would hydrolyze proximal MuB-ATP during dynamic loop formation due to Brownian motion . As propcis-immunity in the life of Mu? Avoiding insertion into regions flanking Mu ends would avoid destroying flanking Mu copies when packaging begins, since the DNA packaging machinery resects on average 100 bp of host DNA flanking the left end and 1.5 kb of DNA flanking the right end. Even though Mu samples the E. coli genome extensively in a distance-independent manner  [Mu ends (L and R) define a boundary separating two modes of MuB binding and immunity . We had  in vivo , 58, hening EST) . We obsecis-immunity more accurately, and show that it persists well into the lytic cycle for WT prophages, but is abolished in ΔMuB strains. We show that Mu genome immunity also persists through the lytic cycle for WT prophages, and is only rarely infringed upon in ΔMuB prophages, showing conclusively the distinction between these two forms of immunity. There is clearly more to be learned about what enables genome immunity.MuB is critical for Mu’s ability to efficiently capture targets for transposition. We show in this study that besides enabling efficient targeting, MuB also makes refractory targets more facile, likely by displacing bound proteins. By weakening/altering boundary features that demarcates the Ter region, MuB allows Mu to access Ter more readily. Transposition patterns in the absence of MuB have allowed us to more accurately measure the Ter boundaries, revealing that this region is larger than previously estimated. Perhaps in a similar manner, MuB also provides access to targets engaged in transcription/translation. We have mapped the range of All experimental strains are derivatives of MG1655 and listed in the strains table. Prophage gene deletions were introduced into specific prophages using P1 transduction and kanamycin resistance selection. Cells were propagated by shaking at 30 °C in M9-Cas minimal media  and appropriate antibiotics for selection.Prophage transposition was induced by temperature shifting to 42 °C for the appropriate time before harvesting genomic DNA. Early stage transposition (EST) experiments were accomplished by a 15 min temperature shift to capture one transposition event in WT cells as determined in a previous study . Late stOligonucleotide primer sequences are provided in Table Shttps://www.ncbi.nlm.nih.gov/sra/PRJNA597349.Target enriched samples were submitted to the Genomic Sequencing and Analysis Facility (GSAF) at UT Austin for sequencing. Libraries were prepped by GSAF using the facility’s low-cost high throughput method. Sequencing was done on an Illumina NextSeq 500 platform using 2X150 paired ends targeting 10 to 15 million reads. All sequencing data discussed in this work is available at https://github.com/dmwalker/MuSeq.Mu transposition targets were identified using lab software entitled Mu Analysis of Positions from Sequencing (MAPS) as described earlier . MAPS haAdditional file 1: Figure S1. Mu transposition outlines several features of the rRNA operons. EST prophages were pooled to analyze the frequency of Mu insertions into the entire rrn operon, for all 7 operons, for both WT (bottom rows) and ΔMuB (top rows) prophages. Insertion maps start at the TSS of the operon and continue for 5.3 kb. Operon maps are provided as a schematic on top, showing the leading 16 s RNA-encoding segment, followed by coding sequence (CDS) of an intervening tRNA, and finally the 23 s RNA-encoding segment. Each CDS in the operon is marked by a blue line that terminates in a flat head. The rrnD and rrnG operons are located on the (−) strand of DNA, while the remaining 5 are on the (+) strand. ΔMuB patterns generally follow similar trends to the WT prophage, but with reduced efficacy to insert anywhere within the rRNA operon. Figure S2. Mu does not transpose easily into tRNA coding regions. The x-axis provides the relative genomic position with respect to the tRNA labeled on the y-axis, and covers a 400 bp span. The + 1 position indicates the first nucleotide in the matured tRNA sequence. The preprocessed 5′ leader coding region would be encompassed in the region between − 200 to + 1. The e position is + 75 bp from the TSS and is the typical size of mature tRNA. The + 150 region is 150 bp from the TSS. For each of the 86 tRNA genes, the number of Mu insertions in and around the gene are tabulated for both the WT and ΔMuB prophages during EST. The transcriptional propensity is nucleotide level resolution of the degree of transcription for that particular nucleotide [Figure S3. Frequency of consensus target sequences for WT and ΔMuB prophage insertions across the E. coli genome. A. The genome for MG1655 from genbank (genid: 545778205) was partitioned into 200 equally sized bins, and the number of times the 5′-CYSRG-3‘ sequence and it’s reverse compliment appeared on the + strand in each bin was tabulated. B. The number of Mu insertions for each consensus sequence was calculated for both the WT (left) and ΔMuB prophages (right). The number of insertions reported is for the consensus sequence as written and the corresponding reverse complement. There are 1024 possible pentamers for Mu to insert, and the sequence identifier ‘other’ accounts for the 1016 sequences not covered by the ‘CYSRG’ consensus sequences and their reverse compliment. Figure S4. Insertion patterns outside Mu ends are nearly symmetrical for the left and right ends of Mu. The frequency of Mu insertions per 100 bp as a function of distance from Mu is plotted individually for each end, the combined data shown in Fig. cleotide . A higheAdditional file 2: Table S1. Oligonucleotides used in this study.