Schizosaccharomyces pombe and compare it with Saccharomyces cerevisiae and the mammalian polycomb system. We present recent structural and mechanistic evidence, which suggests that histone acetylation protects active transcription by disrupting the positive feedback loops used by the heterochromatin machinery and that H2A and H3 monoubiquitination actively drives heterochromatin, whereas H2B monoubiquitination mobilizes the defenses to quench heterochromatin.Eukaryotic genomes are segregated into active euchromatic and repressed heterochromatic compartments. Gene regulatory networks, chromosomal structures, and genome integrity rely on the timely and locus-specific establishment of active and silent states to protect the genome and provide the basis for cell division and specification of cellular identity. Here, we focus on the mechanisms and molecular machinery that establish heterochromatin in  •Heterochromatin-associated complexes are attracted and repelled by histone marks.•Acetylation of specific lysine residues protects euchromatin from silencing.•Methylation of histone H3 lysine 9 and 27 amplifies heterochromatin.•Nucleosome ubiquitination licences and enforces feedback loops. The accurate and timely organization of the genome into active and inactive genes, domains, and compartments is critical for organism development, for determination of cellular identity, and for genome replication, segregation, and protection . In eukaHistone post-translational modifications (PTMs) are ‘read, written, and erased’ by chromatin-modifying complexes to establish their foothold on a genomic target and to attract supporting factors ,6. In adSchizosaccharomyces pombe, heterochromatin dominates the centromeres, telomeres, and mating-type locus. It depends on the intimate cooperation of highly conserved chromatin modifiers and RNAi complexes, which use small RNAs and histone PTMs to establish silent genomic regions in Refs. , 14, 15. of Chp1 ,17  by the histone acetyltransferases Gcn5 and Mst2 is associated with open reading frames of expressed genes [In vitro, H3K14ac is required and sufficient to support nucleosome disassembly by the histone chaperone Nap1 [S. pombe is not clear because H3K14ac is enriched on open reading frames and not in promoters. H3K14ac levels are regulated by the zinc-dependent HDAC Clr3, which shows exquisite specificity for H3K14ac, and by the NAD+-dependent HDAC Sir2, which deacetylates H3K14ac, H3K9ac, and H4 lysines 5, 12, and 16 [In ed genes ,28. H3K1ed genes ,30 . Given  of Clr2 ,44.In summary, Clr3 is regulated on many levels to specifically deacetylate H3K14ac and is recruited as part of SHREC, but also independently ,45. The Recently, a new role for H3K14 emerged in heterochromatin silencing. Using the purified CLRC complex, Oya et al.  have shoSaccharomycescerevisiae and metazoan polycomb silencing mechanisms to show that the concept of using specific histone lysine residues to protect euchromatin is widely used across highly divergent heterochromatin systems.While the positive effect of acetylation in gene expression is often explained by its potential to relax chromatin structure due to loss of positive charges on histone tails, the key role of H3K14 in H3K9 methylation shows that specific acetylation sites function to break the feedback loops that drive heterochromatin establishment and maintenance. In the following, we will analyze S. cerevisiae uses the unique set of silent information regulator (SIR) proteins for heterochromatin formation, which form a complex composed of Sir2, Sir3, and Sir4 that binds heterochromatic regions in a finely tuned balance between SIR proteins and the chromatin substrate  is a mark tightly associated with active gene transcription and has been shown to reduce the compaction of chromatin fibers . However in Ref. ). Bindin in Ref. . Recent  in Ref. , showingall RNAs . H4K16acilencing . Even thlog Jhd2 ,54.FigurDrosophila development and fall into two distinct subfamilies: the PRC1 complexes are ubiquitin ligases that catalyze the monoubiquitination of H2AK119 (H2AK119ub), whereas PRC2 complexes are H3K27 methyltransferases (reviewed in Ref. [Polycomb repressor complexes (PRCs) were discovered as repressors of HOX genes in  in Ref. ). These  in Ref. . The fir in Ref. . It is n in Ref. ,58. Reco in Ref. ,60  contributes to PRC silencing has started to emerge recently ,62, and In the polycomb system, H3K27 acetylation is in direct competition with H3K27 methylation and protects active transcription from silencing. It has been found that NuRD complexes facilitate the silencing of active genes by deacetylating H3K27ac and thereby preparing the chromatin landscape for polycomb repression ,64.Even though organisms use highly divergent systems to segregate their genomes into heterochromatin and euchromatin, the following fundamental concepts are shared between many systems: (1) Acetylation of specific lysines protects euchromatin from the heterochromatin machinery. (2) HDACs prepare chromatin for the establishment of feedback loops that drive heterochromatin formation. (3) Nucleosome ubiquitination guides and enforces the feedback loops to stabilize heterochromatin and euchromatin. The recently discovered signaling through H3K14ub in fission yeast shows that acetylation not only protects the direct methylation targets of the heterochromatin machinery but that acetylation can also control regulatory crosstalk through ubiquitination. In the mammalian system, these regulatory mechanisms remain much less well understood, and we anticipate that exciting discoveries remain to be made regarding the compartmentalization mechanisms of the genome in cell homeostasis and disease.Nothing declared.


Erythropoiesis is a process of enormous magnitude, with the average person generating two to three million red cells every second. Erythroid progenitors start as large cells with large nuclei, and over the course of three to four cell divisions they undergo a dramatic decrease in cell size accompanied by profound nuclear condensation, which culminates in enucleation. As maturing erythroblasts are undergoing these dramatic phenotypic changes, they accumulate hemoglobin and express high levels of other erythroid-specific genes, while silencing much of the non-erythroid transcriptome. These phenotypic and gene expression changes are associated with distinct changes in the chromatin landscape, and require close coordination between transcription factors and epigenetic regulators, as well as precise regulation of RNA polymerase II activity. Disruption of these processes are associated with inherited anemias and myelodysplastic syndromes. Here, we review the epigenetic mechanisms that govern terminal erythroid maturation, and their role in human disease. Erythropoiesis is the process of making red blood cells. At each stage of maturation, erythroblasts are both phenotypically distinct and have a unique transcriptomic profile  and chroDuring the maturation of a committed progenitor to a reticulocyte, the nucleus condenses to ∼1/10th of its original volume prior to enucleation.. This drAcetylation of histone tails is generally associated with open chromatin structure and transcriptional activation, and regulation of histone acetylation is critical for the establishment appropriate patterns of erythroid gene expression and chromatin structure. Studies of friend leukemia transformed erythroblasts suggested that global deacetylation of histone residues is necessary for erythroid chromatin condensation.. Mass spAlthough a global decline in histone acetylation does not accompany human erythropoiesis, deacetylation of select histone modifications likely plays an important role. Several histone deacetylases (HDACs), including HDACs 2, 3, and five are highly expressed in maturing erythroblasts and have essential roles in erythropoiesis.. HDAC5, Dynamic changes in histone methylation also occur during erythropoiesis, with both histone methyltransferases, such as NSD1  and SETDHistone marks that reflect active transcription through gene bodies, such as H3K36me2/3 and H3K72me2 also vary dynamically during terminal maturation, , and plaIn vivo deletion of factors that regulate the transcription cycle including HEXIM1, TIF1γ, TFIIs, JMJD6 and TRIM28, results in severe anemia,  to shape the H3K27me3 landscape.. GATA faKLF1 is essential for both chromatin condensation and enucleation during terminal erythropoiesis.. KLF1 prOther transcription factors also play a role in regulating the erythroid epigenome and chromatin architecture. For example, the transcription factor FOXO3 is upregulated during the late-stage of terminal erythroid maturation and promotes the expression of genes essential for terminal erythroid maturation, with loss of FOXO3 leading to multilobed nuclei and deficient enucleation . The traMutations of transcription factors and epigenetic regulators can lead to both inherited and acquired erythroid disorders. In addition, Genome Wide Association Studies have demonstrated that genetic variants associated with red cell phenotypes often occur in enhancers or other regulatory elements critical for the proper expression of the erythroid transcriptome.. Table 1Mutations in GATA1, or GATA1-target sequences, cause anemia, which can be accompanied by thrombocytopenia, consistent with its role both megakaryopoiesis and erythropoiesis. Highlighting the essential nature of the cross talk that occurs between transcription factors and epigenetic modifiers, mutations that impair the ability of GATA1 to interact with co-activator and co-repressor complexes can lead to severe clinical phenotypes. For example the R216Q mutation, which impairs the ability of GATA1 to recruit the TAL1 activating complex, , leads tMutations in GATA1-binding sites that lead impaired expression of the target gene have been linked with a number of hematologic disorders, , includiDecreased GATA1 protein levels, have also been associated with human disease. DBA is an inherited erythroid failure syndrome that is associated with short stature and predisposition to cancer, most commonly caused by mutations in the ribosomal protein genes.. In DBA,KLF1 is also an essential erythroid transcription factor. . MutatioMutations in epigenetic regulators have also been associated with inherited anemias. As noted above, a point mutation of the histone methyltransferase ASH1L has been associated with a beta thalassemia-like phenotype.. MutatioAcquired mutations in GATA1 and other erythroid transcription factors and epigenetic regulators can also lead to hematopoietic disorders. Intriguingly, the GATA1s mutation is commonly acquired in infants with Down Syndrome, and results in transient myeloproliferative syndrome , highligThe epigenetic and transcriptional control of terminal erythroid maturation is complex, with each stage of erythropoiesis tightly regulated by various interdependent epigenetic mechanisms. Mutations that disrupt the transcriptional and epigenetic control of erythropoiesis lead to inherited anemias, highlighting the critical nature of these interactions. Understanding the fundamental mechanisms that govern erythroid maturation is essential to understanding the how mutations or other genetic perturbations result in disordered erythropoiesis, and to designing rational therapies.


Dinoflagellates have a generally large number of genes but only a small percentage of these are annotated as transcription factors. Cold shock domain (CSD) containing proteins (CSPs) account for roughly 60% of these. CSDs are not prevalent in other eukaryotic lineages, perhaps suggesting a lineage-specific expansion of this type of transcription factors in dinoflagellates, but there is little experimental data to support a role for dinoflagellate CSPs as transcription factors. Here we evaluate the hypothesis that dinoflagellate CSPs can act as transcription factors by binding double-stranded DNA in a sequence dependent manner.We find that both electrophoretic mobility shift assay (EMSA) competition experiments and selection and amplification binding (SAAB) assays indicate binding is not sequence specific for four different CSPs from two dinoflagellate species. Competition experiments indicate all four CSPs bind to RNA better than double-stranded DNA.bone fide transcription factors. We conclude the transcription factor complement of dinoflagellates is even smaller than previously thought suggesting that dinoflagellates have a reduced dependance on transcriptional control compared to other eukaryotes.Dinoflagellate CSPs do not share the nucleic acid binding properties expected for them to function as The online version contains supplementary material available at 10.1186/s12860-021-00368-4. Lingulodinium and Symbiodinium, have revealed a general paucity  of sequences annotated as transcription factors (TF). This is in sharp contrast to the roughly 6% of genes annotated as TF in plants  ATP (PerkinElmer) (see above) and purified using filtration chromatography on a Bio-Gel P10 column (Bio-Rad). One ng labelled probe was incubated with increasing concentrations of CSPs in the binding reactions as described above.Forty nt RNAs were synthesized by T7 RiboMAX RNA production kit (Promega) using dsDNA templates containing the N9 and T7 promoter sequences. Thereafter, RQI RNase-free DNase (Promega) was used for degradation of the dsDNA templates. The in vitro transcribed RNAs were quantitated using spectrophotometry (1.2 μg/μL), end-labeled using [γ-Symbiodinium and Lingulodinium CSPs were cloned and expressed as a fusion protein with a C-terminal GST tag as described above. The BL21 cell lysates were centrifuged and the supernatants containing GST tagged CSPs were incubated with Glutathione Sepharose 4B beads (Promega) for 45 min at room temperature with end-over-end agitation. Beads were washed 4 times in PBS. Immobilized LpCSP1, SkCSP1, SkCSP2 and SkCSP3 were tested for sequence-specific DNA binding activities against a set of degenerate oligonucleotides using a selection and amplification binding assay (SAAB) [y (SAAB) , 36. A sy (SAAB) . DNA wasAdditional file 1: Supplemental Figure S1. Purification of LpCSP1, SkCSP1, SkCSP2 and SkCSP3. A shows recombinant LpCSP1-GST, SkCSP1-GST, SkCSP2-GST and SkCSP3-GST analyzed on an 18% acrylamide SDS-PAGE gel after affinity purification. B shows LpCSP1, SkCSP1, SkCSP2 and SkCSP3 after removal of the GST tag by thrombin digestion and binding to glutathione-Sepharose 4B beads. The sizes of the molecular weight markers (left) are shown in kilodaltons.Additional file 2: Supplemental Table S1. List of primers used for PCR amplification and cloning of LpCSP1, SkCSP1, SkCSP2 and SkCSP3 sequences in pGEX4T2 plasmid.Additional file 3: Supplementary Table S2. List of proteins selected for phylogenetic reconstruction.


The adrenal cortex produces steroid hormones as adrenocortical hormones in the body, secreting mineralocorticoids, glucocorticoids, and adrenal androgens, which are all considered essential for life. Adrenocortical tumors harbor divergent hormonal activity, frequently with steroid excess, and disrupt homeostasis of the body. Aldosterone-producing adenomas (APAs) cause primary aldosteronism (PA), and cortisol-producing adenomas (CPAs) are the primary cause of Cushing’s syndrome. In addition, adrenocortical carcinoma (ACC) is a highly malignant cancer harboring poor prognosis. Various genetic abnormalities have been reported, which are associated with possible pathogenesis by the alteration of intracellular signaling and activation of transcription factors. In particular, somatic mutations in APAs have been detected in genes encoding membrane proteins, especially ion channels, resulting in hypersecretion of aldosterone due to activation of intracellular calcium signaling. In addition, somatic mutations have been detected in those encoding cAMP-PKA signaling-related factors, resulting in hypersecretion of cortisol due to its driven status in CPAs. In ACC, mutations in tumor suppressor genes and Wnt-β-catenin signaling-related factors have been implicated in its pathogenesis. In this article, we review recent findings on the genetic characteristics and regulation of intracellular signaling and transcription factors in individual tumors. Adrenocortical tumors are broadly classified into adenomas and carcinomas based on their potential biological behavior. In addition, adrenocortical adenomas are further subdivided into functional adenomas that secrete excessive steroid hormones and non-functional ones which do not. In this review article, we will review the findings of recently reported studies on genetic alterations and their regulation of intracellular signaling in aldosterone-producing adenoma (APA) as a cause of primary aldosteronism (PA) and cortisol-producing adenoma (CPA) as a cause of Cushing’s syndrome, subclinical Cushing’s syndrome, and adrenocortical carcinoma (ACC). APA is an adenoma producing excessive aldosterone autonomously, and somatic mutations of ion channels located at the cell membrane have been frequently reported, resulting in alteration of calcium signaling and its downstream transcription factors ,2,3,4,5.KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, CLCN2, and CTNNB1 genes have been frequently detected in APA [KCNJ5 encodes a rectifying potassium ion channel (Kir 3.4) that regulates resting cellular membrane potential. The genetic variants and hot spots of somatic mutations in KCNJ5 are well characterized, including L168R, G151R, T158A, G151E, I157del, T152C, and E154Q [NR4A1 (NGFIB), NR4A2 (NURR1), and NR4A3 (NOR1) [KCNJ5 in cell line experiments increased intracellular calcium ion concentration due to depolarization, enhanced expression of NURR1, and induced expression of CYP11B2 [KCNJ5 mutation is mediated by the activation of NR4A family orphan receptors via calcium signaling through the increase in intracellular calcium ion concentration, which in turn induces the expression of aldosterone synthesis genes, including CYP11B2  ,29 and t3 (NOR1) . It has  CYP11B2 . These r CYP11B2 .ATP1A1 encodes a sodium-potassium cotransporter , a protein whose essential function is to export sodium ions out of the cell and transport potassium ions into the cells. Genetic variants of somatic mutations in ATP1A1 include L104R, del100_104, V332G, and G99R [ATP1A1 L104R loses ion selectivity and allows hydrogen ions to enter the cell, resulting in an increase in aldosterone production via cell depolarization or intracellular acidification [ATP1A1 del100_104 is another genetic variant that causes loss of ion selectivity, and it has been reported that the influx of sodium ions, due to the loss of ion selectivity, causes cell depolarization , and somatic mutations such as G403R, S652L, F747L, and R990H have been reported [CACNA1H encodes a T-type calcium ion channel (CaV3.2), and somatic mutations such as I1430T and T4289C have been reported [CACNA1H lead to gain-of-function, which is a decrease in the threshold of the potential for calcium ion influx into the cell, increasing intracellular calcium ion concentration, subsequently triggering depolarization and promoting aldosterone secretion , suggesting its roles in promoting Ca ion accumulation in the ER. Calneuron1 is also reported to be highly expressed in APA [The expression of ntration . TASK isl cortex . PCP4 is[CYP11B2 , it may d in APA , suggestHSD3B2 was more highly expressed in the adrenal cortex than HSD3B1. However, HSD3B2 expression was not significantly increased in normal tissues and APA. In contrast, HSD3B1 is significantly upregulated in APA tissues compared to the normal adrenal cortex, suggesting that HSD3B1 is an important factor in APA [HSD3B1 is also dependent on calcium signaling and increases in response to the transcriptional activity of the NR4A family of transcription factors, which are considered downstream transcription factors of calcium signaling. Therefore, the above calcium signal regulators may be involved in the gene expression of CYP11B2 as well as HSD3B1.r in APA . Gene exCYP11B2 gene contains transcription factor-binding sequences, such as the NGFI-B response element (NBRE), Ad4, Ad5, and Ad1. The NR4A family is an orphan nuclear receptor for which no ligand has been identified, and its expression levels and intracellular signaling may contribute to the expression of downstream genes [CYP11B2 [CYP11B2, still remain unknown. In 2018, we identified poly (ADP-ribose) polymerase 1 (PARP1) as a transcription factor complex of NURR1 in H295R cells [Ad4BP, and is known to be essential for adrenal and gonadal development. SF-1 expression is also reported to be upregulated in APA [The promoter region of the am genes ,46,47. T[CYP11B2 . It is k[CYP11B2 , SRC-1 [[CYP11B2 , and oth[CYP11B2  as well 5R cells   family proteins, to which PDE8B and PDE11A belong, function as cAMP-degrading enzymes. When intracellular cAMP is degraded by PDE family proteins, PKA decreases its enzymatic activity due to a decrease in intracellular cAMP concentration. Mutation of PDE family proteins causes the loss of enzymatic activity, which leads to the loss of intracellular cAMP degradation, an increase in intracellular cAMP concentration, and an increase in PKA activity. It has been reported that PDE8B is predominantly expressed in the adrenal glands and other steroid hormone-secreting organs. H305P decreases the enzymatic activity of PDE8B and increases the concentration of intracellular cAMP ,11. PDE1GNAS) gene, a G-protein alpha-subunit associated with MC2R, have also been reported in CPA. GNAS mutations activate cAMP-PKA signaling, which in turn enhances the expression of the downstream CYP11B1 gene [CYP11B1 gene contains transcription factor binding sequences such as Ad4, Ad5, and Ad1 [Mutations in the guanine nucleotide-binding protein subunit alpha , but its prognosis is poor or dismal due to its rapid clinical growth and progression. There are two types of ACCs: functional tumors that are based on the presence of hormone excess and nonfunctional tumors that do not secrete hormones.TP53 and RB1 [ZNRF3 and CTNNB1 [CDKN2A [DAXX, MED12, and MEN1 [IGF2 (insulin-like growth factor 2), PRKAR1A, RPL22 , CCNE1 (cyclin E1), CDK4 (cyclin dependent kinase 4), TERT (telomerase reverse transcriptase), and TERF2 (telomeric repeat binding factor 2) have also been reported as genetic mutations in ACC [Genetic mutations in adrenocortical malignancies have been detected in tumor suppressor genes such as  and RB1 ,71,72, md CTNNB1 ,21,71, m [CDKN2A , and mutand MEN1 ,71. In as in ACC .Mutations in tumor suppressor genes are also frequently reported in cancers of other organs, and they play important roles in the pathogenesis of ACC.TP53 mutations in ACC are nonsense or frameshift mutations that cause a loss of TP53 function [TP53 gene is also known to be the causative gene of Li-Fraumeni syndrome. TP53 encodes p53 protein. The p53 protein is constantly degraded and accumulates in the cells when it detects DNA damage and suppresses cell growth [RB1 is a tumor suppressor gene detected in retinoblastoma, and RB protein binds to the transcription factor E2F and inhibits its transcriptional activity, thereby suppressing cell growth [function . TP53 gel growth . The dell growth . In ACC,l growth ,18.CTNNB1 mutation was frequently detected in ACC, suggesting its possible contribution to tumor growth or progression. β-catenin is a transcription factor, and its activation depends not only on the accumulation of β-catenin in the nucleus by post-translational modification but also on the effect of transcription factor complexes. Therefore, it is expected that the epigenomic factors that construct the transcription factor complex will be elucidated in the future, in order to be able to develop drugs that specifically target it.Mutations in the Wnt-β-catenin system, an important intracellular signal for cell proliferation, are also important. β-catenin is a transcription factor that is constantly degraded, and its degradation mechanism is regulated by the phosphorylation of β-catenin . Wnt proZNRF3 is a cell surface ubiquitin ligase that regulates Wnt-β-catenin signaling by ubiquitinating and degrading the Wnt protein receptors Frizzled and LRP6 [ZNRF3 loses its activity due to mutation, Wnt-β-catenin signaling is not regulated and β-catenin activity is increased [and LRP6 . When ZNncreased ,71  is a known factor that interacts with RNA polymerase. MED12 is involved in the activity of β-catenin, and its mutation has been reported to increase the activity of Akt and decrease the activity of GSK3B, an upstream regulator of β-catenin, resulting in increased transcriptional activity of β-catenin [MED12 mutations contribute to the growth of malignant tumors [MED12 mutations, remain unclear, and future studies are expected . MEN1 acts as a transcriptional repressor in the epigenome, forming protein–protein interactions with various transcription factors and repressing their transcriptional activity. It has been reported that MEN1 also binds to β-catenin and affects its transcriptional activity, while β-catenin has been reported to activate transcription [MEN1 have been implicated in the pathogenesis of MEN1, and Menin promotes the expression of p27 mutations in MEN1, which have been implicated in the pathogenesis of MEN1. However, the mechanism by which Menin promotes p27 expression and inhibits cell proliferation is thought to be disrupted by genetic mutations [cription ,82. MutaDAXX, a chromatin remodeling factor, affects histone H3.3 and promotes telomere elongation. It also interacts with β-catenin and TCF4 to increase its transcriptional activity [DAXX in ACC are often reported to be deletion type mutations, and their relevance to proliferation is currently unclear.activity . MutatioIGF2 is a key factor in adrenal development, and increased gene expression has been reported in ACC [RPL22, CCNE1, CDK4, TERT, and TERF2 in ACC using RNA sequencing [CCNE1 and CDK4 are cell cycle regulating genes, which control cell proliferation, while TERT and TERF2 are telomere-related genes, which regulate cell senescence and immortality.d in ACC . IGF2 had in ACC , suggestquencing . CCNE1 aLRIG1, ZFPM1, CRIPAK, GARS, and ZNF517 have been discovered [Additionally, ACC has been investigated using next-generation sequencing, and mutations in genes such as scovered . AlthougAs mentioned above, β-catenin, which plays a critical role in adrenal carcinoma, binds to the transcription factors TCF or LEF to form transcription factor complexes. p300 , CBP 8787, and oCYP11B1. The Wnt-β-catenin system is important in ACC, and the suppression of β-catenin transcriptional activity is important in future therapeutic development. Mutations of β-catenin have been reported in adenomas, but β-catenin is mainly involved in tumorigenesis, and in adenomas, it seems to promote steroid production as a secondary effect by affecting the NR4A family and CREB. In addition, the presence of transcription factor complexes is important for the activation of transcription factors, and future studies focusing on transcription factors and transcription factor complexes are warranted in APA, CPA, and ACC.In APA, the cause of PA, the upregulation of aldosterone synthesis genes by calcium signaling is a key cascade that enhances aldosterone secretion by activating transcription factors, mainly the NR4A family. In CPA, the activation of cAMP-responsive transcription factors such as CREB increases cortisol secretion by upregulating the gene expression of


FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of  Tumors represent the assembly of cancer and tumor microenvironment cells (TME). Cancer-associated fibroblasts (CAFs) are prominent cells within the tumor microenvironment and are critically involved in cancer progression. Recently, CAFs have emerged as attractive therapeutic targets for gene therapy ,2,3. HowAfter the successful registration of the gene therapy anticancer drug Imlygic by the Food and Drug Administration (FDA), a huge amount of works in the field of anticancer gene therapy has been published. Currently, great interest in the development of antitumor gene therapy is, where possible, the use of local  drug administration, which reduces the systemic toxicity of drugs ,5. By thWhen local administration of the gene therapy drug is used, a variety of tumor cells might be affected—cancer cells and microenvironment cells including CAFs. Cancer cells are well known for their cellular plasticity, contributing to tumor heterogeneity and therapy resistance . This caFAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, SPARC) as elements for transcriptional targeting of gene expression in tumor fibroblasts.In this study, we investigated the possibility for transcriptional targeting of cancer-associated fibroblasts. Herein, the promoters of certain genes, the expression of whose were increased in CAFs, were used to evaluate their ability for efficient and selective expression of transgenes in these cells. The analysis of transfected cell populations in a tumor in vivo is quite difficult and elusive. Therefore, cancer cell lines of pancreatic adenocarcinoma, which is characterized by the most developed tumor microenvironment, were chosen as a model for in vitro experiments. A primary culture of human fibroblasts was used as CAFs. Such an approach, although it has several limitations, give us the opportunity to examine in a simple model the possibility of using the promoters of the genes with a high level of expression in the stroma  method .We have suggested that the promoters of genes, the expression of whose is increased in the tumor stroma, could be active mainly in the cells of the tumor microenvironment. To test this hypothesis, the following genes FAP, CXCL12, CTGF, and SNAI1 genes is characteristic of CAFs in the TME of pancreatic cancer [FAP gene is selectively expressed in stroma fibroblasts of epithelial tumors. FAP is robust and selective marker for reactive mesenchymal stroma cells associated with pathophysiologic tissue remodeling [CXCL12 gene encodes a chemokine that is involved in inflammatory processes associated with cell migration. In pancreatic cancer, the CXCL12 is predominantly secreted by CAFs, while its expression in cancer cells is virtually absent [CTGF gene is predominantly expressed by TME fibroblasts [SNAI1 gene encodes the SNAIL transcription factor, which regulates the epithelial-mesenchymal transition in the processes of embryonic development, tissue regeneration, and carcinogenesis. Expression of SNAIL in pancreatic cancer is observed mainly in the nuclei of stroma cells, while it is rarely found in cancer cells [According to the literature, the expression of the c cancer ,18,19,20modeling . Due to modeling . The CXCy absent . The CTGroblasts . The SNASPARC gene, which encodes a protein involved in the remodeling of the extracellular matrix in pancreatic cancer, is characteristic of both cancer cells and CAFs, but in the latter, its expression is much higher [IGFBP2 gene is a potential stroma-associated biomarker in pancreatic cancer [JAG1 gene encodes the Jagged-1 protein, one of ligands that interacts with its respective Notch receptor. The activation of ligand-induced Notch signaling pathway in the CAFs is associated with poor prognosis, metastasis, and aggressiveness of the tumor [Expression of the h higher . IGFBP2 c cancer . The JAGhe tumor ,26,27.The selected panel of genes, to a certain extent, reflects the characteristic pattern of gene expression of the pancreatic adenocarcinoma microenvironment, with a predominance of genes associated with extracellular matrix remodeling, angiogenesis, metastasis, and growth factors ,29.FAP, CXCL12. IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes in the primary culture of human fibroblasts IVP-9TS, in pancreatic cancer cell lines  and lung cancer  cell line by quantitative real-time PCR (qPCR). The obtained transcription profiles of the FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes in the studied cancer cell lines are comparable with the values of their expression according to the RNA-seq E-MTAB-2706 database   . This inFAP and CXCL12 genes were detected only in IVP-9TS primary culture of human fibroblasts, which is consistent with the literature data on their predominant expression in CAFs, but not in cancer cells [JAG1 and IGFBP2 genes, a low transcription level was shown in all cell lines under the study. The transcription level of the CTGF gene was high and almost the same in all studied cell lines, except for MIA PaCa-2, in which it was absent. As expected, the transcription level of the SNAI1 gene was low in most cancer cell lines, except for the MIA Paca-2. However, SNAI1 expression was also almost absent in the IVP-9TS stroma fibroblasts. Transcription of the SPARC gene was higher in stroma fibroblasts (IVP-9TS) and lung cancer cells , compared to pancreatic cancer cell lines.The transcripts of the er cells ,22. For CXCL12 and FAP genes.Thus, specific transcription in the primary culture of human fibroblasts IVP-9TS was shown only for the FAP, CXCL12. IGFBP2, CTGF, SNAI1, and SPARC). Whenever possible, the minimal region of the promoter that provide high activity was chosen. These regions contain the necessary and sufficient number of binding sites for transcription factors (TFs), able to provide the activity of the promoter. Based on the literature analysis of data on the deletion analysis of promoters, we selected regions of promoters for obtaining reporter constructs . The proFAP and CXCL12 genes, which were specifically transcribed in fibroblasts, two promoter fragments were chosen: the first, the minimal promoter, and the second, the promoter with distal promoter elements, in order to evaluate their contribution to the possible specific regulation of the promoter activity. For the FAP gene promoter is about 2000 bp. The core promoter controlling FAP expression is located within the 750 bp region, upstream the transcription initiation start. The most critical for transcription of the FAP gene is the proximal region of ~126 bp located upstream the transcription start site. This region provides the minimum level of transcription and contains the most important cis-regulatory elements (CREs) [The size of the s (CREs) . In thisCXCL12 gene promoter is about 1500 bp [CXCL12 gene expression [The length of the  1500 bp . Deletio 1500 bp . A 753 bpression .IGFBP2 gene promoter is less than 1000 bp length and is located in the CpG island. The region of about 640 bp upstream the first exon of the IGFBP2 gene has promoter activity and does not contain TATA and CAAT sequences [IGFBP2 gene promoter; three binding sites for this factor were found in the promoter. In the proximal region of the promoter, there are binding sites for the Sp1 and NF-κB factors, and the E-box is located in distal part [The equences . The tratal part . We haveCTGF gene promoter is about 800 bp; most of the identified binding sites for transcription factors are located in the proximal region extending for about 550 bp upstream the ATG site [The ATG site . Based oJAG1 gene promoter has hardly been studied and there are few data on its structure. Identification of the human JAG1 gene promoter was based on bioinformatic methods [JAG1 gene promoter, thus the total size of the promoter is considered to be about 1700 bp. Our analysis based on the UCSC Genome Browser database showed that this region contains the largest number of putative binding sites for TFs, and we used this region for cloning.In contrast to other promoters used in this work, the  methods . As mentSNAI1 gene promoter contains an E-box essential for the regulation of expression by negative feedback [SNAI1 gene with a length of 929 bp containing most of the necessary regulatory elements was cloned [The feedback , two HREfeedback , Smad elfeedback , NFκB-sefeedback , sites Afeedback . A fragms cloned .SPARC gene requires CREs, the most important of which are purine-rich sequences—GGA-boxes, connected to each other by a 10-nucleotide spacer. The first GGA-box provides maximum promoter activity, while the pyrimidine-rich spacer region is responsible for negative regulation [SPARC gene promoter [Transcription of the gulation . Based opromoter ,42, we cluciferase gene under the control of various promoters  gene (designated as SURV) [Plasmid pGL3-PV containing the standard weak nonspecific SV40 promoter and pGL3-Basic vector, a promoterless plasmid, were used as control constructs in the experiment. To evaluate the relative strength of the promoters, a reporter construct containing a strong viral nonspecific cytomegalovirus immediate-early promoter (CMV) known as the “gold standard” of a highly active promoter was used as a control. A construct containing the well-studied cancer-specific promoter of the human as SURV) ,44, was Obtained results for transient transfection of various cells showed that the studied promoters are active in all studied cell lines . Unexpected results were obtained using constructs containing the CXCL12 and FAP promoters, which showed high specific transcription in the primary culture of human fibroblasts. It turned out that the activity of these promoters is unspecific and is the weakest among all the studied promoters.CTGF gene in these cells (data not shown).We used the human umbilical vein endothelial cells (HUVEC) as a control non-cancer cell line to determine the selectivity of the “tumor on” phenotype of the promoter . All proWith low activity (up to 1000 relative light units (RLU), weaker than the SV40 promoter)—FAP, CXCL12, JAG1.With medium activity —IGFBP2, SPARC, SURV.With relatively high activity —SNAI1, CTGF.Although the luciferase expression by reporter constructs containing regions of various promoters showed different activities in the panel of cell lines, the promoters could be divided into three groups, according to the level of their activity:We were searching for the promoters that are active selectively in CAFs. Our results indicate that there are no such selectivity for promoters under the study. Most likely, transcriptional targeting suffers from the problems similar for molecular targeting of surface markers—the lack of selective CREs. At least if the minimal region of the promoter that provide its high activity was chosen as CRE. Notwithstanding, we noticed that: (i) promoters could be grouped by strength, which could be represented as the median activity of promoter; (ii) universal promoters in CAFs could work better than in cancer cells. We suggest that the ratio of the median activity of promoters relative to each other could be relatively stable for conservative promoters. We chose one promoter from each of three groups  for whose the median activity differs by an order of magnitude to examine our hypothesis. Since we are interested to use promoters for gene therapy, we investigate our suggestion in the mouse model. Currently, little is known about how the activity of promoters in artificial constructs is related in vitro and in vivo. We compared the changes in the median promoter activity for different promoters during the transition from one system to another, as well as compared their activities relative to each other and the strong universal CMV promoter. To ensure that our results can be used in mouse models or translational research, we have determined the luciferase activity levels from previously used constructs containing human gene promoters in both mouse cell lines and in vivo mouse model.For further study, we should choose the promoters with the highest degree of identity between human and mouse in order to avoid the contribution of species-specific transcription factors. To determine the degree of identity of the studied mouse and human promoters of homologous genes, a bioinformatic analysis of the structure of the promoters was performed .Based on previous data, we hypothesized that the ratio of the median activity of promoters relative to each other could be relatively stable for conservative promoters. The CTGF, IGFPB2, and FAP 2.2 promoters were chosen from three groups that differed by orders of magnitude in median activity in human cell lines. The constructs with the strong universal CMV promoter and the cancer-specific SURV promoter were taken as control promoters. We have shown above, that universal promoters can provide a higher level of gene expression in fibroblasts. To examine this, along with the universal nonspecific viral CMV promoter, we took the previously studied universal mammalian promoter PCNA .To examine the possible contribution of species-specific TFs, we determined the activity of the studied promoters in murine cell lines. For this, the mouse lung cancer cell line CMT 167 (the cells could be used to obtain mice with inoculated syngeneic tumor) and the mouse fibroblasts NIH/3T3 were employed. Transfection and data analysis was performed as in the case of human cell lines. The values of the promoter activities are shown in the Analysis of the results obtained showed that all promoters with respect to CMV exhibit low activity. This was expected due to the calculated values of median activity relative to CMV promoter for human cell lines. The activity of all studied promoters was at least an order of magnitude lower than in human cell lines. The activity of promoters in cancer cells and fibroblasts differed little, and can be represented as the median activity. The median activity of the promoters decreases in the row CTGF, IGFBP2, and FAP 2.2, similar to what was shown previously for human cell lines. However, the level of changes in the promoter activity was other; the changes were in 2–3 times, but not by an order of magnitude, as in the case of human cells. We suggest that this could be because the degree of identity of human and mouse promoters is about 65–80%, which reduces the efficiency of human promoters in mouse model.Survivin (BIRC5) gene promoters . It was shown that in the CMT 167 model the transfection level is the highest, which is important, since other promoters exhibit very low activity, and in vivo their activity could be lower than the sensitivity level of the method.6 CMT 167 lung cancer cells at both flanks. When the tumors reached the size of 50–120 mm3, the corresponding constructs were injected intratumorally. A polyplex previously examined by us for gene therapy purposes was used as a non-viral vector for the delivery of constructs into cells [In total, seven groups of mice were formed (3 mice per group)—one group for each construct and a control group of animals. C57BL mice were subcutaneously inoculated with 10The activity of promoters decreases in the series of CTGF, IGFBP2, and FAP 2.2/SURV. The activity pattern of these promoters completely repeat the activity patterns observed by us during in vitro transfection of mouse cell lines. The ratio of the activity of these promoters is conserved both among themselves and relative to the activity of the universal CMV promoter.CTGF gene is activated by hypoxia [An exception is the activity of the PCNA promoter, which in the case of in vitro showed higher values of activity than in vivo. We hypothesize that this may be either due to contact inhibition of cell growth in the tumor (PCNA is a well-known proliferation marker), or due to tumor hypoxia, it is assumed that the activity of the PCNA promoter is inhibited during hypoxia ,48. On t hypoxia . Thus, iThe rapid development of gene therapy drugs with non-viral vectors is restrained by such factors as ineffective transfection of target cells and a low level of transgene expression. Intratumoral administration of such drugs can lead to transfection of all types of tumor cells—the cancer cells, immune cells of the tumor, and cancer-associated fibroblasts. The latter are of interest as a long-term source for the synthesis of therapeutic genes. The possibility of transcriptional targeting of transgene expression in CAFs is a poorly studied area. Little is known about which tumor cells are primarily subjected to gene therapy transformation and, as far as possible, the implementation of transcriptional targeting. The analysis of transfected populations of cells in a tumor in vivo is quite complex and elusive. Therefore, as a model for in vitro study, the primary culture of human fibroblasts and cancer cell lines of different lineage, were chosen.CXCL12 and FAP genes that showed high and specific transcription in fibroblasts. Transcription of the IGFBP2, CTGF, JAG1, and SPARC genes was less specific, but in most cases it was increased in fibroblasts.The aim of our study was to reveal the possibility to use promoters of the genes with increased expression in the TME for transcriptional targeting of transgene expression in CAFs. During the study of gene transcription in a panel of human cancer cell lines and primary culture of human fibroblasts, we identified the CXCL12 and FAP genes was observed; by pattern of gene expression, they corresponded to fibroblasts, and the SURV promoter of the cancer-specific gene Survivin (BIRC5) worked in it worse than in cancer cells. Thus, we conclude that IVP-9TS was a relevant and sufficient model of CAFs.It is well known that the population of CAFs is highly heterogeneous, which is due to both the different origin and stage of differentiation of cells, as well as because of functional heterogeneity of CAFs subpopulations in the tumor stroma . All of luciferase gene. For example, previously published works indicate the activity of the FAP promoter as part of an artificial construct in FAP-positive cell lines, where the required TFs are present [We hypothesized that promoters of genes with increased expression in stroma may maintain selective promoter activity in CAFs. We used artificial constructs, where such promoters were used to control the expression of the reporter  present ,52. To tCXCL12, FAP, and SPARC genes, which showed high transcription levels in IVP-9TS as compared to cancer cell lines. For the promoters of the CXCL12 and FAP genes, the transcripts of whose were detected specifically in IVP-9TS fibroblasts, an extremely low and nonselective promoter activity was shown both in IVP-9TS and in cancer cell lines. This was unexpected, especially in the case of the FAP gene promoter. Despite the fact that some works indicate the importance of the distal CREs of the FAP gene promoter for its tissue-specific activity [FAP gene promoter.We have demonstrated that almost all of the studied promoters (except for CTGF and SNAI1) exhibit extremely low levels of promoter activities. We found selective promoter activity in the primary culture of human fibroblasts for none of the studied promoters. Even in the case of the promoters of the activity ,52, we dCTGF is tightly regulated [It is worth noting that the CTGF promoter activity was highest among all studied promoters. The observed activity of the CTGF promoter in the endothelial cell line HUVEC may suggest that the promoter could belong to endothelial cell-specific promoter class . These tegulated . The higSurvivin gene promoter in the reporter constructs do not correlate with the high level of the native Survivin protein in corresponding cell lines [We did not find any correlations between the high level of endogenous gene transcription and the activity of the corresponding promoters in the reporter constructs. This generally confirms the previously published work, which showed that the high activity of the ll lines . When analyzing the activities of promoters on human cells, we had divided the studied promoters into several groups according to the level of activity. In the absence of a pronounced selectivity of the promoters, we suggested that each of the studied promoters could be characterized by its own median level of activity in all used cell lines (median activity). We believe such median activity could be a characteristic of each promoter, which is possibly conserved in various types of cells, and depends little on their lineage. The median activity of a promoter can serve as its characteristic, which depends on the structure of the promoter, the composition of binding sites for TFs. Thus, the most important for the median activity of the promoter is the possibility of using a wide range of transcription factors. Really, the universal viral promoters CMV and SV40 showed a higher level of activity in fibroblasts as compared to cancer cells. This fact, as well as the shown low activity of most promoters, suggests that the universal promoters may be suitable for providing expression in CAFs, although this may sacrifice the selectivity for promoter activity. Other drawbacks of many universal promoters is silencing, even when administered as part of artificial constructions by non-viral vectors ,56. ThusWe have shown that the concept of median promoter activity is retained in vivo, at least partially and upon short-time transgene expression. This is a fundamental point. We speculate that the median promoter activity compared to CMV activity (or other known promoter) may offer insights for the promoter activity in vivo. The degree of identity and features of the regulation of the promoter should also be taken into account. Thus, it is difficult to expect high promoter activity for weak promoters such as FAP, but it is possible for strong promoters like CTGF and PCNA. In addition, the results obtained suggest that additional CREs should be used to provide selective promoter activity. CXCL12 and FAP) have the lowest and nonspecific activity when used in artificial reporter constructs.No correlation was found between the high level of native gene transcription in cells and the activity of the corresponding promoters in reporter constructs during transfection of the same cells. The promoters of genes that showed the highest transcriptional specificity in fibroblasts (The promoters used in this work could be divided into three different groups by their strength (the median activity characterizing each promoter). The CTGF promoter was the strongest promoter among the studied promoters. The ratios of the promoter activity for different promoters in reporter constructs can persist both when used in vitro and in vivo, including for different species. We have shown this in our study under conditions of short-term expression, at least for conservative promoters (a relatively high degree of identity of human vs mice promoters).To ensure transcriptional targeting it is not enough to use only promoters. Other CREs  should also be used.Intratumoral administration of therapeutic constructs can lead to transfection of all types of tumor cells. Based on our in vitro results, we suggest that the level of transgene expression in fibroblasts compared to cancer cells can be higher when using universal promoters .The results obtained showed that:TERT and BIRC5 genes [Apparently, the search for promoters providing transcriptional targeting in CAFs may be in vain when it is required to provide selective transgene expression in them. In general, we have to admit that transcriptional targeting of CAFs has limitations similar to the targeting of surface markers. It is possible to achieve a high level of transgene expression in fibroblasts, but sacrifice selectivity. As in the previous work , we assuC5 genes . Possiblfibroblast activation protein (FAP) gene. For cloning and further study, we used two fragments of the FAP promoter, FAP 0.75 (750 bp) and FAP 2.2 (2144 bp) with coordinates −2026/+118 and −632/+118 relative to the transcription start site (TSS), respectively.The FAP used was the promoter of the human C-X-C motif chemokine ligand 12 (CXCL12) gene. For cloning and further study, we used two fragments of the CXCL12 promoter, CXCL12 0.7 (753 bp) and CXCL12 1.5 (1517 bp) with coordinates −1397/+120 and −633/+120 relative to the TSS, respectively.The CXCL12 used was the promoter of the human insulin-like growth factor binding protein 2 (IGFBP2) gene. For cloning and further study, we used a fragment of the IGFBP2 (634 bp) promoter with coordinates −531/+103 relative to the TSS. The IGFBP2 used was the promoter of the human connective tissue growth factor (CTGF) gene. For cloning and further study, we used a fragment of the CTGF (408 bp) promoter with coordinates −365/+43 relative to the TSS.The CTGF used was the promoter of the human jagged canonical Notch ligand 1 (JAG1) gene. For cloning and further study, we used a fragment of the JAG1 (1724 bp) promoter with coordinates −1238/+486 relative to the TSS.The JAG1 used was the promoter of the human snail family transcriptional repressor 1 (SNAI1) gene. For cloning and further study, we used a fragment of the SNAI1 (929 bp) promoter with coordinates −868/+61 relative to the TSS.The SNAI1 used was the promoter of the human secreted protein acidic and cysteine rich (SPARC) gene. For cloning and further study, we used a fragment of the SPARC (1234 bp) promoter with coordinates −1182/+52 relative to the TSS.The SPARC used was the promoter of the human baculoviral IAP repeat containing 5 (BIRC5) gene. For further study, we used a fragment of the SURV (1498 bp) promoter, that was earlier studied in our laboratory [The SURV used was the promoter of the human boratory ,44 and iproliferating cell nuclear antigen (PCNA) gene. In our study, we used a PCNA short promoter  that was earlier studied in our laboratory [The PCNA used was the promoter of the human boratory ,57 and iThe CMV used was CMV Pr/Enh promoter containing an AseI/BglII fragment of the promoter of early cytomegalovirus genes from plasmid pEGFP-N1 (Clontech Laboratories (Takara Bio), Shiga, Japan) .To obtain reporter constructs, chosen regions of promoters were amplified from the human genomic DNA using specific primers  and Q5 HTo obtain FAP 2.2-luc-pGL3 plasmid the promoter FAP 2.2 was amplified by PCR using the primers FAP-for-2145/FAP-NcoI-Rev and subcloned to the pAL-2T vector (Evrogen) by TA-ligation. Fragment containing FAP 2.2 was excised from the pAL-2T vector by SacII (blunted)/NcoI restriction endonucleases and cloned into a pGL3-Basic Vector digested with HindIII (blunted)/NcoI restriction endonucleases by sticky-blunt ligation. To obtain FAP 0.75-luc-pGL3 plasmid the promoter FAP 0.75 was amplified from FAP 2.2-luc-pGL3 plasmid by PCR using the primers FAP-HindIII-for/FAP-NcoI-Rev and cloned into a pGL3-Basic vector digested with HindIII/NcoI restriction endonucleases by sticky-end ligation.To obtain CXCL12 1.5-luc-pGL3 plasmid, the promoter CXCL12 1.5 was amplified by PCR using the primers CXCL12-HindIII-for/CXCL12-NcoI-Rev and cleaved by HindIII/NcoI restriction endonucleases. The excised fragment was cloned into a pGL3-Basic vector digested with HindIII/NcoI. To obtain CXCL12 0.7-luc-pGL3 plasmid, the promoter CXCL12 0.7 was amplified from CXCL12 1.5-luc-pGL3 plasmid by PCR using the primers SDF1-F630/CXCL12-NcoI-Rev. The amplification product was cleaved by NcoI restriction endonuclease and cloned into a pGL3-Basic vector digested with SmaI/NcoI restriction endonucleases.To obtain CTGF-luc-pGL3 plasmid the promoter CTGF was amplified by PCR using the primers CTGF-HindIII-for/CTGF-NcoI-Rev and cleaved by HindIII/NcoI restriction endonucleases. The excised fragment was cloned into a pGL3-Basic vector digested with HindIII/NcoI.To obtain JAG1-luc-pGL3 plasmid the promoter JAG1 was amplified by PCR using the primers JAG1-HindIII-for/JAG1-NcoI-Rev and cleaved by HindIII/NcoI restriction endonucleases. The excised fragment was cloned into a pGL3-Basic vector digested with HindIII/NcoI.The fragment of the SNAI1 promoter was taken from modified plasmid pGL3 kindly provided by the laboratory of Dr. Antonio Garsia de Herreros (Spain). To obtain SNAI1-luc-pGL3 plasmid the fragment containing SNAI1 promoter was excised by KpnI/HindIII restriction endonucleases and cloned into a pGL3-Basic vector digested with KpnI/HindIII. To obtain SPARC-luc-pGL3 plasmid the SPARC promoter was amplified by PCR using the primers SPARC-F2-For/SPARC-R72-NcoI-Rev, subcloned to the pAL-2T vector (Evrogen) by TA-ligation. Fragment containing SPARC promoter was excised by NcoI restriction endonuclease and cloned into a pGL3-Basic vector digested with NcoI.The SURV-luc-pGL3 plasmid with the SURV promoter was obtained earlier ,44. The All restriction endonucleases were purchased from Thermo Fisher Scientific . All plasmids were isolated by Endofree Plasmid Midi or Giga Kit  according to the manufacturer’s protocol. The structure of all plasmids obtained was confirmed by restriction analysis and sequencing. All sequences of the promoters used could be found in ® CRL-1420), PANC-1 (ATCC® CRL-1469), AsPC-1 (ATCC® CRL-1682); human umbilical vein endothelial cells—HUVEC (ATCC® CRL-1730); non-small-cell lung cancer cell line Calu-1 (ATCC® HTB-54™) and mouse embryonic fibroblast cell lines NIH/3T3 (ATCC® CRL-1658™) were obtained from the American Type Culture Collection . Mouse lung carcinoma CMT 167  was obtained from European Collection of Cell Cultures . Cancer cell lines derived from human pancreatic cancer—MIA PaCa-2  .2 and 37 °C.MIA PaCa-2, PANC-1, AsPC-1, NIH/3T3, Calu-1, CMT 167 cell lines, and IVP-9TS fibroblasts were cultured in DMEM/F12 (1:1) medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin and 10% fetal bovine serum. The media and supplements were purchased from Gibco (Thermo Fisher Scientific). HUVEC cell line were cultured by BEGM Bronchial Epithelial Cell Growth Medium BulletKit  supplemented with 2% fetal bovine serum. Cells were maintained in a humidified atmosphere at 5% COluciferase gene and an internal control plasmid pRL-TK  in the weight ratio of 9:1. In 48 h after transfection, the activity of firefly and Renilla reniformis luciferases was measured in cell extracts using a Dual-Luciferase Reporter Assay System (Promega) and a GENios Pro luminometer . In parallel experiments, cells were transfected with a promoterless pGL3-Basic plasmid or a PV-pGL3 plasmid, containing only the SV40 promoter. For each construct under the study, at least three independent transfections were performed. The luciferase activity was normalized to that of BV-pGL3 Basic vector activity. Calculations were made using the MS Excel 2013 program.Cells were transfected in 24-well plates using Lipofectamine 2000  according to the manufacturer’s recommendations. Transfection was done with 1 mkg (per well) mixture of a reporter plasmid carrying the firefly To isolate total RNA cells were trypsinised and washed twice with phosphate-buffered saline (PBS). Then, the cell pellet was collected and stored at −70 °C prior to isolation of RNA.Total RNA was isolated from 1 million cells using an RNeasy Mini Kit (Qiagen) followed by treatment with DNAse RQ1 (Promega) according to the manufacturer’s protocol. The quality of RNA was analyzed by electrophoresis in a 1% agarose gel containing ethidium bromide. The amount of RNA was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) at the absorption wavelength of 260 nm.The transcription level of the genes under the study in different cell lines and IVP-9TS primary culture was evaluated by qPCR using a qPCRmix-HS SYBR reaction mixture (Evrogen). The first cDNA strands were synthesized using hexanucleotide primers and Mint reverse transcriptase (Evrogen) according to the manufacturer’s protocol. For this purpose, total RNA was isolated from the cell pellets of the corresponding cell lines. A gene specific primer pair see , was use6 of the CMT 167 cells were injected subcutaneously into both flanks of the lower part of the mouse body. This was done in accordance with the 3R principles to reduce the number of animals in the experiment. After 10 days, when the tumors reached the size of 50–120 mm3, a copolymer-DNA complexes containing corresponding plasmids were injected intratumorally in dose of 0.08 mkg DNA/mm3. A polyethylenimine (PEI)-polyethylene glycol (PEG)-TAT peptide copolymer (PPT) was used to obtain copolymer-DNA complexes, as described earlier [The C57BL mice (6–8 weeks) were obtained from the Pushchino Animal Breeding Facility . The studies using mice were reviewed and approved by the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Institutional Animal Care and Use Committee . All animal manipulations were performed according to the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, Council of Europe (ETS 123). For tumor generation, 100 μL of PBS solution containing 10Each tumor was placed in Lysing Matrix A tube  and 0.5 mL of PLB 1X buffer (Promega) was added. The tumor was grinded and lysed by FastPrep-24 machine . The aliquots of the lysates were used to determine the luciferase activity and amount of protein by the Bradford method.To measure the amount of protein, the samples were mixed with a dye reagent in the ratio of 4:1, and incubated at room temperature for 10 min with constant stirring. The absorbance was measured with the Benchmark™ Plus Microplate Reader  at 595 nm. The activity of firefly luciferase in cell extracts was measured using a Dual-Luciferase Reporter Assay System (Promega) and GENios Pro  luminometer. The luciferase activity was normalized to that of 1 mg of protein. Calculations were made using MS Excel 2013 program.To evaluate the degree of identity for human and mouse homologous genes and their promoters corresponding sequences were extracted from UCSC Genome Browser database. The following database assembly was used—Human Dec. 2013 (GRCh38/hg38) and Mouse Jun. 2020 (GRCm39/mm39).Alignment of the human and mouse promoter sequences was performed for region upstream the untranslated regions (UTR) using the AlingX Vector NTI program. First, the 2.5 kb regions upstream the UTR were aligned and the region of maximum identity was found. After that, the regions corresponding to the fragment of the used promoter were aligned. The coordinates were determined using a fragment of the promoter of the corresponding human gene, then the same coordinates were used for the mouse promoter, alignment was performed, and then the zone of maximum overlap was found—“max identity”. These are the best matching overlapping promoter regions.To align the gene sequences, the CDS (coding sequence) of the corresponding genes were taken. If there were several transcript variants, the closest ones were selected. Predicted Protein sequence from UCSC Genome Browser was taken to align proteins. Alignment was performed by NCBI BLASTP tool (Identities).


The nucleolus harbors the machinery necessary to produce new ribosomes which are critical for protein synthesis. Nucleolar size, shape, and density are highly dynamic and can be adjusted to accommodate ribosome biogenesis according to the needs for protein synthesis. In cancer, cells undergo continuous proliferation; therefore, nucleolar activity is elevated due to their high demand for protein synthesis. The transcription factor and universal oncogene MYC promotes nucleolar activity by enhancing the transcription of ribosomal DNA (rDNA) and ribosomal proteins. This review summarizes the importance of nucleolar activity in mammalian cells, MYC’s role in nucleolar regulation in cancer, and discusses how a better understanding  of aberrant nucleolar activity in cancer cells could lead to novel therapeutics. The nucleolus, one of the largest membraneless organelles, is located in the nucleus and is the home of all of the steps necessary for ribosomal biogenesis including rDNA transcription, ribosomal RNA (rRNA) processing, and the assembly of ribosomes. The lack of a membrane allows the nucleolus to be very dynamic, as its size and composition can adjust to the demands for protein synthesis. For example, in cases of hyperproliferation, a higher rate of protein synthesis and ribosome biogenesis must be achieved to allow for cell growth, and the nucleolus increases in size and density to accommodate those needs ,2.Nucleolar morphology and function are tightly regulated during the cell cycle. Nucleoli are most active in G2, the cell cycle phase that precedes cell division and when protein synthesis capacity is the highest . ConversMammalian cells may contain one or more tripartite nucleoli, and each comprise three morphologically and biochemically distinct compartments that perform specific roles to produce ribosomal subunits: the fibrillary center (FC), the dense fibrillar component (DFC), and the granular component (GC). The FCs are encased in DFCs, which are surrounded by the GC . These tThe initial step of ribosome biogenesis begins in the FC when upstream binding factor (UBF) and selectivity factor 1 (SL1), also known as TIF-1B, and TIF-1A, bind to RNA polymerase I (RNAPolI) at the rDNA promoter. This initiates the transcription of the 47S pre-rRNA precursor, giving rise to 28S, 18S, and 5.8S mature rRNAs ,18,19. TThe rRNAs undergo several steps of processing including methylations and pseudouridylations ,23. ThesErrors in rDNA transcription, rRNA processing, and ribosome assembly are the cause of several disorders collectively named ribosomopathies. These include degenerative diseases such as abdominal aortic aneurysm ,29 and PThe presence of nutrients and growth factors in the cellular environment leads to the activation of signaling pathways that promote cell growth, including the production of new ribosomes. Hyperproliferative cancer cells often display activated growth factor signaling that can induce elevated RNAPolI–mediated rDNA transcription ,39,40, lDue to the high demand for ribosomes, transformed cells hyperactivate nearly all steps in ribosome biogenesis including rDNA transcription, rRNA processing, and the expression of RPs . These cv-myc) that caused myelocytomatosis, leukemia, and sarcoma [v-myc was acquired from a cellular oncogene dubbed c-myc, now referred as MYC [MYC was originally discovered as a viral oncogene  that regulate key biological processes; specifically, MYC and MYCN are essential for embryonic development ,57. MembAll MYC variants contain highly conserved regions called MYC boxes (MB) C that arMBII is a crucial region where histone acetyltransferase (HAT) complexes bind. HATs promote histone acetylation, which opens chromatin, facilitating access by the transcription machinery and ultimately activating gene transcription ,85,86. MDrosophila, as well as in vertebrates [TFIIIB, critical for the activity of RNAPolIII [For proliferating cells to meet the ribosome and protein synthesis demand, rRNA production must be maintained at a high rate . The regtebrates . MYC aidtebrates . Second,tebrates ,56, whictebrates  and SL1 tebrates . Therefotebrates . AdditioNAPolIII ,96,97,98myc-/- fibroblasts expressing empty vector or reconstituted with MYC, we found that MYC promotes the expression of the regulators of nucleolar activity (nucleolin (NCL), which is needed for the processing of 47S pre-rRNA [nucleophosmin (NPM1) and fibrillarin (FBL). Due to their biophysical properties, NPM1 and FBL separate into immiscible phases, contributing to the assembly of the GC and DFC, respectively  and of its heterodimeric partner aryl hydrocarbon receptor nuclear translocator (ARNT) and promotes their expression [nucleolar and coiled-body phosphoprotein 1 (NOLC1), rRNA processing such as periodic tryptophan protein 2 homolog (PWP2), and protein synthesis such as 2-oxoglutarate and iron dependent oxygenase domain containing 1 (OGFOD1) in MYC-expressing cells [AHR in MYC-expressing cells causes decreased proliferation, nucleolar disassembly, and reduced protein synthesis. This suggests that MYC regulates nucleolar activity, and thus protein synthesis, partly through the transcriptional regulation of AHR. Moreover, AHR also regulates the production of nucleotides that are necessary for the elevated rate of rRNA production in cancer cells [Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), dihydroorotate dehydrogenase (DHODH), and uridine monophosphate synthetase (UMPS), encoding for the enzymes of the de novo pyrimidine biosynthesis pathway [DHODH and UMPS and to cooperate with MYC to transcribe these genes. Knocking down AHR in MYC-expressing cells decreases the expression of DHODH and UMPS and the levels of the pyrimidine nucleotide uridine 5-monophosphate (UMP), which affects the number of pyrimidines needed for rRNA synthesis [de novo pyrimidine biosynthesis to partially support nucleolar activity. MYC was also found to regulate the transcription of genes encoding for factors responsible for translation elongation and initiation factors such as eif4e [In addition to directly regulating the transcription of rDNA and RPs, MYC promotes the transcription of additional transcription factors that regulate one or more aspects of nucleolar function. For example, MYC binds to the promoter of the transcription factor pression . AHR in ng cells . Consequer cells ,109. Car pathway , were pr pathway ,111,112.ynthesis ,108. Theynthesis  activateas eif4e ,113,114.as eif4e .NPM1 [eif4e, ncl, and cdk4 [As discussed above, MYC was shown to regulate multiple aspects of nucleolar function, including transcription of rDNA and RPs genes . ConversNPM1  and NPM1and cdk4 ,116,117.ucleolus ,117. FBXoduction .Additionally, a feedback loop between MYC and ribosomal proteins such as RPL5 and RPL11 was also documented. MYC activity was found to be altered by RPL11, which is capable of binding to MYC, thereby inhibiting its transcriptional activity . MoreoveAs a result of the extensive effects of MYC on ribosome biogenesis, MYC-expressing cells generally have larger and hyperactive nucleoli . TherefoInterestingly, some drugs, including cisplatin, oxaliplatin, and doxorubicin, were shown to have off-target effects that inhibit RNAPolI activity, altering rRNA production ,122,123.de novo nucleotide biosynthesis, which limits nucleotide availability and thus impairs rRNA production affecting nucleolar activity. For instance, work with small-cell lung cancer (SCLC) models showed that SCLC with elevated MYC expression were more sensitive to the inhibition of the de novo purine biosynthesis enzyme inosine-5′-monophosphate dehydrogenase (IMPDH1/2), which affected rRNA production and thus, cell proliferation [de novo pyrimidine biosynthesis enzyme DHODH has been shown to decrease the production of rRNA, to induce nucleolar disassembly, and to stabilize p53 in glioblastoma, breast, and colon cancer cells, leading to a decrease in proliferation and an increase in apoptosis [Interfering with rRNA and ribosome production can alter the homeostasis and integrity of the nucleolus, activating nucleolar stress that causes cell cycle arrest and apoptosis. The best-known mechanism of nucleolar stress is mediated by p53 . Under nferation ,134. Inhpoptosis ,109. Addpoptosis . During poptosis ,136. WheIn recent years, the concept of ribosome heterogeneity has emerged , and undThe concept that ribosomes can be specialized may lead to a new research avenue for the development of drugs that target cancer-specific ribosomes. Whether MYC or other oncogenes regulate the production of tumor-specific ribosomes is yet to be determined. If such ribosomes exist, this will provide a completely new area of investigation with potential for novel strategies to target tumor cells.


Bacteria usually contain several different σ factors to switch genes to be expressed. While the primary σ factor is responsible for housekeeping genes transcription and therefore essential, alternative σ factors are needed for the expression of genes induced in response to particular environmental stress or during development process. σ factors competitively bind to the core enzyme to form holo RNAP. To save cellular resources, they should be activated at an appropriate extent and timing. Therefore, the understanding of how the activity of σ factors is regulated and which genes are controlled by σ factors are important for understanding cellular responses to environmental changes.The transcription of bacterial genes driven by DNA-dependent RNA polymerase (RNAP) is the first step in gene expression. Holo RNAP consists of multi-subunit, catalytic core enzyme, formed by the αGuerreiro et al. reviewed the signal transduction cascade that activates SigB and the role of the SigB-mediated general stress response in the virulence of the pathogen Listeria monocytogenes. The multi-protein complex stressosome is an essential stress sensor, conserved in Firmicutes, which include Bacillus subtilis and L. monocytogenes. It is composed of the RsbS and RsbR paralogs and the serine/threonine kinase RsbT. RsbT is released from the complex upon sensing of stress and controls phosphorylation states of the anti-anti-σ factor RsbV, thereby releasing SigB from the anti-σ factor RsbW. The SigB regulon composed of more than 300 genes are important to resistance to stresses, including acid and osmotic stress, and bile salt, during infection of L. monocytogenes. Veyrier et al. show a novel functional aspect of an anti-σ factor, i.e., activating function. Natural mutations observed in the anti-σ factor RskA of the σ factor SigK in clinical isolates of mycobacteria conferred such an activity. Truncation of the C-terminal part identified the region required for the activator function of RskA. This activating function of the anti-σ factor was also demonstrated in different bacterial genera, indicating underappreciated functions of anti-σ factors. In addition to the step of the formation of holoenzyme, the step of transcription initiation is the target of transcription factors interacting with σ factors. Vishwakarma and Brodolin review such transcription factors regulating σ factors and the holo RNAP. In contrast to anti-σ factors, Mycobacterium tuberculosis RbpA and Escherichia coli Crl act as activators by stabilizing the interaction of a σ factor with the core enzyme and promoting the formation of open promoter complex for transcription initiation. They also review phage proteins repressing σ factor function of the host. The effects depend on promoter types.This Research Topic contains three original research and three review articles. The half of the articles discuss how the activity of σ factors is regulated. The switching of a σ factor to form the holo RNAP is mainly regulated by the inhibitory interaction between σ factors and their cognate negative regulators, so-called anti-σ factors, which are modified and/or degraded in response to environmental stimuli. 70-family. Most of the members in this group are involved in the response to environmental stress. Temperature-dependent differential regulation of genes in an operon by two ECF σ factors in Pseudomonas aeruginosa was demonstrated by Bouffartigues et al. While heat shock induced all genes in the cmaX-cfrX-cmpX operon, encoding Mg2+-dependent channel, small protein of unknown-function, and mechanosensitive channel, respectively, by the σ factor AlgU, cold shock induced the latter two cfrX-cmpX from the SigX-dependent promoter located upstream of cfrX (intragenic region of cmaX). As the operon components encode proteins affecting membrane tension, which is influenced by cold shock, authors also showed that the drug valinomycin targeting the membrane induced SigX-mediated expression of the operon.The other three articles discuss the physiological function of σ factors. Extracytoplasmic function (ECF) σ factors comprise the largest group, Group 4, of the four groups in the σSoutourina et al. determined 1,562 potential TSSs for Clostridioides difficile genes using 5′ end RNA-seq. σ factors were assigned based on comparison of the sequences preceding the TSSs with the consensus patterns of the promoters already identified in C. difficile. This analysis allowed refining the promoter consensus sequences. Moreover, in this topic, only this study treated a σ factor belonging to the σ54 family, represented by RpoN in E. coli. The σ54-type σ factors, including C. difficile SigL, recognize promoters consisting of −24 and −12 regions, not −35 and −10 regions as the σ70-type ones, with respect to the TSS, and require cognate transcriptional regulators for transcriptional activation. Another high throughput sequencing technology to identify promoter sequences is ChIP-seq. The complex of the target σ factor and DNA fragments encompassing its binding chromosomal regions is recovered by using the antibody specific to the σ factor. The recovered DNA fragments are amplified, sequenced, and mapped. RpoS orchestrates general stress response in E. coli, like SigB in L. monocytogenes described above. Schellhorn reviews the RpoS regulon, which is determined by transcriptome, proteome, and ChIP-seq, of E. coli strains. Based on the regulon components, RpoS functions as a metabolic switch. As described by Guerreiro et al., the regulons of the σ factors involved in general stress response are not only essential for metabolic adjustment and stress response but also represent burden for the growth, leading to the paradigm Self-Preservation and Nutritional Competence (SPANC). This may explain how, in many situations, mutations leading to RpoS loss of function can contribute to improved nutrient scavenging.Transcriptional start site (TSS) determination is the first step to identify a promoter sequence. Recent high throughput sequencing technologies enable researchers to map the TSSs at the genome-wide level, identifying promoters of genes expressed under the growth conditions. The articles in this special issue represent a part of roles of σ factors as molecular switches for physiological adaptation to intra/extracellular environmental changes. Although not specifically covered by this issue, recent structural studies revealed the molecular mechanisms of the “switches” in more detail (Glyde et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Eukaryotic mRNA 3´-end processing is a multi-step process beginning with pre-mRNA transcript cleavage followed by poly(A) tail addition. Closely coupled to transcription termination, 3´-end processing is a critical step in the regulation of gene expression, and disruption of 3´-end processing is known to affect mature mRNA levels. Various viral proteins interfere with the 3´-end processing machinery, causing read-through transcription and altered levels of mature transcripts through inhibition of cleavage and polyadenylation. Thus, disruption of 3´-end processing contributes to widespread host shutoff, including suppression of the antiviral response. Additionally, observed features of read-through transcripts such as decreased polyadenylation, nuclear retention, and decreased translation suggest that viruses may utilize these mechanisms to modulate host protein production and dominate cellular machinery. The degree to which the effects of read-through transcript production are harnessed by viruses and host cells remains unclear, but existing research highlights the importance of host 3´-end processing modulation during viral infection. Viruses disrupt host transcription termination by interfering with the mRNA 3´-end processing machinery.This disruption leads to the production of read-through transcripts called Downstream-of-Gene (DoG) transcripts that include the upstream mRNA sequence but are retained in the nucleus and are therefore non-coding.Influenza A virus protein NS1 and herpes simplex virus-1 protein ICP27 inhibit canonical cellular mRNA cleavage, polyadenylation, or both through interference with CPSF subunits.Disruption of transcription termination in virus-infected cells occurs on a genome-wide scale and affects the antiviral response.Viruses selectively disrupt host 3´-end processing while viral transcription and 3´-end processing remain intact.via read-in transcription.Read-through transcription has a multitude of effects, including downregulation of genes that experience readthrough and inhibition of downstream genes Transcription of eukaryotic genes continues until termination, marked by cleavage of the pre-mRNA and discontinuation of RNA synthesis by RNA polymerase II (Pol II). Termination of eukaryotic pre-mRNAs and 3´-end processing, which refers to the endonucleolytic cleavage process followed by polyadenylation –6. EndonPolyadenylation is mediated by poly(A) polymerase (PAP), which catalyzes the addition of 50-100 adenine nucleotides to the exposed 3´OH of the mRNA after cleavage, forming the poly(A) tail , 10. WhiFOXP3 gene lead to downregulation of FOXP3, a key transcription factor for regulatory T cells, resulting in IPEX syndrome, a fatal autoimmune disorder linked to defective development of regulatory T cells  protein ICP27. Read-through transcription causes production of Downstream-of-Gene (DoG) transcripts that are retained in the nucleus and thus not translated. Such genome-wide disruption of transcription termination affects thousands of genes, including those involved in the innate antiviral response. Remarkably, the ability of viruses to disrupt host 3´-end processing specifically, while preserving their own transcription and 3´-end processing, allows for efficient viral gene expression and simultaneous host shutdown.Viruses disrupt host mRNA processing using various mechanisms –35, 37. Influenza virus is known to affect host RNA processing negatively through several mechanisms, including disruption of both 3´- and 5′-end processing , 38–41. There is conflicting evidence regarding the necessity of NS1 in viral disruption of transcription termination. Read-through transcription also occurs independently of CPSF-NS1 interaction in influenza strains producing NS1 proteins that do not bind CPSF . AlternaAdditionally, NS1 interferes with polyadenylation through binding to PABP2 using a region distinct from that which binds CPSF. This interaction leads to nuclear redistribution of PABP2, as well as disruption of its nucleocytoplasmic shuttling, and a consequent decrease in polyadenylation of host transcripts .via its immediate-early protein ICP27 (Infection with herpes simplex virus-1 (HSV-1) causes disruption of transcription termination in host genes in ICP27 , 47, 48.pro) causes in vitro cleavage of CstF-64 , a picornavirus, also affects host transcription and 3´-end processing factors , 50. Wennslation . This obHuman immunodeficiency virus-1 (HIV-1) Tat protein stimulates Pol II transcription of viral genes . Tat proFor other viruses, little has been reported regarding disruption of 3´-end processing, read-through transcription, and downstream effects of these processes during infection. However, other viruses use a multitude of methods to suppress host gene expression at several stages of the RNA production and maturation pathway, including interference with 5´-end processing and dysregulation of splicing , 33, 35.The extent of host RNA processing defects in virus-infected cells had remained unclear as understanding the relevant viral mechanisms was initially limited to the examination of sample genes or the use of reporter constructs. More recently, with genome-wide analysis through next-generation sequencing, it has become evident that viral disruption of mRNA processing affects cellular genes globally across the transcriptome. For instance, 7-8 hours after HSV-1 infection, 64% of cellular genes experienced greater than 15% of their total transcripts reading through . In factIn another example, during infection with an influenza strain (A/BM/1/18) that expresses NS1 expected to interact with CPSF, more than 600 host genes were downregulated . ImportaViruses also inhibit the production of critical antiviral mRNAs through induction of read-through transcription, though it is not yet known whether specific immune pathways are selectively targeted. Salient genes in the innate antiviral response are affected. For example, in influenza strain A/Udorn/72, NS1 binding to CPSF30 inhibits 3´-end processing of the mRNA for IFN-β, a key regulator of the type I interferon response , 59. Whevia manipulation of 3´-end processing to ensure successful infection of the host cell. Whether viruses preferentially suppress antiviral mRNA production via this mechanism remains to be further examined.In HSV-1 infection, the gene with highest level of readthrough is interferon regulatory factor 1 (IRF1) . IRF1 isGlobal analysis of read-through transcription has revealed that despite the extensive 3´-end processing defects in virus-infected cells, viruses evade disruption of 3´-end processing of their own transcripts , 61, 62.Viral infections promote failure of transcription termination at many cellular genes, but the roles of the act of read-through transcription versus the resulting transcripts remain unclear. Interestingly, production of read-through transcripts has also been observed in a variety of cellular stress conditions, including oxidative and osmotic stress –66. OnlyInfluenza NS1 overexpression leads to a decrease in pre-mRNA cleavage, and the resulting read-through transcripts are polyadenylated at lower frequency than mRNAs in NS1-mutant overexpressing cells . The assIn HSV-1-infected cells, Hennig et al. reported that read-through transcripts are enriched in nucleoplasmic and chromatin fractions as compared to the cytoplasmic fraction . ConsistTranscriptional readthrough during HSV-1 infection is both pervasive and potent, affecting many host genes to a great extent. Specifically, by 8 hours post infection (h.p.i.), 53% of all cellular genes experienced greater than 35% readthrough, whereas the median read-through levels at peak induction were 6-15% in cells subject to hyperosmolarity or heat shock , 60. ConIn the case of influenza virus infection, it was also proposed that host genes with decreased protein production were read through at high levels and that termination failure contributed to their low expression . ReadthrRead-through transcription is also associated with alterations to canonical splicing patterns in virus-infected cells. This includes aberrant splicing patterns in the gene body region upstream of the PAS at which readthrough occurs, as well as the downstream-of-gene regions that are read through  grants R01GM140735 (to JS) and F30AI157301 (to AP). JS was supported by the Howard Hughes Medical Institute.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Many diseases that involve malignant tumors in the elderly affect the quality of human life; therefore, the relationship between aging and pathogenesis in geriatric diseases must be under-stood to develop appropriate treatments for these diseases. Recent reports have shown that epigenetic regulation caused by changes in the local chromatin structure plays an essential role in aging. This review provides an overview of the roles of telomere shortening on genomic structural changes during an age-dependent shift in gene expression. Telomere shortening is one of the most prominent events that is involved in cellular aging and it affects global gene expression through genome rearrangement. This review provides novel insights into the roles of telomere shortening in disease-affected cells during pathogenesis and suggests novel therapeutic approaches. Telomeres are repetitive sequences that are located at the ends of human chromosomes and are known to constitute ribonucleocomplexes that play a vital role in genome stability. One of the most prominent events that can occur due to the imperfect telomere structure would be an end-to-end fusion between telomeres. Telomere fusion is closely related to the early development of cancer cells, and it is known to induce aneuploidy in cancer cells through the rearrangement of chromosomes ,2,3. It Telomeres are sequences that exist at the end of chromosomes and are composed of TTAGGG repeats in vertebrates . The lenThe primary function of telomeres is to stabilize chromosome integrity. Incomplete DNA replication in human cells causes a constant loss of terminal telomeric repeats. Thus, the existence of telomeres at the very end of the human genome allows for them to serve as a protective nucleoprotein structure that prevents the loss of information of sub-telomeric genes . TelomerThe unique structure at the end of the telomere is assisted by internal complementary interactions between repeats and trans-acting proteins such as TRF1, TRF2, and POT1 ,22. ThesThe continuous loss of telomeres induces M1 senescence, which causes cell division arrest, followed by an M2 crisis that involves further extensive telomere attrition in the absence of cell cycle checkpoints ,29. TherTelomeres are essential cellular components that must be maintained for the continuous division of cells. Therefore, stem cells prevent the loss of telomeres by expressing telomerase reverse transcriptase, which induces elongation of the telomeres . TelomerThe regulation of gene expression is another function of telomeres that has recently attracted attention. Telomeres are a type of constitutive heterochromatin along with centromeres, and they form a huge complex that inhibits gene transcription by means of unique histone modifications ,39,40. HSaccharomyces cerevisiae yeast model [The human genome has many sub-telomeric genes that are distributed within a range of tens to hundreds of kilobases from the loci of the telomeres. The expression of these genes can be regulated through the telomere position effect (TPE), a fact best exemplified by the st model . In 2001st model , and thiChanges in gene expression of human cells due to aging and alterations in physiological functions are the main phenomena that are associated with aging . CellulaTERT that are 1.2 mega bases away from chromosome 5p by forming telomere loops in CD4+ T lymphocytes and promyelocytic cancer cells [TERT gene by promoter mutation is frequent in human cancers [Telomeres, being constitutive heterochromatin domains, also perform the crucial function of strongly suppressing the expression of nearby subtelomeric genes. This is called the telomere position effect (TPE). The phenomenon was extensively studied in the yeast model at first , but it er cells . Since t cancers ,59,60, f cancers ,62.DUX4 gene, which could be a cause of FSHD with other transcription factors by regulating downstream target genes [DUX4 is associated with epigenetic dysregulation of subtelomeric D4Z4 microsatellite at chromosome 4q35 [SORBS2 (Sorbin and SH3 domain-containing protein 2) is also regulated by telomere length in the muscles of FSHD patients [Another representative disease that is related to the altered gene regulation by telomere shortening includes FSHD , and the role of TPE during the late onset of the FSHD in aged patients has been well characterized . Studieset genes ,65. The ome 4q35 . Anotherpatients . TherefoEpigenetic regulatory mechanisms, such as histone modification, DNA modification, and chromatin remodeling are known to play crucial roles in complex mammalian gene regulatory networks ,69. ChroIt also has been recently shown that telomeres, which are repetitive sequences at the ends of chromatin, make looping structures that are complementary to internal genomic regions that might be several mega bases away from the chromatin ends ,78,79,80The loss of telomeres due to cell division causes gradual structural changes in the genome. The changes that are accumulated over time can completely change the position of each component of the genome that is related to gene expression because regulatory elements such as promoters, enhancers, silencers, and insulators could work in a three-dimensional way . Since tTERT and the chromosome 5p telomere, TRF2 promoted the interaction between the 5p telomere and internal telomeric sequences located near the hTERT locus [The expression of specific genes is regulated by changes in telomere length because the three-dimensional folding structure of the genome changes through consecutive telomere attrition . The pheRT locus . FurtherMany cell divisions over time cause a critical shortening of telomere length, followed by an increase in the expression of certain genes by disengaging the looped complex between the telomere and target loci. However, the molecular and cell biological mechanisms underlying this phenomenon are still largely unknown.Another important question may be whether TPE-OLD occurs only inside the same chromosome. All of the reports to date that suggest the physical association of telomeres with specific loci have only shown binding within the same chromosome ,79,80.Since eukaryotic chromosomes are confined to discrete regions (chromosome territories) during the interphase , this seThis non-canonical function of telomeres is highly important in age-related diseases because telomere shortening disengages the looping interactions and this is followed by the activation of pathogenic genes ,78. TeloTERT) or treatment with 6-thio-2′-deoxyguanosine (6-thio-dG) inhibited the formation of colonies.Short telomere length is associated with tumors, Alzheimer’s disease, and diabetes mellitus ,88,89,90TERT transcription is related to the length of the telomeres, suggesting that TPE-OLD may be involved in the acquisition of telomerase reactivation during the development of GCTB. Another significant disease in which TPE-OLD is likely involved is triple-negative breast cancer (TNBC). Transcriptional coactivator with PDZ-binding motif (TAZ) is highly expressed in TNBC and is involved in the Hippo signaling system to regulate cell growth and death [TERT transcription was found only in late passages of TAZ knockdown cells with shortened telomeres, which indirectly suggests that TPE-OLD may be one of the major mechanisms in the development of certain types of breast cancer [Mechanisms related to TPE-OLD also have been reported recently based on a study of giant cell tumor of bone (GCTB), which is a rare bone tumor . The autnd death . The reat cancer . In prevt cancer ,95, implt cancer , studyinThe role of local genome structure change by telomere shortening at each chromosome end might explain why senescent cells show significant alterations in gene expression, followed by a decline in cell functions that are related to proliferation and survival. Research on the specific alterations in genome architecture in terms of long-range interaction between telomeres and particular loci during human aging will expand the understanding of the fundamental biology of geriatric diseases.Considerable technical difficulties in simulating the complex and multifaceted processes of cellular aging hinder the study of the roles of the telomere position effect from being able to clarify the mechanisms that are involved in the onset of aging. In particular, an increase in population doubling during continuous cultivation of cells (in vitro aging) affects many components of aging factors so that the results could be significantly different from human aging in vivo ,98. ContConformational changes in chromatin, DNA modification, and histone modification are considered to be key factors in various human pathologies ,101,102.It has also been shown that telomere shortening causes local genomic structural changes, which is followed by global gene expression perturbation. The development of various cancers, such as colorectal and prostate cancers, is closely related with telomere shortening ,106,107.TERT promoter mutation occurs in various cancers and is known to play an important role in the survival of tumor cells through telomerase reactivation [TERT locus and the 5p telomere may be related to mutation and hypomethylation of the hTERT promoter. A recent report suggests that hypomethylation of the mutated hTERT promoter might be cooperatively induced by disengaged telomere looping, which could cause significant differences in CpG island methylation in hTERT-promoter mutant cell lines [TERT promoter mutation in the early development of cancer, thus making it necessary to conduct extensive research in this area [TERT, 20–40% of the hTERT locus was still bound to the telomere in short telomere or aged fibroblasts [C1S, ISG15, and SORBS2 [TERT through promoter mutation in cancer cells, unlike most genes that exhibit biallelic expression [TERT in young and long telomere cells. This research will facilitate the understanding of the mechanisms of cancer development and telomerase reactivation, which is essential for developing therapeutic agents.For example, the htivation ,108,109.ll lines . Many sthis area ,112,113.roblasts  contraryd SORBS2 ,79. Thispression . It may The phenomenon of telomere looping that results in the inactivation of target genes was recently discovered ,80. Howe