Shigella spp., and provides the backdrop to the regulatory cascade that controls virulence by silencing many genes found on the large virulence plasmid. H-NS and its paralogue StpA are present in all four Shigella spp., but a second H-NS paralogue, Sfh, is found in the Shigella flexneri type strain 2457T, which is routinely used in studies of Shigella pathogenesis. While StpA and Sfh have been proposed to serve as “molecular backups” for H-NS, the apparent redundancy of these proteins is questioned by in vitro studies and work done in Escherichia coli. In this review, we describe the current understanding of the regulatory activities of the H-NS family members, the challenges associated with studying these proteins and their role in the regulation of virulence genes in Shigella.The histone-like nucleoid structuring protein (H-NS) has played a key role in shaping the evolution of  Shigella spp. are gram-negative, intracellular bacterial pathogens in the γ-proteobacteria that cause bacillary dysentery in humans. Four Shigella species have been described: S. dysenteriae, S. flexneri, S. boydii and S. sonnei. Each of these species is primarily transmitted by the fecal–oral route and invades cells of the colonic epithelium. The destruction of the colonic epithelial barrier and the severe inflammation that follows causes the symptoms of this disease . Shigella spp. are estimated to cause more than 88 million cases of shigellosis each year [ach year , and morach year . These sShigella spp. are close phylogenetic relatives of Escherichia coli. All four Shigella species harbor a large virulence plasmid (~180–220 kb) [–220 kb) , which i–220 kb) . Evidenc–220 kb) ,6,7.E. coli, many of which are involved in the transcription, translation and the production of cell envelope components needed for adaptation to varying environments [Shigella, this H-NS-dependent process is thought to have allowed the initial acquisition and long-term maintenance of the large AT-rich (up to 70% in many ORFs) virulence plasmid without the newly acquired genes compromising the organism′s fitness. The eventual integration of these genes into existing transcriptional networks is also predicted to have been aided by H-NS-mediated silencing . Todironment . In contironment . ConsequShigella spp. contain up to two H-NS paralogues. The first paralogue, StpA, is chromosomally encoded and found in all Shigella spp. [S. flexneri type strain, 2457T [Shigella in mutants that lack hns. However, different nucleic acid binding activities and expression profiles of these proteins  rter macrodomain of the chromosome of both E. coli and Shigella spp. In these two closely related organisms, H-NS is 100% identical, making in vitro studies on the E. coli H-NS protein directly applicable to the Shigella protein. H-NS is small (15.4 kDa), highly abundant  and funcultures . Each prcultures ,22; FiguE. coli [E. coli proU operon, 5′ AATATATCGA 3′ [The ability of H-NS to transcriptionally silence genes can be explained by its DNA binding preference and the nucleoprotein complexes that form. High-affinity binding sites for H-NS have been found in E. coli ,25, leadE. coli . InteresE. coli , which iATCGA 3′ ,25, is pATCGA 3′ , explainATCGA 3′ .Shigella [Once bound to a high-affinity region, H-NS oligomerizes along DNA into regions with lower affinity ,28, leadShigella ,33,34.Shigella virulence plasmid, including those encoding the transcriptional regulators of the virulence gene cascade , H-NS silences many genes encoded by the cascade  and mxi (PipgD) operons have been more thoroughly characterized [icsB promoter was initially found to be H-NS-responsive using a PicsB-lacZ transcriptional reporter in an E. coli background. Subsequently, electrophoretic mobility shift assays (EMSAs) and DNase I footprints revealed that H-NS binds the icsB-ipgD intergenic region, protecting sequences between +60 to −220 relative to the icsB transcription start site (TSS) [icsB requires a VirB-binding site centered 120 bp upstream of the icsB TSS. Based on biochemical and structural studies [ipgD, evidence for H-NS-mediated silencing comes from two different lacZ transcriptional reporters: an ipgD-lacZ reporter in E. coli [lacZ reporter located in the second to last gene of this fifteen gene operon in S. flexneri [S. flexneri [icsB promoter are also required for the regulation of the ipgD promoter, this does seem likely given the close proximity of these two promoters.Indeed, all three VirB-regulated promoters found within the at 30 °C , but thecterized ,57. The te (TSS) . Other ate (TSS) . The tra studies ,58,59, a studies ; Figure flexneri , as wellflexneri . While iipa-mxi-spa region is icsP. Promoter activity of this gene was measured using a plasmid-borne PicsP-lacZ reporter in both E. coli and Shigella backgrounds [icsP promoter reveal that the DNA sequences located between 800 and 350 bp upstream of the primary icsP TSS are required for H-NS-mediated repression [icsP TSS [icsP promoter occurs from such remote sites.An example of a VirB–regulated gene that lies outside of the kgrounds ,52. Whilpression , the ViricsP TSS . To datemxi operon because mxiE encodes the last transcriptional regulator of this cascade  and Sfh , —share a similar domain organization to H-NS [[E. coli, restoring Bgl (ability to use β-glucosides) and mucoidy phenotypes to those exhibited by wild type [Shigella [stpA and sfh transcripts are significantly elevated in an hns mutant background, whereas hns transcript levels are not significantly altered in stpA or sfh null mutants [hns gene or the acquisition of AT-rich DNA by horizontal gene transfer [Shigella [E. coli or Salmonella [Shigella spp., we will review these activities before describing our current understanding of the role that StpA and Sfh play in Shigella virulence gene regulation.The two other H-NS family members found in to H-NS [; Figure to H-NS [. In addiild type , leadingShigella ,66,67. I mutants . As “moltransfer ,67,68. AShigella ,66, the lmonella ,30,69,70E. coli, StpA serves to supplement the H-NS pool through its transient expression during exponential growth [stpA in E. coli, Salmonella and Shigella strains strongly suggests that this paralogue arose through an hns gene duplication event that predates the divergence of these bacterial clades from one another [td- intron mutant from bacteriophage T4 when overexpressed in vivo [E. coli, where StpA destabilizes the sRNA MicF. Since MicF functions to block translation of the OmpF porin, the actions of StpA make it a negative post-transcriptional regulator of MicF. Interestingly, H-NS also negatively regulates MicF levels but does so by repressing transcription and hence the production of this sRNA [In l growth ,71. The  another . Like H- another ,30,73,74 another , suggest in vivo , subsequ in vivo ,77,78. I in vivo . Anotherhis sRNA ,81. ThusS. flexneri strain serotype 2a, 2457T [Salmonella enterica Typhimurium suggests that Sfh facilitated the acquisition of this AT-rich plasmid by preventing native pools of H-NS being titrated away from AT-rich sequences located on both the Shigella chromosome and virulence plasmid [sfh was introduced into S. enterica Typhimurium, the expression of over 300 genes was altered when compared to the wild type plasmid [sfh plasmid [In contrast to StpA, much less is known about the Sfh protein. Sfh shares higher amino acid identity with StpA than H-NS . Unlike StpA, Sfh is encoded by the R27-like conjugative plasmid found exclusively in a, 2457T . While i plasmid ,70. In s plasmid ,70. Doub plasmid . Regardl plasmid .S. flexneri strain 2457T raises questions about their expression profiles. Protein levels of each H-NS family member found in 2457T have been measured in wild type cultures grown in LB broth at 37 °C [Shigella. Furthermore, they allow hypotheses to be generated about when heteromeric interactions between family members are most likely to occur (if they occur), and when Shigella virulence gene expression is likely regulated by individual proteins or combinations thereof, at least under the conditions tested.The presence of three H-NS family members in at 37 °C ,17. WhilShigella is the nature of the hns mutant being used. The hns mutant alleles used in Shigella studies  are notns::Knr; ,50,66,83ns::Knr; ,66, thesns::Knr; . Consequckground . These fShigella is relatively rudimentary compared to H-NS and is limited to analyses of the H-NS-silenced and VirB-anti-silenced mxi operon. These analyses were carried out in a 2457T derivative named BS184 [mxiC::lacZ transcriptional fusion in the large mxi operon located on the virulence plasmid. As expected for an H-NS-regulated locus, lacZ production in BS184 is low at 30 °C, but elevated at 37 °C in stationary phase cultures, consistent with the well-characterized temperature-dependent regulation of virulence genes in Shigella [hns dominant-negative mutant allele showed elevated levels of lacZ activity at 30 °C [icsP promoter (hns dominant-negative allele. In contrast, lacZ production in BS184 derivatives carrying clean deletions of sfh, stpA or both was similar to that observed in the BS184 wild type background at 30 °C and 37 °C. This finding strongly suggests that StpA and Sfh do not play a regulatory role at this locus in the presence of H-NS [hns dominant-negative allele was combined with the sfh deletion, lacZ activity increased at 30 °C above levels observed in the hns mutant, suggesting that Sfh may participate in the regulation of the mxi locus, but only in the absence of functional H-NS.Our understanding of the involvement of StpA and Sfh in virulence gene regulation in ed BS184 , which cShigella . Furtherat 30 °C , consistat 30 °C  and datapromoter . This ph of H-NS . InteresstpA or sfh in the BS184 background led to a decrease in lacZ expression at 37 °C [virB promoter, the virF promoter or both. This decrease in ipgD promoter activity may have been caused by the paralogues themselves or through a loss of VirB anti-silencing. Support for the indirect modulation of the mxi operon by StpA and Sfh was provided by EMSAs using DNA fragments taken from the virB and virF promoters. Recombinant H-NS, StpA and Sfh proteins were shown to bind to these DNA sequences in a concentration-dependent manner. It is surprising that DNA fragments taken from the ipgD promoter were not used in this experiment because this promoter controls the mxiC::lacZ transcriptional reporter in BS184. It is also unfortunate that the stability of the >9 kb mxi transcript generated prior to the lacZ fusion was not examined in the presence and absence of the paralogues because StpA, at least, has been shown to modulate RNA stability [In the same study, overexpression of either at 37 °C . Under ttability ,80.Shigella virulence gene expression.In summary, it is clear that StpA and Sfh can be a substitute for H-NS, but it is not known whether the silencing imparted by these proteins is mechanistically identical to that mediated by H-NS. Furthermore, it remains unclear if the backup role exhibited by StpA and Sfh in these assays is the only regulatory effect that these proteins impart in the context of Shigella counter-silencing protein VirB. To address this, we tested whether VirB can alleviate silencing mediated by either of the two H-NS paralogues StpA and Sfh at the icsP promoter. To do this, an E. coli hns dominant-negative background was used to avoid some of the complications that arise when using similar mutant alleles in Shigella, including artifacts caused by the instability of the virulence plasmid and indirect effects caused by the up-regulation of both virF and virB genes. In the E. coli hns dominant-negative mutant background, overexpression of hns, stpA or sfh leads to silencing of the icsP promoter (virB from an inducible promoter relieved this silencing (Shigella physiology remains unclear, evidently VirB does have the ability to relieve silencing mediated by StpA or Sfh if required to do so.The observation that StpA and Sfh can silence virulence genes, albeit in the absence of H-NS, not only raises questions about the silencing activities of these proteins, but also about their interplay with the Shigella virulence was established [Shigella virulence plasmid do not overlap promoter elements [Shigella virulence genes.Over 25 years ago, the importance of H-NS as a regulator of ablished . Despiteelements  may indielements ,63,75 orelements . ConsequShigella, a global in vivo approach will be required. Indeed, the identification of sites bound by H-NS in vivo at 30 °C and 37 °C and in the presence and absence of virB would provide valuable insight into virulence gene regulation in Shigella and provide a foundation for studies that seek to clarify mechanisms of silencing and anti-silencing. On a separate but related topic, the recent finding that H-NS family members are phosphorylated in S. flexneri [E. coli [In this regard, if we are to more thoroughly understand H-NS-mediated silencing in flexneri  and E. c[E. coli  raises tShigella spp. contain one and sometimes two H-NS paralogues adds another layer of complexity to virulence gene regulation in this pathogen. Most of the evidence gathered in Shigella suggests that the paralogues act as a functional substitute for H-NS, a finding that is further supported by VirB alleviating either StpA- or Sfh-mediated silencing of the icsP promoter (S. flexneri strain 2457T [hns mutants bearing dominant-negative alleles in Shigella research. Nevertheless, through careful experimentation, additional roles of StpA and Sfh and their regulatory effects on virulence gene expression may be identified in Shigella.The observation that promoter . In contin 2457T . It is pE. coli, mutants lacking all genes encoding H-NS family members are prohibitively sick [Shigella will be achieved.In order to get a clear view of the role of each H-NS family member, it will be important to study each protein in the absence of the other family members. The use of null mutants, alone or combination, may elucidate how these proteins function independently and coordinately to regulate virulence genes. The risk of these studies is that the remaining protein may or may not be present at levels found in wild type cells due to the fact that H-NS family members are capable of negative auto- and cross-regulation . This woely sick ,88. NeveShigella is lacking. The tendency of these proteins to form heteromers, however, is an important consideration for investigators because heteromeric complexes may form when two or more H-NS family members are expressed together at high levels. Furthermore, it is possible that the resulting heteromers will have different activities than their homomeric counterparts.A further layer of complexity arises from the finding that all three H-NS family members are capable of forming heteromers with each other, at least in a yeast two-hybrid assay . CurrentShigella presents a fascinating opportunity to study the relationship between H-NS, StpA and Sfh and their role in the regulation of virulence gene expression in this important human pathogen. The presence of two or more of these nucleoid structuring proteins in all Shigella species, pathogenic E. coli and Salmonella strains and many other bacterial pathogens elevates interest in this family of proteins that are intricately tied to bacterial physiology and virulence. Since loss of H-NS family members causes severe growth defects in some bacteria, H-NS and its paralogues may constitute prime molecular targets for novel antibacterial therapies.To conclude,


Tcrb gene. However, the coordinated action of trans-acting factors in the activation of Eβ during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the Eβ function. A single mutation at one of the two Runx binding motifs within the Eβ severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCRβ expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated Eβ activity, demonstrating that Runx complex binding is essential to initiate Eβ activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCRβ expression, whereas Eβ itself is continuously required for TCRβ expression. These findings imply that Runx complexes are essential for Eβ activation at the initiation phase, but are not necessary for maintaining Eβ activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for Eβ activity are differentially regulated, depending on the developmental stage and cellular activation status.A TCRβ enhancer, known as the Eβ enhancer, plays a critical role in V(D)J recombination and transcription of the  Enhancers are classically defined as DNA elements that can activate the transcription of their target loci irrespective of their orientation or distance from the transcriptional start siteTranscriptional control of the spatio-temporal expression of developmentally regulated genes involves dynamic communication between DNA regulatory elements (Tcr) genes and immunoglobulin (Ig) genes are assembled from variable (V), diversity (D) and joining (J) gene segments by a process referred to as V(D)J recombination2Tcrb locus occurs first in CD4−CD8− double-negative (DN) thymocytes, and then Tcra rearrangement follows during transition into CD4+CD8+ double-positive (DP) thymocytes only after the functional assembly of a Tcrb allele is secured by a process known as β selection. At the Tcrb locus, the expression of functionally assembled Tcrb alleles prevents further V to DJ recombination on the second allele to ensure the mono-specificity of the antigen receptor cis-regulatory elements and trans-acting factors.During T and B lymphocyte development, antigen receptor genes such as T cell receptor , located at the 3′ side of the Cβ2 region, has been shown to play an essential role in the recombination and transcription of DβJβ clusters6Tcrb alleletrans-acting factors that bind to the enhancer.Within the ~670 kb trans-factors for Eβ. Mutagenesis studies with reporter transfection assays and transgenic substrate have demonstrated that both Ets-1 and Runx binding motifs are essential for Eβ enhancer activity11Runx1 in DN thymocytes by the Lck-cre transgene resulted in a decrease of DN4 thymocytes, while DN3 cell numbers were not affectedEts-1 and Runx transcriptional factors have been implicated to function as Tcrb locus, illustrating distinct regulation of Eβ activity at the initiation versus maintenance phases by trans-acting factors.In this study, we report that Eβ-mediated TCRβ locus activation in T cell progenitors requires Runx binding sites, but the Eβ enhancer becomes independent of Runx complexes to maintain TCRβ expression in mature T cells. Thus, the functional requirements of Runx complexes for Eβ activation are distinct at different stages of the Runx1f/f:Lck-cre miceTcrb activation. In contrast, an equivalent level of surface TCRβ on cells lacking Cbfβ proteinTcrb expression at distinct stages prompted us to examine the roles of Runx complexes to control Eβ function, as well roles of Eβ in Tcrb expression during T cell development, particularly at later developmental stages. We therefore first addressed Runx sites (5′-PuACCACG/A-3′) within the Eβ for their requirement for enhancer function by targeting mutations by homologous recombination in mouse embryonic stem (ES) cells (Accumulation of DN3 cells expressing a lower percentage of intracellular TCRβ chain (i.c.TCRβ) in the thymus of S) cells . M1 and S) cells . In the EβΔ/Δ) in the germline resulted in a loss of surface TCRβ expression on thymocytes (+CD8+ DP thymocytes in the EβΔ/Δ mice has been shown to be dependent on the expression of γδTCR complexEβM3/Δ mice showed a severe reduction in thymocyte number and a complete loss of surface TCRβ+ cells (EβΔ/Δ and EβM3/Δ mice. These phenotypes were completely recapitulated in EβM3/M3 mice (data not shown). In the thymus from the EβM1/Δ and EβM2/Δ mice, thymocyte numbers were reduced with an increase in the CD4CD8− DN cell proportion, suggesting a partial block at the CD4−CD8− DN to CD4+CD8+ DP transition. In contrast to EβM3/Δ mice, a significant number of CD4+CD8+ DP thymocytes expressing surface αβTCR complex and mature thymocytes were detected in both EβM1/Δ and EβM2/Δ mice. Furthermore, the levels of surface αβTCR expression on T cells in the peripheral lymphoid tissues from these mice were similar to that from control animals J recombination6trans-factors onto Eβ, thus acting as important mediators for holocomplexes formation between the pDβ promoters and the Eβ enhancer. Presumably, decreased affinity of Eβ with Runx complexes upon the loss of one docking site results in an unstable Runx binding, leading to a reduced probability of recruitment of sequential factors, including RAG-1/2trans-acting factor complexes that bridge the Eβ enhancer to pDβ promoters could be formed even with one Runx docking site for a certain amount of time sufficient to induce successful Dβ to Jβ recombination.The Tcrb locus9Lck-Cre led to a significant decrease of Vβ to DJβ joining. Furthermore, the removal of Eβ by Cd4-Cre after Vβ to DβJβ assembly quickly erased surface TCRβ expression from DP thymocytes. These results suggest that, after the relocation of Vβ to juxtapose Eβ, the activity of pVβ promoters becomes Eβ-dependent. Thus, our findings shed new light on Eβ function in the control of the activity of pVβ promoters. It is unclear how the dependency of pVβ promoters on Eβ is altered at a distinct developmental stage. Recent studies proposed the presence of a barrier element upstream of the Dβ1Jβ1 cluster with features of H3K4me3 accumulationWhether the Eβ enhancer is involved in the activation of Vβ promoters remains to be clarified. Previous studies showed that the deletion of the Eβ enhancer had no significant effect on germline transcription, histone acetylation at the 5′ Vβ regions or long-range interaction of the Vβ segments with Dβ under germline configuration of the Tcrb expression. Along with sustained TCRβ expression from the EβM1 and EβM2 allele in mature T cells as well as sustained TCRβ expression in T cells lacking Runx complex functionTcrb expression from the rearranged Tcrb gene is. To secure mono antigen specificity on each T cell, once a functional TCRβ chain is produced, further recombination at the other allele is inhibited at the stage of the Vβ to DβJβ assembly, known as allelic exclusion. Although some models assuming distinct accessibilitiesEβ removal from DN cells by Lck-Cre suggested the involvement of Eβ in this reaction in addition to maintaining transcription from pVβ promoters. Given that Runx complexes are involved in holocomplex formation at Dβ to Jβ joining reactionTcrb gene can be maintained. It is important to further clarify how Runx complex controls the Eβ activity and whether Eβ plays any role in controlling Vβ to Dβ rearrangement.Because TCRβ expression on DP thymocytes was quickly lost upon Eβ removal, Eβ is likely to be a sole, or at least indispensable, enhancer to maintain BamHI-BamHI fragment corresponding to the 5′ long arm of our targeting vector was cut from a plasmid containing genomic DNA from the Tcrb locus (a gift from Dr. Bories). The 3′ short arm and the Eβ region were PCR amplified from the same plasmid using primers containing the desired restriction enzyme sites at the both ends. For the construction of the Eβ flox targeting vector, these fragments were ligated sequentially into the plasmid harboring a thymidine kinase (TK) gene and a neomycin resistance gene (neor) cassette flanked by two loxP sequences. The M1, M2 and M3 mutation were created by an overlapping PCR, and were sequenced to confirm the mutations. Each mutant Eβ fragment containing an HpaI site at the 5′-end and a SalI site at the 3′-end was replaced with the wild-type Eβ fragment by ligation into HpaI/SalI digested targeting vector. Each targeting vector was linearized by NotI digestion, and was transfected into the E14 ES cells as described previouslyneor gene. Mutant mouse strains harboring the EβΔ, Eβ flox, EβM1, EβM2, or EβM3 mutation were established through germline transmission from chimera mice.The 3.0 kb E8I-Cre transgene, a 1.9 kb LCR/TE (locus control region and thymocyte enhancer) regionXbaI site at the 5′end, and was cloned into the pCR-TOPOII vector (Invitrogen). The 1.6 kb HindIII-HindIII fragment of the core E8I enhancer was excised together with the Cd8a promoter fragment from the plasmidEcoRV and XhoI digestion. The 1.9 kb XbaI-EcoRV LCR/TE fragment and 2.1 kb EcoRV-XhoI E8I/Cd8a promoter fragment were ligated into the XbaI and XhoI digested pBluescript vector by trimolecular ligation, generating a pTE/E8I vector. The 6 kb XhoI-XhoI fragment containing the intronic region from the mouse Cd4 locus followed by Cre-ires-GFP sequences was cut out from the previously described Cd8-Cre transgene plasmidXhoI cleaved pTE/E8I vector. The E8I-Cre transgene was separated from the vector by NotI digestion, and was microinjected at Japan SLC inc. All mice were maintained in the animal facility at the RIKEN IMS, and all animal procedures were in accordance with protocol approved by the institutional guidelines for animal care.For the construction of the All monoclonal antibodies used for cell staining were purchased from BD Biosciences. Intracellular staining was performed as previously described+ DN cells purified using MACS (Miltenyi) microbeads. Cδ transcript was amplified by primers; 5′-agccagcctccggccaaaccatc-3′ and 5′-ctcttgggccatagcaaggctc-3′. DNA-PCR for analyzing Dβ to Jβ rearrangement and Vβ to DβJβ rearrangement were performed with 5000 sorted CD25+CD44− DN3 thymocytes. Primers used for RNA-PCR and DNA-PCR were identical to those described previously7RT-PCR assays for germline transcription of the Dβ1 region were performed using total RNA from CD25+ T cells were stimulated with 2 μg/mL immobilized anti-CD3ε (553058: BD Biosciences) and 2 μg/mL soluble anti-CD28 antibody (553295: BD Biosciences) during the first two days. Cells were then maintained in the medium supplemented with 20 U/ml rIL-2 (11271164001: Roche) for additional days.FACS sorted CD8−CD3−CD4−CD8−TN thymocytes according to the protocol provided by the manufacturer of the ChIP assay kit (Upstate Biotechnology). Purity of cells after purification was at least over 98%. Rabbit anti-Runx1 serum used for Runx1-ChIP was previously describedEβ14, Cd4 silencer (S4)Tcrb locusChromatin-DNA was prepared from purified Lineage How to cite this article: Seo, W. et al. Distinct requirement of Runx complexes for TCRβ enhancer activation at distinct developmental stages. Sci. Rep.7, 41351; doi: 10.1038/srep41351 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Drosophila system in which the non-canonical pathway was initially characterized, to compare putative canonical versus non-canonical transcriptional targets across the genome. We analyzed microarray expression patterns of wildtype, Jak gain- and loss-of-function mutants, as well as the Stat loss-of-function mutant during embryogenesis, since the contribution of the canonical signal transduction pathway has been well-characterized in these contexts. Previous studies have also demonstrated that Jak gain-of-function and Stat mutants counter heterochromatin silencing to de-repress target genes by the non-canonical pathway.The Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway has been well-characterized as a crucial signal transduction cascade that regulates vital biological responses including development, immunity and oncogenesis. Additionally to its canonical pathway that uses the phosphorylated form of the STAT transcription factor, recently the non-canonical pathway involving heterochromatin formation by unphosphorylated STAT was recently uncovered. Considering the significant role of the JAK/STAT pathway, we used the simple p = 0.004). Furthermore, putative canonical and non-canonical transcriptional targets identified displayed some differences in biological pathways they regulate, as determined by Gene Ontology (GO) enrichment analyses. Canonical targets were enriched mainly with genes relevant to development and immunity, as expected, whereas the non-canonical target gene set mainly showed enrichment of genes for various metabolic responses and stress response, highlighting the possibility that some differences may exist between the two loci.Compared to canonical target genomic loci, non-canonical targets were significantly more associated with sites enriched with heterochromatin-related factors  contains supplementary material, which is available to authorized users. Drosophila /TM3 males. Third instar larval progenies were heat-shocked at 37 °C for 2 h daily over 3 to 4 days, and resulting adult females of the genotype hsp70-flp; FRT82BStat92E06346/FRT82B which were used to produce embryos lacking in maternal Stat92E gene products, as described previously in the dominant female-sterile “germline clone” technique [Stat92Emat– and control w1118 were collected between 1 and 2 h after egg laying on apple agar plates with yeast paste. The embryos were washed twice with deionized water and total RNA was prepared using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s manual. RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit .All crosses were carried out at 25 °C on standard cornmeal/agar medium. Fly stocks of echnique . Stat92EE. coli RNase H and DNA ligase) to convert single-stranded cDNA into double-stranded DNA (dsDNA), which was then used as a template for in vitro transcription in a reaction containing biotin-labeled UTP and T7 RNA Polymerase to generate biotin-labeled antisense RNA (aRNA). Twenty μg of labeled aRNA was fragmented and 15 μg of the fragmented aRNA was hybridized to Affymetrix Drosophila Genome 2.0 Array Chips according to the manufacterer’s Manual . Array Chips were stained with streptavidin-phycoerythrin, followed by an antibody solution (anti-streptavidin) and a second streptavidin-phycoerythrin solution, performed by a GeneChip Fluidics Station 450. The Array Chips were scanned with the Affymetrix GeneChip Scanner 3000. For the numerical conversion to expression intensity and Present/Absent calls employing MAS5 [To prepare microarray samples from the RNA prepared, 200 ng of total RNA was used to prepare biotin-labeled RNA using Ambion MessageAmp Premier RNA Amplification Kit . Briefly, the first strand of cDNA was synthesized using ArrayScript reverse transcriptase and an oligo(dT) primer bearing a T7 promoter. Then DNA polymerase I was used , #323 (S2 cells), #955 (OregonR 3rd instar larvae), #2074 (S2 cells), #2665 (OregonR 2–4 h embryo), #2666 (BG3-c2 cells), #2668 (S2 cells) and #3956 (OregonR 14–16 h embryo), for Su(var)3–9 binding sites, #952 (BG3-c2) and #2673 (S2), and for H3K9me3 enrichment, #971 (yellow cinnabar brown speck 0–4 h embryo) and #4939 (OregonR 14–16 h embryo). In our study, if one or more enrichment sites were found to overlap with the annotated genomic locus indicated by the microarray data, the gene was deemed to be relevant to heterochromatin. For the overlap between probe sets upregulated by hopTum-l/+ and downregulated by hop3/+, probe sets overlapping with downregulated Stat92E06346/+ was categorized as putative canonical targets, whereas those upregulated were categorized as non-canonical targets. Similarly, Stat92Emat- was also analyzed by taking into consideration, overlaps with hopTum-l/+ upregulated and hop3/+ downregulated probe sets. Probe sets where the Stat92E06346/+ and Stat92Emat- showed opposite trends were removed from the analyses, as well as hopTum-l/+ and hop3/+ overlaps without significant Stat92E06346/+ or Stat92Emat- differential expression.HP1a and Su(var)3–9 binding and the heterochromatic H3K9me3 enriched sites were obtained from the publicly available modENCODE ChIP-seq database for comparison with genomic sites associated with the relevant probe sets identified from the microarray analysis . The folThe two-tailed Fisher’s exact test was used to determine significance between the difference in the number of probe sets for which their relevant sites overlapped with HP1a, Su(var)3–9 and/or H3K9me3 enriched sites and transposable elements comparing putative canonical versus non-canonical targets. Database for Annotation, Visualization and Integrated Discovery (DAVID), version 6.8 Beta was used for functional annotation and assessing the top 20 Fold Enrichment of Gene Ontology terms of putative canonical and non-canonical sets .Additional file 1:Normalized dataset. (XLSX 3589 kb)Additional file 2:Significant up- and down-regulated fold change probe sets of each mutant genotype. (XLSX 308 kb)Additional file 3:Overlaps of significantly changed mutant probe sets and putative canonical/non-canonical transcriptional target classification. (XLSX 138 kb)Additional file 4: Fig. S1.hopTum/+ embryo samples. RT-PCR was conducted on 0–12 h W1118 wildtype control, or hopTum/+ embryo collection to assess the upregulation of previously known JAK-STAT target genes. (JPEG 27 kb)Validation of transcriptional upregulation of known target genes in Additional file 5:Heterochromatin marker modENCODE enrichment and transcriptional target overlaps. (XLSX 56 kb)


The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix–loop–helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response  Whereast manner .cis-element may compete , C-box (GACGTC), and G-box (CACGTG) sequences are bound more preferentially  motif CACATG   for each hexamer, we computed the permutation P-value by P=(m+1)/(M+1), where m is a number of recorded P-values not greater than the meta P-value.In the third step, we determined the statistical significance of selected hexamers by a permutation test. In each permutation, we mixed promoters between genes so that each promoter was used exactly once . Then weP-value <0.005.We considered the association between the presence of the hexamer and the auxin responsiveness as significant with the permutation Bonferroni-corrected cis-regulatory elements using TOMTOM . We considered the matches with an E-value <0.05 as significant. For hexamers without significant matches, we additionally screened the literature.We compared the detected hexamers with known P-values of enrichment with Bonferroni multiple testing correction. We considered over-representation as significant at a family-wise error rate (FWER) below 0.05.To study if the obtained hexamers are enriched within ARF-binding regions, we used available whole-genome data on ARF6 ChIP-Seq  the number of positions which this hexamer occupies across all upstream regions of 21 098 genes relative to the total number of all possible positions; and (ii) the similar one across all promoter segments associated with a specific chromatin state.Thirdly, we tested if the hexamers were enriched in the upstream regions located within specific chromatin states of auxin-responsive genes (positive set) relative to the same promoter segments of non-regulated genes (control set). For each hexamer, we compared two proportions via one-tailed Fisher’s exact test: (i) the number of positions this hexamer occupies in the promoter segments of a specific chromatin state within a positive gene set relative to the cumulative number of all possible positions in these segments; and (ii) the same proportion for the control gene set.P-values of enrichment with Bonferroni multiple testing correction, considering the association as significant at FWER <0.05.In each step, we adjusted To expand our knowledge on the scope of transcriptional regulation in auxin response, we aimed to detect putative AuxREs from meta-analysis of auxin-responsive transcriptome data sets without prior assumptions on the transcription factors binding these.A. thaliana , which were substantially enriched in upstream regions of auxin-responsive genes . We consWe found the canonical AuxRE core TGTCTC and its analog TGTCCC enriched in upstream regions of auxin-up-regulated genes, thus confirming previous findings , were alcis-elements with more than five A/Ts in a row  to be the most abundant and the most significant in our search , 3. Two-Analyzing the A/T-rich hexamers against the data on Arabidopsis transcription factor-binding sites generated by Among non-A/T-rich hexamers, TOMTOM found significant matches with MYB-binding sites for GATAAG and AGGGTT, a FUS3-binding site for CATGCA, TCP-binding sites for TGGGCC and GTCCCC, and a number of ACGT-containing sequences, which resemble the binding sites for several transcription factors families  . A closeWhat could over-representation of these hexamers in auxin-responsive regulatory regions mean? The identified hexamers could be the core sequences for transcription factor-binding sites mediating primary or secondary response. They could be the coupling hexamers for TGTC-containing AuxRE, constituting with it the composite element and bound by ARF partner transcription factors  hexamers is significantly enriched within ARF2-binding regions , which pAvailable whole-genome maps . Upstream regions of both auxin up- and down-regulated genes appeared to be enriched with the chromatin in state 4 (FWER <0.001). Up-regulated genes additionally possess significantly higher portions of chromatin state 1 (FWER <0.001) and state 3 (FWER <0.05) in the upstream regions comparing non-responsive with auxin-responsive genes. Down-regulated genes were additionally enriched with chromatin states 2 (FWER <0.01) and 5 (FWER <0.001).Analyzing the distribution of putative AuxREs  over difWe next tested if putative AuxREs  are specMany A/T-rich putative AuxREs are specifically over-represented in chromatin states 2 and 5 of both auxin-activated and repressed genes, with the repressed genes showing a greater variety . Most ofNegatively and positively auxin-responsive genes differed by non-A/T-rich hexamers associated with specific chromatin states . All chrInterestingly TGTC-containing cores were not enriched in any chromatin state. Recently we showed that ethylene-responsive genes possess an EIN3-binding site within specific chromatin state 4  found by the TOMTOM tool  of putative AuxREs with the transcription factor-binding sites identified by TOMTOM (Fig. S2. Distribution of A/T-rich putative AuxREs along the upstream regions of auxin-responsive genes.Supplementary Figures_S1_S2Click here for additional data file.Supplementary Tables_S1_S4Click here for additional data file.


PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A ( During hematopoiesis mature blood cells are generated from hematopoietic stem cells (HSC), which reside in the bone marrow. HSCs produce progeny of intermediate repopulation potential, the progenitor cells. Meanwhile, maintaining a finite stable pool of stem cells by self-renewal. Hematopoietic progenitor cells undergo further differentiation into mature cells of the lymphoid and myeloid branches. This differentiation process is influenced by internal and external signalling events and by the activity of transcription factors at the endpoint of these signalling pathways . TranscrTAL1 (T-cell acute lymphocytic leukemia 1) is one of the central transcription factors for HSC development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation –8. FurthPRKACB gene encodes the beta subunit of PKA. There are four isoforms of PRKACB described and in addition several splice variants, each of the isoforms has a separate promoter and a distinct N-terminus [To examine the role of TAL1 during megakaryopoiesis, we performed chromatin precipitation and promoter ChIP array analysis to define megakaryocytic target genes of TAL1. We identified a number of TAL1 target genes, which had recently been associated with megakaryocytic differentiation of K562 cells . In partterminus , 25.PRKACB (Cβ3) together with the hematopoietic transcription factors RUNX1 and GATA1 (GATA-binding protein 1). Our data show that GATA1 acts as an activator of Cβ3 isoform expression. Furthermore, RUNX1 and TAL1 contribute to the repression of Cβ3 expression. Repression of Cβ3 during megakaryocytic differentiation is triggered by the loss of coactivator binding and by recruitment of corepressor proteins to the Cβ3 promoter.We found that TAL1 binds to the promoter of isoform 3 of glycophorin A (GYPA) 10-fold better than with a TAL1 antibody, despite more stringent washing conditions with Strep-CP , GCC2 (GRIP and coiled-coil domain containing 2), PCBD2 , Dicer, PIP5K1a , CBL (casitas B-lineage lymphoma), PRKACB , WNT9  and CTNNB1 (catenin (cadherin-associated protein) beta 1)  Figure .IL-19, Dicer and PRKACB were upregulated, whereas WNT9 was downregulated upon Tal1 knockdown. The other genes did not exhibit expression changes upon TAL1 knockdown under these conditions   was increased, whereas the Cβ3 isoform was decreased  Figure . To anald Figure . The decR Figure .Cβ3 isoform and identified several potential binding sites for TAL1 (E-Boxes) 5′ of the transcription start. Furthermore, RUNX1 and GATA1 sites were present in this area   software  we analya Figure . We clonr Figure . Interes1 Figure . The exp1 Figure . Subsequ1 Figure . Furthery Figure . These d) Figure .PRKACB expression . PKA-signalling is important at the megakaryocytic/erythroid branching [PRKACB (Cβ3) is a direct target gene of the hematopoietic transcription factors TAL1, RUNX1 and GATA1. Interestingly, expression of PRKACB Cβ3 is upregulated during erythroid differentiation and downregulated during megakaryopoiesis. In this respect, Cβ3 behaves like an erythroid gene. Recently, we reported that the transcription factor RUNX1 is a suppressor of erythroid TAL1 target genes such as KLF1 and the microRNA miR-144/451 [PRKACB Cβ3 isoform. On the other hand, our data suggest an activating role of the transcription factor GATA1 on Cβ3 isoform expression.The transcription factor TAL1 is an important regulator of gene expression at the erythroid/megakaryocytic branching. To better understand the function of TAL1 at this important differentiation point, we identified target genes of TAL1 using a biotin-tagged TAL1 in K562 cells. For more detailed analysis we focused on the ranching . Furtherranching , 36. In -144/451 , 17. SimαII integrin promoter [KLF1, and during megakaryocytic differentiation epigenetic corepressors are recruited dependent on the presence of RUNX1 [We showed that TAL1, GATA1 and RUNX1 were all already present at the promoter before megakaryocytic specification and remained there during and after differentiation. However, TAL1 and GATA1 binding was decreased upon megakaryocytic differentiation. It had been described that TAL1 and GATA1 can be found together on GATA1/E-box composite elements, which assembles a multiprotein complex with GATA1, LDB1, LMO2, TAL1 and E2A in erythroid cells –43. Simipromoter . Furtherpromoter . Interesof RUNX1 .Cβ3 promoter. Decreased Cβ3 expression correlates with reduced presence of the coactivator proteins WDR5 and p300  , nuclearell type , 55. It ntiation . Howeverntiation , 56, 57.PRKACB isoforms could connect aberrant transcription factor function with altered PKA signalling in leukemia. This would be supported by the observation that PRKACB regulation by the important oncogene c-Myc is involved in the control of proliferation [PRKACB isoform expression and the modification status of TAL1 in differentiation and leukemia.It was also discussed that cAMP signalling plays a role in malignant hematopoiesis . Therefoferation . Our obsin vitro under serum-free conditions and subjected to erythroid or megakaryocytic differentiation. Expansion took place for 6 days in StemSpan medium (Stem cell Technologies) supplemented with Flt-3 ligand (100 ng/ml), SCF (100 ng/ml), IL-3 (20 ng/ml) and IL-6 (20 ng/ml). For erythroid differentiation cells were transferred into StemSpan with SCF (20 ng/ml), IL-3 (5 ng/ml), dexamethasone , estradiol  and erythropoietin . For megakaryocytic differentiation cells were treated with SCF (1 ng/ml), TPO (30 ng/ml), IL-9 (13.5 ng/ml), and IL-6 (7.5 ng/ml) for 4-6 days [K562 cells (ATCC) were maintained in RPMI with 10% FCS and 1% penicillin/streptomycin. To induce megakaryocytic differentiation, K562 cells were treated with 30 nM phorbol 12-o-tetradecanoylphorbol-13-acetate (TPA). BirA-K562 cells were established as described . HEK293 4-6 days .Knockdown of TAL1, RUNX1 and GATA1 was achieved with shRNAs present in the pGIPZ vector (Open Biosystems) . The accCβ3 isoform was amplified from genomic DNA using phusion polymerase (New England Biolabs) and inserted in the luciferase vector pGL4.10 (Promega). Luciferase activity was measured 48h after transfection and normalised to the activity of a cotransfected CMV-promoter driven β-galactosidase vector. Data represent three independent experiments, performed in duplicates. Error bars show the standard deviation. Transfections were performed with metafectene according to the manufacturer's instructions.The 5′-region of the 7 K562 cells were harvested and resuspended in 10 ml RPMI medium and cross-linked for 15 min with 0.75% formaldehyde. The cross-link was stopped by adding glycine to a final concentration of 0.125 M, the cells were washed with phosphate-buffered saline and lysed in 1.2 ml lysis buffer for 30 min on ice . Chromatin was sheered using a bioruptor sonification device (Diagenode) resulting in fragments with an average of 500 bp length. Subsequently, 450 μl RIPA buffer , 5 μl blocked protein-G magnetic beads  and 2-10 μg antibody (or IgG as control) were added to 150 μl of chromatin and incubated over night at 4°C on a rotating wheel. One sample was spared for input control. The beads were captured using a magnetic rack and washed three times with wash buffer  for 10 min on a rotating wheel at 4°C and once with final wash buffer  for 15 min. DNA was eluted using elution buffer  at room temperature for 15 min with shaking. The cross-link was reversed in 400 μl elution buffer with 5 μl proteinase K  for 5-6 hours at 65°C. The DNA was extracted using phenol-chloroform and precipitated over night at −20°C and washed with 70% ethanol. Antibodies used: anti-RUNX1 from Abcam (ab23980), anti-TAL1 from Abcam (ab75739), anti-PRMT6 from Santa Cruz (sc-55702), anti-p300/CBP from NeoMarkers (MS-586), anti-LSD1 from Abcam (ab17721), anti-WDR5 from Santa Cruz (sc-100895), anti-GATA1 from Santa Cruz (sc-1233) and anti-RNApol IIa from Abcam (ab5408). For the ChIP-Assay shown in Figure 1×10Streptavidine/biotin-Chromatin-Precipitation (Strep-CP) was performed as described . FollowiTotal mRNA was prepared using the RNeasy purification kit (Qiagen) and synthesis of cDNA was performed with omniscript reverse transcriptase (Qiagen). Specific primers were used for quantitative RT-PCR. PRKACB . For isoform specific PRKACB RT-PCR forward primer were designed from the isoform specific 5′-regions and the specific exons and one reverse primer was used for all isoforms . Primer pairs used for qRT-PCR are given in


Escherichia coli central carbon metabolism with more than 30 known transcription factors. Using a library of fluorescent transcriptional reporters, we comprehensively quantify the activity of central metabolic promoters in 26 environmental conditions. The expression patterns were dominated by growth rate‐dependent global regulation for most central metabolic promoters in concert with highly condition‐specific activation for only few promoters. Using an approximate mathematical description of promoter activity, we dissect the contribution of global and specific transcriptional regulation. About 70% of the total variance in promoter activity across conditions was explained by global transcriptional regulation. Correlating the remaining specific transcriptional regulation of each promoter with the cell's metabolome response across the same conditions identified potential regulatory metabolites. Remarkably, cyclic AMP, fructose‐1,6‐bisphosphate, and fructose‐1‐phosphate alone explained most of the specific transcriptional regulation through their interaction with the two major transcription factors Crp and Cra. Thus, a surprisingly simple regulatory program that relies on global transcriptional regulation and input from few intracellular metabolites appears to be sufficient to coordinate E. coli central metabolism and explain about 90% of the experimentally observed transcription changes in 100 genes.Transcription networks consist of hundreds of transcription factors with thousands of often overlapping target genes. While we can reliably measure gene expression changes, we still understand relatively little why expression changes the way it does. How does a coordinated response emerge in such complex networks and how many input signals are necessary to achieve it? Here, we unravel the regulatory program of gene expression in  Approximately 15% of the tested promoters, mostly consisting of TCA cycle promoters (e.g. sdhC), were activated on carbon sources supporting growth rates below 0.8/h, but were not activated by chloramphenicol treatment that led to similarly low growth rates. The activity of the majority of tested promoters, however, was consistently related to the growth rate supported by each condition  that regulates the respective promoter with its promoter‐specific parameters Kli and αli. Upon transformation into log space and normalization, for example, to a reference condition (marked with Δlog in the subsequent text), this equation can be approximated to  with its promoter‐specific parameters Kequation : (2)Δloget al, et al, As a result, log‐normalized promoter activity can be described by a linear combination of global  and specific  transcriptional regulation. We hypothesized that global regulation would manifest as a common promoter activity pattern across conditions, similar to the aforementioned scaling factor when comparing two conditions  of them are true signals. Conversely, most metabolites appear to be irrelevant for explaining specific transcriptional regulation of any of the promoters, and few metabolites were inferred to regulate more than one promoter. For example, F1P and FBP were identified as potential regulatory signals for several glycolytic promoters, and cyclic AMP was identified as a potential regulatory signal for a third of the tested promoters, in particular in TCA cycle and carbon uptake systems , could not be explained by either global or specific transcriptional regulation, presumably because we did not quantify the underlying metabolic signal , for which transcriptomics and promoter activity data deviated substantially, possibly because some of the regulatory elements in its complex promoter , PEP likely constitutes a false‐positive signal.Finally, we asked which transcription factors mechanistically establish the inferred regulatory links between metabolites and promoters. Based on the known transcriptional regulatory network of talA and pck promoters by Crp‐cAMP, in contrast to previous reports using reporter plasmids in Crp deletion  or Crp overexpression strains (pck), respectively, which did not account for global regulation  and examined the distribution of correlation coefficients across all reported target promoters with and without removal of global regulation  governed by three regulatory metabolites , was sufficient to explain the majority of changes in promoter activity across conditions  and fluorescence  were performed at 37°C with shaking using a plate reader  at 6‐ to 10‐min (steady‐state experiments) or 10‐min (dynamic experiments) intervals. Diauxic shift experiments were performed as above, using M9 medium with 0.5 g/l glucose and 2 g/l succinate that was inoculated from M9 glucose precultures. Cultivations for the quantification of intracellular metabolite concentrations were performed as follows: M9 medium batch cultures in 96‐deep‐well format plates  were inoculated 1:50 from LB precultures and incubated overnight at 37°C under shaking. Subsequently, 96‐deep‐well plate cultures were inoculated with overnight cultures to a starting OD600 of 0.03  and incubated at 37°C under shaking. Culture OD600s were monitored by OD600 sampling from parallel wells on the same deep‐well plate and subsequent OD600 measurements using a plate reader .All experiments were performed using M9 minimal medium  at −20°C , and stored at −80°C until measurements. Before measurements, samples were resuspended in 100 μl water, centrifuged for 5 min  to remove residual particles, and transferred to V‐bottomed 96‐well sample plates (Thermo Fisher Scientific). Measurement, data acquisition, and data analysis were performed as described previously  and dln(OD)/dt by two‐point finite difference numerical approximation. Promoter activity was corrected for fluorescence background by subtracting the corresponding signal of the promoter‐less plasmid reporter strain p139  promoter activity data was performed using the Pearson correlation coefficient as the distance metric between promoters (cutoff: 0.225).All data processing steps were performed with custom MATLAB software. Promoter activities and corresponding growth rates were determined as described previously . Promoters were treated as variables, and conditions were treated as observations, yielding singular vectors that have the same dimension as the conditions. The first singular vector, which captures most of the data set's variability, was defined as global regulation. The specific transcriptional regulation component of each promoter was quantified by subtracting this first singular vector. Note that for the normalized data used here, singular value decomposition and principal component analysis  in equation corr. Conditions in which a metabolite was used as a carbon source, or secreted by the cells, were omitted. Identification of pairwise regulatory metabolites was performed by systematic linear regression of each promoter's specific transcriptional regulation component based on the equation s = p1 × metabolite1 + p2 × metabolite2, where p1 and p2 denote the promoter‐ and metabolite‐specific parameters to be determined in the regression. The goodness of fit was again determined as the Pearson correlation coefficient between measured and predicted specific transcriptional regulation. To assess whether any metabolite pair can explain the respective promoter's specific regulation component better than the best single metabolite, we calculated the difference in Akaike information criterion (AIC), which penalizes the number of parameters when comparing different models . The AIC of each promoter–metabolite (or metabolite pair) combination was calculated as described before , and K denotes the number of parameters in the respective model . See Where et al, et al, Inference of potential transcription factor–metabolite interactions was performed based on the reported transcriptional regulatory network (as reported in RegulonDB; Salgado The parameters of each identified promoter–metabolite interaction see Fig  were re‐The data generated in this work are available as Tables KK, LG, and US designed the study. KK and LG performed experiments and computational analyses. SFB and DC assisted with experiments. YVN performed additional experiments not included in the manuscript. KK and US wrote the manuscript, with contribution from LG.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Table EV4Click here for additional data file.Table EV5Click here for additional data file.Table EV6Click here for additional data file.Table EV7Click here for additional data file.Table EV8Click here for additional data file.Review Process FileClick here for additional data file.


INK4ap16 is a potent inhibitor of the G1/S-phase transition of the cell cycle. INK4ap16 governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress INK4ap16 expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate INK4ap16 expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of INK4ap16 regulators in human epidermal SC self-renewal, aging and cancer.The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment.  The epidermis is a self-renewing tissue characterized by several compartmentalized layers of keratinocytes in stages of progressive differentiation by virtue of a temporal and spatial gene regulation: Namely, the basal layer is composed by proliferating keratinocytes; the upper layers are composed by viable and differentiated cells; the horny layer is composed by terminally differentiated cells . EpidermThe hierarchical model suggests the existence of two distinct proliferative cell compartments consisting of slow-cycling SCs and committed progenitor cells. They are hierarchically arranged and contribute to the homeostasis of discrete regions of epidermis. Indeed, slow-cycling cells give rise to actively cycling progenitors that persist for various periods of time in a transient-amplifying (TA) phase and generate differentiated cells . This viThe stochastic model suggests that the basal epidermis is composed of a single type of population of actively cycling progenitors functionally equivalent, having 50% probability to differentiate or divide. Thus, they directly generate differentiated spinous cells without entering a TA phase . DiffereMouse and human skin have significant differences in cellular architecture and physiology, making it difficult to apply these concepts to human epidermis. Mouse skin has a dense array of hair follicles, whereas human skin displays larger areas of interfollicular epidermis characterized by downward projections of the epidermal rete ridges. Moreover, the human epidermal compartment is thicker with more cell layers than mouse skin. Indeed, human epidermis turnover occurs every 40–56 days, whereas mouse epidermis turns over every 8–10 days . HoweverFor self-renewal, SC has to enter into the cell cycle and divide, and at least one daughter cell must be maintained in an undifferentiated state. Human clonal evolution from SCs to TA cells is a continuous unidirectional process, is instrumental in building up the epidermal structure since TA cells may generate postmitotic terminally differentiated keratinocytes, which migrate upwards and form suprabasal epidermal layers . The SC INK4ap16 as an intrinsic human clonal evolution regulator. Indeed, it increases during in vitro clonal conversion and its forced expression or downregulation impairs this process [INK4ap16 governs the processes of SC self-renewal and its deregulation may result in aging or tumour development [Several studies indicate the tumor suppressor  process ,18,19. Ielopment .The SC self-renews, taking advantage of the cell cycle machinery to divide. Mitogenic signals induce the expression of D-type cyclins, which bind to and activate cyclin-dependent kinase 4 (CDK4) or CDK6. These complexes inactivate the pRb proteins through phosphorylation that induces pRb-E2Fs dissociation. The E2F transcription factors promote the G1- to S-phase transition through the transcription of their target genes .INK4ap16 is a potent inhibitor of the G1-phase transition of the cell cycle and is considered a tumor suppressor gene. Following several stress stimuli , INK4ap16 directly binds CDK4/6, inhibiting its kinase activity and preventing pRb phosphorylation. Thus, pRb remains associated with E2Fs in the cytoplasm, preventing the E2F-mediated transcription and resulting in cell cycle block [INK4ap16 (or p21) pathway, may be considered the initial step of cellular senescence [INK4ap16 mediated senescence results in chromatin reorganization or senescence-associated heteochromatin foci (SAHFs) which are related to the repression of genes regulated by E2F1 [le block . The tranescence . p16INK4 by E2F1 .INK4ap16 gene (CDKN2A) is contained within the Inhibitor of cdk4/ Alternate Reading Frame (INK4/ARF) locus , which acts as an epigenetic regulator of the INK4/ARF locus [The F) locus A, a compRF locus ,24,25.INK4ap16 to inhibit cellular proliferation must be tightly controlled. To this aim, the regulation of INK4ap16 expression is complex and involves multiple transcription factors and a finely tuned epigenetic control [To maintain tissue homeostasis, the ability of  control .Epigenetic regulators have specific enzymatic activities, which modify DNA accessibility and chromatin structure, and, in turn, control gene expression A.INK4ap16 [DNA methyltransferase (DNMT) and ten-eleven translocation (TET) family enzymes—Conversion of cytosine DNA bases to 5-methyl-cytosine (5mC) is one of the best-characterized epigenetic modifications, which occurs predominantly in CpG islands, and is mostly associated with transcriptional repression. Indeed, 5mC may inhibit transcription by preventing the transcription factor binding to DNA or it may recruit methyl-DNA-binding proteins that facilitate the assembling of chromatin repressor complexes . Howeverp16INK4a . Specifip16INK4a ,29.INK4/ARF transcription. When they repress the transcription, SCs may enter in the cell cycle [INK4/ARF locus, likely carrying out different biochemical functions. Moreover, the subunit composition is cell type specific [INK4/ARF genes. The recruitment of PRC1 and PRC2 to the INK4/ARF locus is mediated by the long noncoding RNA, ANRIL. ANRIL binds SUZ12 subunit of PRC2 to induce the Ezh2-mediated H3K27me3 and consequent silencing of the INK4/ARF locus. ANRIL binds the CBX7 subunit of PRC1, allowing the recognition of H3K27me3 necessary for the H2AK119ub1 and maintenance of silencing. Therefore, ANRIL transcription modulation affects the repressing ability of PcG proteins [Polycomb group (PcG) protein and Jumanji protein families—In mammals, PcG proteins belong to two classes of complexes, Polycomb Repressor Complex 1 and 2 (PRC1 and PRC2), which operate a transcriptional repression via histone modifications and chromatin compaction. PRC2 functions as an initiator of transcription repression and PRC1 functions as a repressor maintenance complex. These complexes are critical for the homeostatic regulation of the ll cycle ,31. The ll cycle . Multiplspecific . Canonicspecific . PRC1 cospecific ,30,32. Tproteins ,34.INK4ap16 expression must be induced to prevent uncontrolled cell cycle progression. The expression of H3K27me3 demethylases of the Jumanji family proteins (JMJD3 and UTX) increases following oncogenic stimuli and removes the repressive histone marks from the INK4ap16 promoter [INK4ap16 expression. In this setting, PRC1 is not able to recognize the unmethylated H3K27, the chromatin in the region surrounding INK4ap16 promoter becomes decondensed and accessible for transcription factor activity [On the contrary, in the presence of stress signals, promoter . Jun dimactivity . In addiactivity . Thus, tINK4ap16 expression [The histone methyltransferase enzymes—The enzyme SET domain containing (lysine methyltransferase) 8 (Setd8)/lysine methyltransferase 5A (KMT5a) catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1) that characterizes transcriptionally active genes. Indeed, inhibition of Setd8 induces the reduction of H4K20me1 at several loci, such as INK4a/ARF, and, in turn, increases pression .INK4a/ARF locus and mediate INK4ap16 induction during replicative and oncogene-induced senescence, counteracting PRC-mediated repression [The enzyme, histone-lysine N-methyltransferase 2A (KMT2A) or myeloid/lymphoid or mixed-lineage leukemia 1 (MLL1), trimethylates the histone H3 at lysine 4 (H3K4me3), a mark associated with transcriptional activation. MLL1 and the E3 ubiquitin ligase DDB1-CUL4-ROC1 complex directly bind to the pression .INK4ap16 regulation mediated by the HAT p300 mainly depends on availability of several transcriptional co-factors. Indeed, the transcription factors SP1 and HMG box-containing protein 1 (HBP1), recruit p300 to the INK4ap16 promoter. Both chromatin acetylation and HBP1 foster chromatin decondensation and subsequent gene transactivation [INK4ap16 repressor [INK4ap16 promoter. Most of these interactions depend on activity of transcription factors such as Lymphoid Specific Helicase (LSH), HLX1, ZBP-89, and YY1. For instance, HDAC3 and HDAC4 are recruited to INK4ap16 promoter by YY1 to repress its expression. HDAC2 may also directly bind the INK4ap16 promoter [Histone acetyltransferases (HATs) and histone deacetylases (HDACs)—Histone acetylation is a dynamic process controlled by two large families of enzymes with opposed activities: HATs and HDACs. HATs catalyze the acetylation of ε-amino groups of lysine residues within histone tails, facilitating chromatin relaxing and subsequent gene transactivation. The tivation . p300 isepressor . HDACs rpromoter ,39.INK4ap16 promoter to activate transcription. Moreover, SWI/SNF displaces PcG silencing complexes from INK4ap16 promoter and reduces its DNA methylation [INK4ap16 and INK4ap16/BRG1 interaction negatively modulates the chromatin remodelling activity of BRG1 itself. However, BRG1 is not required for INK4ap16-induced cell cycle inhibition. Taken together, these findings indicate a putative feedback loop between INK4ap16 and the SWI/SNF complex [Chromatin remodelers—The multisubunit SWI/SNF family complexes contain SWI2/SNF2-like ATPases that hydrolize ATP to disrupt histone-DNA interactions within the nucleosome. This structural alteration results in an open chromatin state suitable for transcription machinery access . As a suhylation . Among S complex .INK4ap16 expression by recruiting HDAC1 that deacetylates the H3 histone to establish a repressive chromatin structure at the INK4ap16 promoter and delays cell senescence [LSH, a member of the SNF2/helicase family, regulates DNA methylation and transcriptional silencing . LSH openescence .INK4ap16 promoter [Nuclear architecture remodelers—The specialized AT-rich binding protein 1 (SATB1) is a genome organizer that interacts with AT-rich sequences and recruits chromatin-remodeling enzymes to regulate chromatin structure and gene expression . Thus, dpromoter .INK4/ARF locus, regulating both chromatin compaction and gene expression. CTCF interaction with the chromosomal boundary is important for INK4ap16 expression maintenance [INK4ap16 promoter and, in turn, INK4ap16 downregulation. Furthermore, CTCF inhibits epigenetic silencing through chromatin remodeling of the INK4ap16 locus [CCCTC-motif binding factor (CTCF)—CTCF is a zinc finger transcription factor that binds the ntenance . Indeed,4a locus ,50,51.INK4a/ARF allow an overall regulation by the same chromatin remodelling event(s) following specific stimuli. However, a permissive chromatin state is necessary but not sufficient for gene expression. Indeed, transcription requires the binding of activation factors to specific enhancer and promoter elements and the subsequent recruitment of RNA polymerase. Transcription factors model the precise gene expression pattern required for specific functions, binding cooperatively to DNA in the form of cell-type-specific multiprotein complexes [INK4ap16 gene is finely controlled by several transcription factor antagonistic pathways , a helix loop helix (HLH) transcription factor, binds Ets transcription factors and inhibits their activity [Ets-binding site-mediated regulation—The E-box binding transcription factors E-26 transformation-specific (ETS) proteins (Ets1 and Ets2) are activated by the RAS-MAP kinase pathway following dangerous stimuli. Ets 1 and Ets 2 bind to roblasts . To suppactivity . Thus, tINK4ap16 expression. ID1 may influence the transactivation activity of E47 acting as dominant negative following heterodimerization. TWIST1 inhibits INK4ap16 transcription by decreasing E47 expression [INK4ap16 promoter to upregulate its expression in human cells [E-box-mediated regulation—E47 is a basic helix-loop-helix (bHLH) protein that homo- and heterodimerizes with other HLH proteins and binds two consensus sequences in the E-box element to activate pression . Myc is an cells .INK4ap16 transcription through direct association with its promoter region, resulting in the cell growth promotion and cellular senescence prevention [Y box-mediated regulation—Y box-binding protein 1 (YB-1) is a transcription factor that represses the evention .INK4ap16 promoter contains at least five putative GC boxes that represent the putative binding target sites for Sp transcription factors [INK4ap16 promoter harbors a GC box for Sp1 binding [INK4ap16 transcription by direct binding to DNA and recruiting P300 to the INK4ap16 promoter [Sp1-binding site-mediated regulation—The GC-rich region within the  factors ,56,57. AINK4ap16 promoter contains a HBP1 binding site. This is a downstream effector in the Ras/MAPK signaling pathway positively regulating INK4ap16 transcription by direct binding to DNA and recruiting P300 to its promoter [INK4ap16-specific and global DNA hypomethylation changes [HBP1-binding site-mediated regulation—promoter . Of note changes .INK4ap16 promoter harbors a negative regulatory element that contains a binding site for B-Myb, a transcription factor involved in the regulation of cell survival, proliferation, and differentiation [INK4a transcription silence element (ITSE)-mediated regulation—The ntiation .INK4ap16 transcription by binding to three identified AP1-like sites within the INK4ap16 promoter. Conversely, c-Jun acts as a JunB antagonist, downregulating INK4ap16 expression. Interestingly, INK4ap16 is able to bind to JNKs and inhibit c-Jun phosphorylation and AP-1 activity [Ap1 site-mediated regulation—AP1 proteins are homodimers and heterodimers composed of basic region-leucine zipper (bZIP) proteins, including Jun proteins , Fos proteins , Jun dimerization partners (JDP1 and JDP2), and the closely related activating transcription factors . JunB activates activity ,60.INK4ap16 promoter and interacts with Sp1, enhancing INK4ap16 expression. PPARγ phosphorylation represses its transactivation function [Peroxisome proliferator response element (PPRE)—Peroxisome proliferator-activated receptor alpha (PPARα) specifically binds to the canonical PPRE region of the function .INK4ap16 repression [INK4/ARF gene transcription is associated with SC marker induction [A key requirement for SC maintenance throughout life is the pression . Indeed,nduction .INK4ap16 expression is undetectable during embryogenesis and in young tissues. In human epidermis, INK4ap16 is undetectable in primary human keratinocyte cultures from young donors [INK4ap16 is a key regulator of clonal conversion and its inactivation in epidermal SCs is necessary for maintaining their stemness [INK4ap16 is repressed, CDK4-cyclin D1complex is in the active state and allows E2F-mediated transcription of gene involved in cell cycle progression through inhibition of pRb. Indeed, the CDK4-cyclin D1complex regulates keratinocyte proliferation, but is not implicated in calcium-induced keratinocyte differentiation [INK4ap16 promoter [g donors ,18,19. pntiation ,63,64. Spromoter .INK4/ARF locus activation plays a limited role in skin development and homeostasis [INK4ap16 expression [INK4ap16 may be considered a master sensor of aberrant chromatin status that rapidly drives cell cycle exit, inhibiting CDK4-cyclin D1 complex activity [eostasis . Howeverpression ,69,70,71activity . The pRbactivity . Rb protactivity . Beyond activity ,75,76. TINK4ap16 epigenetic regulators (described in paragraph 3) cooperatively modulate the balancing between epidermal SC maintenance and differentiation . On the contrary, the INK4ap16 transcription activators may also promote keratinocyte terminal differentiation acting on epidermal differentiation complex (EDC) genes  [Multiple ntiation . Specifi, SATB1) ,77,78,79INK4ap16 and differentiation genes. Epidermis becomes thickened, likely due to aberrant basal cell differentiation [INK4ap16, and loss of the tissue self-renewal capacity. However, depletion of DNMT1 in human keratinocytes is not sufficient to induce the loss of cell identity. Thus, DNMT1 displays a critical role in maintaining epidermal progenitor cells and tissue renewal [DNA methylation is relevant for tissue homeostasis as it provides functional variability to maintain the equilibrium between SC proliferation and differentiation. In skin SCs, the differentiation gene promoters are methylated and lose this repressive mark following cell fate commitment. Indeed, DNMT1 is mainly expressed in the epidermal basal layer, and decreases with keratinocyte differentiation ,80. Cond renewal . Genes a renewal . Of note renewal .INK4a/ARF locus, impairing proliferation and hair follicle development [The transcriptional repressor PcG proteins are essential to control SC identity and proliferation by preventing unscheduled induction of non-lineage and differentiation genes. Indeed, in keratinocyte SCs, non-epidermal and differentiation genes are H3K27me3-repressed whereas this repressive mark is lost during cell differentiation. In committed cells, stemness genes display high levels of H3K27me3 mark . The rolelopment ,69,83. Lelopment . Jarid2,elopment .INK4ap16 expression is required for proper development, SC maintenance, and homeostasis in several tissues [INK4a/ARF locus in epidermal progenitor cells through its canonical PRC1 activity. On the other hand, it controls both proliferation of basal keratinocytes and inhibition of their premature differentiation by its SUMO E3 ligase activity [PRC1 functions have been investigated in human keratinocytes and the most studied proteins are the PCGF4 subunit/Bmi-1 and Cbx4. Bmi-1-mediated regulation of  tissues . However tissues ,88,89,90 tissues ,90,91 an tissues ,91, sugg tissues ,69,90. H tissues . Cbx4 abactivity . In humaactivity . The remactivity .INK4ap16, fails to differentiate and remains in single-layer, similarly to p63-null mice. Thus, HDAC1/2 mediates repressive functions of p63 in epidermal development, and suppress p53 activity [INK4ap16 increase [HDAC1 and HDAC2 play a critical role in the gene expression control of the epidermal progenitor cells and epidermal differentiation. They are expressed in all epidermal layers but display a higher nuclear expression level in terminally differentiated cells . Conditiactivity . Moreoveactivity . Of noteincrease , as wellincrease .INK4ap16 expression and preventing differentiation. Furthermore, balancing these enzymatic activities regulates the transition between epidermal SC quiescence and activation.Altogether, these data indicate that DNMT1, PRC1, PRC2, and HDAC proteins work in concert to protect epidermal cells from senescence by repressing INK4ap16 expression. Setd8 is a Myc target and is weakly expressed in skin. Deletion of Setd8 during embryogenesis results in p63 loss and a failure to form the epidermis. Setd8 activity is mainly present in dividing basal progenitor cells and is required for normal tissue homeostasis [p63 directly represses HBP1 and, in turn, INK4ap16 expression. HBP1 is also required for differentiation and stratification processes [INK4ap16 transcription and maintain basal progenitor proliferation. The p63-target SATB1 plays a key role in epidermal morphogenesis, controlling tissue-specific chromatin remodeling and INK4ap16 repression in epidermal progenitor cells [p63 directly regulates the expression of the SWI/SNF complex BRG1 that displays a critical role in skin homeostasis. It remodels the higher-order chromatin structure of the EDC for the efficient expression of differentiation genes in epidermal progenitor cells [These epigenetic mechanisms may be regulated by specific transcription factors, such as p63, which is considered a master regulator of epidermal morphogenesis and SC maintenance ,98. It ieostasis . Indeed,eostasis . In the rocesses . Moreoverocesses  and DNMTor cells ,103. p63or cells . BRG1 isor cells ,105. Indor cells . The remor cells .INK4ap16 promoter transcription factor regulators (described in paragraph 3) may have a role in epidermal proliferation and differentiation. Ets1 is expressed in the epidermal basal layer, blocks keratinocyte terminal differentiation, and induces expression of matrix metalloproteases and innate immune mediators [p63 upregulation [INK4ap16 repressor Id-1, Id-2, and Id-3 proteins are expressed in proliferating human keratinocytes and downregulated in differentiated cells. Of note, a cytoplasmic Id-1 expression and nuclear Id-2 and Id-3 expression has been observed in the epidermal basal layer [INK4ap16-negative regulator B-Myb plays an important role in maintaining the undifferentiated phenotype of keratinocytes in the basal epidermal layer [INK4A/ARF locus and the terminal differentiation by preventing the recruitment of AP-1 to promoters of genes governing barrier formation [Several ediators . Ets1 trgulation . Ets2 isal layer . The p16al layer . The ubial layer . Of noteormation . Specifiormation . PPARα iormation .Aging is a complex process characterized by accumulation of macromolecular damage and compromised tissue renewal. Such decrease of tissue regenerative capacity is not necessarily due to a SC-number or self-renewal decline but rather to a reduced ability to produce progenitors and, in turn, differentiated effector cells . EpidermOne aging hallmark is cellular senescence since the number of senescent cells increases in tissues during organismal lifespan. Cellular senescence is triggered by several different intrinsic and extrinsic stimuli that induce growth arrest and distinctive phenotypic alterations, including considerable chromatin and secretome changes. In young organisms, cellular senescence prevents the uncontrolled proliferation of damaged cells. However, in old organisms the continuous and cumulative damages and the deficient clearance of senescent cells results in their accumulation with deleterious effects on tissue homeostasis ,122,123.INK4ap16 has a well-established role in mediating and maintaining cellular senescence during both replicative and stress-induced senescence processes. Thus, it may be considered an effector of aging [INK4ap16 is undetectable in unstressed and healthy tissues of young mammals [INK4ap16 accumulates during tissue aging and therefore is considered one of the most robust aging biomarkers characterized to date [INK4ap16 may be a marker of biological rather than chronological aging [INK4ap16 expression in humans [INK4ap16-null mice are tumor-prone [INK4ap16 it causes a decline of regenerative capacity of hematopoietic, neural SC and intestinal cells of young adult mice, rapidly inducing several features of premature aging. De-induction of INK4ap16 revealed that these features are strikingly reversible [INK4ap16-positive senescent cells in a progeroid mouse model delays the onset of premature aging phenotypes in different tissues, such as adipose tissue, skeletal muscle, and eyes; attenuates the progression of already present age-related disorders; and extends healthspan [of aging ,123. Ind mammals ,125. p16 to date ,127. Speal aging . Similarn humans ,130. Assor-prone . Howeverversible ,127,131.althspan .INK4ap16 expression directly correlates with chronological aging of human skin in vivo. The number of INK4ap16 positive cells increases with age in both epidermal and dermal compartments [INK4ap16 positive cells in human skin is also a marker of biological age in middle-aged individuals with longevity propensity compared to their age-matched partners [INK4ap16 expression at first culture passage has been observed in human keratinocyte from elderly donors [INK4ap16 levels increase in the epidermal basal layer in the aged model of skin equivalent in concomitance with a proliferation marker decrease. In genetically-modified skin equivalents, INK4ap16 overexpression in cultures from young donors results in a decrease of proliferation markers whereas INK4ap16 silencing results in morphology improvement and proliferation markers restoration in cultures from old donors [artments . Furtherpartners . A strony donors . p16INK4d donors ,133.INK4ap16 promoter regulation that represses the INK4a/ARF locus. Indeed, oxidative stress induces demethylation of INK4ap16 promoter and, in turn, human keratinocyte senescence [INK4a/ARF genes [INK4ap16 in human keratinocytes from old donors strongly correlates with Bmi-1 downregulation. What is more, Bmi-1 modulates both INK4ap16 levels and clonal conversion in primary human keratinocytes from elderly donors. Specifically, Bmi-1 overexpression inhibits INK4ap16-promoter activity and decreases the protein expression. These Bmi-1 overexpressing keratinocyte cultures display a delay of cell senescence and a restoration of keratinocyte clonal ability, showing a phenotype similar to that observed in age-matched healthy control subjects [INK4ap16, since the deletion of pRb from cells resulted in histone H3K27 trimethylation loss that impairs the Bmi-1 recruitment to the INK4ap16 locus [INK4ap16 induction during replicative and premature senescence [p63, induces cellular senescence in human proliferating keratinocytes [p63 heterozygous mice [p63-targets LSH and SATB1 play a key role in senescence and aging. Indeed, LSH-deficient mice exhibit accelerated aging and enhanced cellular senescence [INK4a16 [INK4a16 and the pRB/E2F pathway are critical for SATB1-induced cell cycle arrest. Indeed, SATB1 causes anchorage-independent growth and invasive phenotype in INK4a16 deficient cells [INK4ap16 expression. During senescence BRG1 is overexpressed and interacts with pRB to drive SAHF formation. On the other hand, this interaction is dispensable for upregulating INK4ap16 and p21 by BRG1, which depends upon its chromatin remodeling activity [INK4a16 expression and leads to subsequent cell cycle arrest and senescence [An additional common age marker is genomic and epigenomic instability due to damage accumulation throughout life. Alterations in DNA methylation or histone acetylation/methylation, as well as other chromatin-associated protein modification, can induce epigenetic changes . Thus, enescence . The expnescence ,87. PRC2RF genes . Indeed,subjects . Of note4a locus . The enznescence . The chrnescence . Deficieinocytes  and leadous mice . Also, tnescence . E2F1 renescence . SATB1 e[16INK4a . Of notent cells . The chractivity . Interesnescence .INK4a16 transcription [INK4a16 expression in mouse and rat tissues has been attributed mainly to Ets-1 expression [INK4a16 levels observed in senescent human fibroblasts is consistent with the reciprocal reduction of Id-1 and accumulation of Ets-1 [INK4a16 promoter in primary human keratinocytes, no correlation between their expression and 16INK4a expression or SC depletion during skin chronological aging has been detected [INK4a16 transcription [INK4a promoter [INK4a16-repressor B-Myb is regulated by cyclin D1. Thus, its decrease following stress may regulates entry into senescence [ANRIL expression is suppressed. Thus, cell-cycle inhibitors ARFp14, INK4bp15, and INK4a16 are upregulated, likely by reduced interaction with PRC complexes, and senescence is triggered [Following acute stress stimuli, the Ras/MAPK pathway induces Ets1 and HBP1 upregulation that triggers premature senescence via cription ,140. Thepression . Moreoveof Ets-1 . Howeverof Ets-1 ,142. Desdetected . Ras/MAPcription . Moreovepromoter . The 16Inescence . During riggered .Non-melanoma skin cancers (NMSCs) are the most frequent cancers in the Caucasian population. NMSCs, mainly squamous-cell carcinomas (SCCs) and basal-cell carcinomas (BCCs), originate from keratinocytes and are the most common age-associated malignancies ,146. Thep53 and INK4a16/pRb pathways [INK4a16 and Hedgehog signaling pathway is observed in the BCC pathogenesis [Cutaneous SCCs (cSCCs), especially less well-differentiated tumors, have metastatic potential. They represent an interfollicular epidermis neoplasia and may originate from premalignant actinic keratoses (AKs) and “in situ” SCC (ISSCCs). The most important alterations in cSCC etiology involve RAS, pathways . BCCs rapathways . Howeverogenesis .INK4a16 display uncontrolled proliferation bypassing G1/S transition checkpoint. The INK4a16 gene is frequently mutated in human cancers [INK4a16 are prone to spontaneous or induced tumorigenesis [INK4a16 expression. Some reports correlated higher malignancy with INK4a16 loss [INK4a16 overexpression [INK4a have been found in AKs and cSCCs, such as homozygous gene deletions, hypermethylation of its promoter, and mutations in the INK4a/ARF locus. Loss of heterozygosity in at least one locus of the 9p21 region (containing the CDKN2A gene) is observed in 20% of AKs and 46% of cSCCs, suggesting that INK4a16 inactivation may have a critical role in skin lesion progression [INK4a16 gene may be mutated in up to 24% of cSCCs and these mutations may be hereditary or acquired following UV-induced damage [INK4a16 may also be inactivated apart from loss of heterozygosity, promoter hypermethylation, and/or mutations in the INK4a/ARF locus [INK4a16 expression in ISSCCs compared to AKs and in poor differentiated cSCC compared to well differentiated [INK4a16 expression correlates with a functional pRb pathway, suggesting that upregulation is the result of senescence induction following stress stimuli [INK4a overexpression may be a compensatory feedback effect of the inability to control cell proliferation and has been suggested as marker for HPV-associated SCCs [INK4a16 expression in the absence of HPV [INK4ap16 upregulation might occur as a response to pRb pathway dysfunction. Indeed, INK4a16 expression correlates with the lack of pRb phosphorylation in ISSCCs and invasive cSCCs [INK4ap16 due to CDK4 sequestration [INK4a16 expression is observed in BCC [INK4ap16 is overexpressed at the infiltrative front followed by ceased proliferation, suggesting that INK4ap16 could be involved in tumor invasion independent by proliferation effects. Indeed, INK4a16 is implicated in the regulation of matrix-dependent cell migration [INK4ap16 and Ki67 is associated with both BCCs and cSCCs, their expression does not seem to correlate with the degree of proliferation and malignancy. Instead, INK4ap16 overexpression is significantly associated with sun-exposed areas, suggesting a possible induction of INK4ap16 overexpression by UV radiation [INK4ap16 expression.Cells with dysfunctional  cancers  and miceigenesis ,152. HowK4a loss ,154 and pression ,158,159.gression . 16INK4ad damage . In BCC,RF locus . On the entiated ,163,164. stimuli . Human p stimuli ,167. HPV stimuli , thus 16ted SCCs . Howevere of HPV ,170. p16ve cSCCs . Alteratstration . Alternastration ,172. Mord in BCC . In highigration ,174. Howadiation , suggestINK4ap16 expression in-depth in NMSCs, independently from HPV infection. Here, we examined the expression of 50 genes, including p16INK4, downstream pRb pathway proteins and INK4p16 promoter modulators, as reported in human skin cancer datasets available at Oncomine (www.oncomine.org) as previously described [p < 0.01).Further analyses concerning expression of epigenetic modulators and transcription factors (described in paragraph 3) are needed to study escribed ,177. Twoescribed ,179 fromescribed ,182,183 INK4p16 decrease is observed in one dataset from HNSCCs , which are downstream targets of the Ras pathway, is significantly increased  and oral cavity SCCs (oSCCs) (A  (oSCCs) . InformaINK4a16 [INK4ap16 upregulation observed in these datasets may be independent of HPV infection. The pRb pathway effectors, E2F1 and E2F3, significantly increase in NMSCs. E2F2 displays a similar trend  expression is significantly increased in NMSCs . Indeed,The p63 activator Setd8 is upregulated only in SCCs. According to literature, p63 expression is significantly over-expressed in SCCs, BCCs, HNSCCs, and oSCCs . Indeed,The HAT p300 is mainly upregulated in NMSCs whereas the HDACs are globally upregulated in all analyzed pathologies . The p30INK4p16 upregulation in cSCCs and BCCs.Among the other p63 targets, BRG1 is significantly upregulated in NMSC specimens, differently from HNSCCs and oSCCs . BRG1 isB-myb is slightly upregulated in cSCCs and downregulated in the other specimens. CTFC expression is significantly increased in BCCs and cSCCs from one dataset .INK4p16 repressor Id1 is significantly upregulated in AKs, cSCCs, HNSCCs, and oSCCs whereas Id2 and Id3 increase only in AKs and HNSCCs . Ets2 is a major driver of SCCs. Ets2 binds to the super-enhancers and reprograms gene expression to induce inflammation and promote the tumor growth and development . The p16d HNSCCs . In cSCCINK4ap16 expression and epidermal senescence. However, the dominant-negative JunB mutant promotes tumorigenesis [The transcription factor AP-1 promotes the invasive growth and metastasis of various tumors such as SCCs . AP-1 isigenesis ,205. Recigenesis . On the igenesis ,205.c-Fos and FosB are downregulated in BCCs, cSCCs, and oSCC whereas they are strongly upregulated in HNSCCs. FRA1 and FRA2 are mainly upregulated in AKs and cSCCs . FedericClinical studies define ANRIL as a potential diagnostic and prognostic biomarkers in various cancers . ANRIL cINK4p16 upregulation seems a compensatory mechanism to suppress tumor development by senescence. However, senescence of the non-epithelial component of developing skin tumours might enhance growth and invasion of the pre-malignant epidermal cells via other upregulated pathways, such as p63 and Ras. For instance, p63 is induced by oxidative stress. In the analyzed cutaneous tumors p63 targets  are globally upregulated. Ras is a transducer of extracellular mitogenic signals. Of note, in all analyzed tumors Ras targets  are upregulated. Moreover, some of these proteins are also able to induce INK4p16 expression to block uncontrolled growth.In these specimens, As new data sets are deposited, it will be possible to analyze larger data sets and get more in-depth information.INK4ap16 is a potent inhibitor of the G1/S-phase transition of the cell cycle and exhibits a fundamental role in epidermal homeostasis, aging, and tumorigenesis.INK4ap16 expression, protecting cells from senescence and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate INK4ap16 expression that orchestrates cell cycle exit and senescence response. Cellular senescence is a potent tumor suppressor mechanism, acting in coordination with the immune system to clear potentially malignant cells from the tissues. Thus, it may contribute to aging by depleting functional cells required to maintain tissue homeostasis. However, aging and cancer might be considered related endpoints of accumulating damages within self-renewing compartment. Indeed, age-associated changes in chromatin remodeling and gene expression are responsible for the deterioration of multiple cellular functions, including the senescence response. Accumulation of INK4ap16 positive senescent cells in the aged tissue may be due to synergic contribution of the genomic instability, senescence-associated secretory phenotype dysregulation, and decline in the immune system function, and may contribute to worsening the senescence response efficacy in tumor suppression.Keratinocytes are equipped with several enzymes that shape the epigenetic signatures of different epidermal layers, allowing a dynamic and coordinated expression change of gene sets involved in self-renewal and differentiation. In the basal layer, epigenetic modifiers and transcription factors converge their activity to repress INK4ap16 expression in human epidermal cell types may provide new targeted approaches to protect the epidermis from regenerative capacity decline and premature aging and new protocols for regenerative medicine. However, balancing the expression of INK4ap16 and its regulators has to be carefully tuned to avoid the risk of malignant transformation, given that the response to oncogenic stimuli changes at advanced ages. Indeed, better understanding of INK4p16 regulation in skin tumors may be relevant to standardizing new diagnostic or prognostic tools and in designing new cancer therapies.Unveiling the precise mechanisms regulating