BV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (P Geminiviridae are among the most important pathogens of crop plants worldwide [Plant viruses of the family orldwide ,2,3,4. Eorldwide ,6. An inorldwide . As a reGeminiviruses all have characteristic, two-headed particles that encapsidate single-stranded (ss), covalently circularized DNA genomes, mostly under three kilobases (kb) in size . These sThe mechanism of the AC2-mediated trans-activation of late genes, especially those encoded by DNA-B, is of great research and application interests, because, once understood, it offers the potential of engineering begomovirus resistance that is activated by the invading virus itself. For example, antiviral defense factors, such as resistance genes (R genes) or silence-inducing double-stranded (ds) RNA, can be placed under the control of promoters activatable by AC2, so that they would only express when the corresponding virus replicates in the cell to produce AC2. The particular mechanism adopted by the tomato golden mosaic virus (TGMV) has been extensively examined by Sunter and colleagues. Sunter and Bisaro were among the first to show that AL2 of TGMV, which corresponds to AC2 in other bipartite begomoviruses, activated BR1 (BV1 in other viruses) expression at the transcription level ,14. NoteThe mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects important legume crops such as mungbean, soybeans, and lima beans . Its twoBV1 hereafter) to nt positions 26–419  of MYMV DNA-B. Based on the results obtained by Sunter and colleagues using the TGMV system, we hypothesized that the 394-nt PBV1 contained conserved promoter motifs recognizable by certain plant TFs that also interacted with AC2. Here, we report a systematic dissection of MYMV PBV1, leading to the identification of multiple sequence motifs previously shown to be critical for plant responses to abscisic acid (ABA). The current study set out to determine how expression of the BV1 gene of MYMV reacts to AC2-mediated transcriptional activation. Our study was inspired by a previous report , which mBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within the first 70-nt region. Eliminating these ABRE motifs through either deletion or site-directed mutagenesis caused dramatic losses in the PBV1 responsiveness to AC2-mediated activation. ABRE, as well as CE1 and CE3, are also present in more than a dozen bipartite begomoviruses of the Old World lineage. These results strongly suggest that one or more ABA-responsive TFs are probably recruited by AC2 to confer the DNA-binding specificity needed for the transcriptional activation of MYMV PBV1. ABA is a well-characterized plant hormone that plays critical roles in multiple stages of the plant life cycle, including maintaining seed dormancy and spearheading plant responses to drought and cold stresses during vegetative growth ,24. ABA The Core35S-GFP construct was based on pAI101, a binary vector modified from pCambia1300 ,29. An eAgrobacterium tumefaciens strain C58C1 with electroporation [Agrobacterium suspensions were mixed together and delivered into Nicotiana benthamiana leaves as described [Agrobacterium strain was included in all combinations to alleviate RNA silencing-mediated mRNA degradation. In experiments testing the promoter responsiveness to abscisic acid (ABA) treatment, a 50-mM ABA stock solution was prepared by dissolving ABA powder  in dimethyl sulfoxide (DMSO). It was then added to the Agrobacterium suspensions to a final concentration of 20 µM. Accordingly, the same Agrobacterium suspensions containing 1:2500 diluted DMSO were used as the controls for the ABA treatment. Upon verification, the constructs were introduced into the poration . In mostescribed ,30,31,32N. benthamiana leaves following a published protocol [Proteins were extracted from the agro-infiltrated protocol  and subjConfocal microscopic observations were carried out using a Leica Confocal microscope (TCS SP5) at the Molecular and Cellular Imaging Center, the Ohio Agricultural Research and Development Center, The Ohio State University. The samples were mounted under a 10× lens, and the GFP fluorescence was excited with a 488-nm Argon blue laser. The images were captured with an emission wavelength of 517 nm. The resolution pixels of the original images were 1024 × 1024. BV1 indirectly by recruiting one or more plant TFs to confer the DNA-binding specificity. Since different families of TFs are known to bind to different conserved promoter motifs, TFs that bridge AC2 and PBV1 could be inferred from the enrichment of a specific class of conserved DNA sequence motifs in MYMV PBV1. To begin to delineate the conserved DNA motifs in MYMV PBV1, we took advantage of the study of Shivaprasad and colleagues [BV1 to a 394-nt region  region, which we will call Q1 (meaning the first one-quarter of the PBV1) from here on  needed for TF binding and, hence, AC2-mediated transcriptional activation. This hypothesis was tested by fusing various portions and combinations of MYMV and SYMV PBV1 with a GFP reporter , led us to hypothesize that the Q1 region of the MYMV Preporter B. The reltration . The expltration . The silltration , in all BV1 (PBV1-M) promoter was used to drive the GFP transcription without AC2. By contrast, green fluorescent cells were numerous and easily detectable when AC2 was included  drove a substantially higher level of basal transcription than the PBV1-M  likely resided in the highly homologous Q1 portion of the two promoters  of the CaMV 35S promoter (Core35S) that did not contain an ABRE. Notably, the transcription driven by Core35S was close to five times stronger than the AC2-activated PBV1 and approximately 40 times stronger than PBV1 alone , this was likely due to the stimulatory effect of DMSO. Similar conclusions could be drawn for the samples in lanes 17 and 18 (compared with lane 6). In summary, although the three ABREs within PBV1 were clearly needed for AC2-mediated transcriptional activation, the corresponding TFs were not upregulated by the ABA treatment in N. benthamiana leaf cells (see Discussion). However, ABA failed to elicit a significant stimulation of the transcription from the PThe question of how AC2, a protein encoded on the DNA-A of bipartite begomoviruses, transactivates the late genes encoded on the DNA-B, a separate genome segment, is important to resolve, as the answer could offer insights on how genimiviruses coordinate the expression of early and late genes to balance the optimal genome replication with an efficient whole-plant spread. It could also inform us of novel strategies on controlling these viruses. The findings made using the TGMV model strongly suggest that AC2/AL2 acts on the BV1/BR1 promoter by recruiting PPD2, a putative TF of the plant host that confers the DNA-binding specificity ,18,19. HWhile the importance of ABA-responsive promoter elements in BV1 expression has not been recognized before, the involvement of the closely related G-box motif in the transcription of AC1/AL1 (Rep) has been reported . SeparatN. benthamiana, making transcriptional activation less efficient in our system. Consistent with this idea is the fact that the BV1 promoter of the SYMV exhibited much higher basal transcription than the MYMV BV1 promoter in our assay, suggesting virus- and host-dependent effects. An unexpected finding of our experiments is that the MYMV BV1 promoter is very weak. Although we expected to see low transcription without AC2 activation, we were nevertheless surprised to realize that its activity was still many folds weaker than the Core35S promoter upon AC2 activation. Several reasons might account for this low activity. First, the activity detected with our system may only account for a fraction of the BV1 transcription in actual MYMV infections. This is because, in actual infections, BV1 and other late genes are expressed after viral replication from the many progeny genomes newly synthesized in the cells. As a result, BV1 transcription is expected to occur simultaneously on many copies of DNA-B. By contrast, the amount of nonreplicating DNA templates in our nonreplicating system is expected to be very limited. Second, optimal BV1 transcription may demand an ideal host cell environment not provided with our experimental system. The MYMV is a virus of legume plants. Hence, the TFs accessible to AC2 in legumes may differ substantially from their orthologs in N. benthamiana leaf cells at low levels, but their corresponding genes were not responsive to ABA in these particular cells. This scenario is possible, because ABA responses are known to be highly coordinated, often occurring in dividing cells at root and shoot growth points [N. benthamiana cells we used were mostly nondividing. Nevertheless, our system did detect a substantial level of AC2 activation, permitting the interrogation of the ABREs in the MYMV BV1 promoter. In conclusion, our study revealed a previously unrecognized class of promoter elements that mediate the AC2 transcriptional activation of a BV1 promoter of a bipartite begomovirus and paved the way for the identification of the specific TF(s) involved in the process.The lack of response to ABA treatment by the MYMV BV1 promoter is also noteworthy, given the requirement of ABREs for its activation by AC2. This could be explained if the particular ABA-responsive TF(s) were present in h points . Consist


Human papillomaviruses (HPV) are a large family of viruses which contain a circular, double-stranded DNA genome of approximately 8000 base pairs. The viral DNA is chromatinized by the recruitment of cellular histones which are subject to host cell–mediated post-translational epigenetic modification recognized as an important mechanism of virus transcription regulation. The HPV life cycle is dependent on the terminal differentiation of the target cell within epithelia—the keratinocyte. The virus life cycle begins in the undifferentiated basal compartment of epithelia where the viral chromatin is maintained in an epigenetically repressed state, stabilized by distal chromatin interactions between the viral enhancer and early gene region. Migration of the infected keratinocyte towards the surface of the epithelium induces cellular differentiation which disrupts chromatin looping and stimulates epigenetic remodelling of the viral chromatin. These epigenetic changes result in enhanced virus transcription and activation of the virus late promoter facilitating transcription of the viral capsid proteins. In this review article, we discuss the complexity of virus- and host-cell-mediated epigenetic regulation of virus transcription with a specific focus on differentiation-dependent remodelling of viral chromatin during the HPV life cycle. Papillomaviridae (PV) family of viruses is composed of over 450 distinct types of human papillomavirus (HPV) and over 200 animal papillomaviruses (http://pave.niaid.nih.gov). Each PV type is defined by a > 2% difference in sequence from any other know type. PV types are arranged into distinct genera that share > 60% identity in the L1 open reading frame (ORF). HPV types are phylogenetically arranged in five genera; alpha, beta, gamma, mu and nu [At the time of writing, the E: P97 - HPV16, P105 - HPV18; PL: P670 - HPV16; P811 - HPV18), the origin of replication to which the E1 protein binds, as well as multiple binding sites for the viral E2 protein. Relevant to this review was a study in the 1970s that showed that the HPV genome does not exist in a naked state in an productive lesion but as a nucleoprotein complex containing cellular histones [The genome of all HPV types has a similar arrangement characterized by an approximately 8000 base pair circular doubled-stranded DNA genome encased in a non-enveloped icosahedral capsid of about 55 nm in diameter . The virhistones Fig. 1b)E: P97 - E) that is active in undifferentiated basal keratinocytes, the late promoter (PL) that lies within the E7 ORF and becomes active in differentiated keratinocytes, and the PE8 promoter present within the E1 ORF which controls the expression of an E8^E2 protein that regulates viral transcription and viral DNA replication. Alternative RNA splicing leads to the production of multicistronic viral messenger RNAs transcribed from one strand of the genome. Early transcripts initiate from PE and terminate at the early polyadenylation (PolyA) site (PAE) situated at the end of the E5 ORF  present in the URR. The PE8 promoter is active throughout the infectious cycle and unlike PE and PL, constitutive activation of PE8 is not controlled by the viral enhancer elements situated within the URR [Several promoters within the HPV genome have been identified, but for the purposes of this review, we will mention here only the early promoter . The ovHPVs infect basal keratinocytes, the proliferative compartment of squamous epithelia, through wounds and micro-abrasions in the epithelium Fig. . KeratinThe maintenance phase of HPV DNA replication occurs in synchrony with the host DNA which ensures equal partitioning of the viral episomes to daughter cells. E2 plays a central role in this process by tethering the viral genomes to host chromatin to ensure efficient inheritance of the viral DNA. The viral genomes are tethered to different regions of mitotic chromosomes and this involves interaction of E2 with different host factors, including the epigenetic reader bromodomain protein Brd4, the DNA helicase chromosome loss-related 1 (ChlR1) and topoisomerase binding protein 1 (TopBP1) (reviewed in ). E2-med4CGGT that each binds an E2 dimer [E2 protein function is also central to virus transcription control. There are four conserved palindromic E2 binding sites within the URR of high-risk alpha-HPVs with the consensus sequence ACCG(N)E2 dimer   spliced into the 3′ exon of the E2 gene such that E8^E2 protein contains a novel N-terminal domain fused to the DNA-binding domain of E2 [E8 is constitutively active and independent of enhancer elements with the HPV URR, E8^E2 is able to strongly inhibit its own promoter but E2 weakly activates it. Such fine tuning of E8^E2 expression requires E2/E8^E2 binding within the URR and is thought to represent a mechanism whereby HPV controls viral copy number [The E8^E2 protein is encoded by a transcript that originates from the Pin of E2 . As suchin of E2 . E8^E2 iy number .E and PL activities. E5, E6 and E7 protein functions synergize to promote cell cycle re-entry and proliferation, and survival of post-mitotic keratinocytes to orchestrate viral DNA replication competent cells. They do so by targeting the components of key growth control pathways including p53 and retinoblastoma, as well as pathways that enable the virally infected cells to avoid immune detection [Once the infected cell moves from the basal lamina, the normal entry into differentiation is delayed and early gene expression increases with stimulation of Petection . The viretection . To avoietection . Once vietection .DNA in the nucleus of eukaryotic cells is wrapped around octameric complexes of proteins called histones, composed of dimers of H2A, H2B, H3 and H4. Each histone core is wrapped by ~ 147 base pairs of DNA  to form Through epigenetic modification, chromatin exists in different physical states to regulate transcription. Tightly packed, closed chromatin known as heterochromatin is transcriptionally repressed, whereas open chromatin known as euchromatin is permissive for gene transcription as it contains much less densely packed and dynamically associated histones allowing transcription factors to access regulatory elements and drive transcription. These general forms of chromatin are dictated by post-translational modification (PTM) of histones or by direct, covalent methylation of DNA. Heterochromatin is generally characterized by enrichment of repressive epigenetic marks such as H2K4Me2/3, H3K9Me1, H3K27Me2/3 and H4K20Me3. Acetylation of histones decreases their affinity to DNA and as such open euchromatin is enriched in epigenetic marks including H3K4Ac and H3K27Ac . The divDNA can be directly methylated on the 5′ position of the cytosine pyrimidine ring creating 5-methylcytosine (5mC). This covalent modification most often occurs on cytosines preceding a guanine (CpG), and is catalysed by DNA methyltransferases (DNMTs) which catalyse either maintenance or de novo DNA methylation (reviewed by ). While The association of histone complexes with encapsidated HPV DNA was first described by Favre and colleagues in 1977 . ElectroHPV has been shown to interact with several HAT and HDAC family members to regulate viral transcription. CREB-binding protein (CBP) and its paralogue p300 are transcriptional coactivators that bind DNA-bound transcriptional regulators and acetylated histones. Once bound to a promoter, CBP/p300 recruit the basal transcription machinery to activate transcription. CBP/p300 also have intrinsic HAT activity and can acetylate histones , therebyIn the context of an HPV infection, E2 functions to repress E6/E7 transcript production. In an siRNA screen designed to identify cellular factors that contribute to E2-mediated repression of the HPV18 URR, EP400, a component of the NuA4/TIP60 histone acetylase complex was identified . AcetylaSirtuins (SIRT1-SIRT7) are a protein family of class III HDACs that function in DNA damage repair and apoptosis. The stable maintenance of HPV16 and HPV31 episomes within human foreskin keratinocytes results E6/E7-dependent elevation of SIRT1 expression. This increase is maintained within differentiated keratinocytes . SIRT1 pThe viral episome in undifferentiated keratinocytes exists in a repressed chromatin state in part by the recruitment of polycomb repressor complexes 1 and 2 (PRC1/2) which catalyse deposition of repressive H3K27Me3 and H3K119Ub . While tThe histone methyltransferase SETD2 is a writer of trimethylation of histone 3 lysine 36 (H3K36me3), a mark of active transcription. High-risk E7 mediates the post-transcriptional stabilization of SETD2 resulting in increased levels in HPV31 and HPV16 containing human foreskin keratinocytes. SETD2-dependent H3K36me3 deposition is apparent throughout the viral genome and enriched at the 3′ end of the early gene region in both undifferentiated and differentiated keratinocytes and is essential for both maintenance and productive viral replication . InteresThe first evidence of epigenetic modification of HPV DNA was in the form of covalent methylation of CpG dinucleotides on HPV1 DNA , 57. It Modulation of CpG methylation of HPV DNA is important during carcinogenesis. Several studies have demonstrated a correlation between increased CpG methylation within the late gene region of integrated viral sequences and disease progression –67. HighStimulation of keratinocyte differentiation results in an increase in transcripts that originate from the early protomer and the appearance of transcripts that are initiated from within the E6/E7 ORFs around the late promoter , 70, 71.YY1 is a strong repressor of the HPV keratinocyte specific enhancer , 75 and Chromatin binding architectural proteins such as the zinc-finger CCCTC-binding factor CTCF are fundamental in the three-dimensional organization of chromatin. CTCF is a ubiquitously expressed DNA-binding protein that binds tens of thousands of sites in the human genome  and funcCTCF has been demonstrated to regulate transcription of several large DNA viruses including the γ-herpesviruses Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) –96. SimiIt is feasible that loop formation in the HPV genome occurs via loop extrusion between the CTCF and YY1 anchor points but whether this loop is stabilized by cohesin is currently unknown although phosphorylated SMC1 (pSMC1), a structural component of cohesin rings, binds to HPV31 episomes that appears to be important for viral genome amplification . WhetherInterestingly, ChIP-Seq data available from the ENCODE project  for HeLaEpigenetic regulation of HPV transcription is necessary for episome establishment, genome maintenance and completion of the productive HPV life cycle. The complex interplay of positive and negative epigenetic regulation of HPV transcription is inextricably linked to the differentiation status of the infected cell; the viral genome exists in an epigenetically repressed state in the undifferentiated basal cells with low-level gene expression such that the episome can replicate but prevent immune activation. As cells enter a programme of differentiation, epigenetic repression of the viral genome is alleviated and the viral chromatin structure is maintained in an active state, resulting in increased expression of viral replication proteins and activation of the late promoter and capsid protein production. This complex transcriptional programme, requiring a plethora of host cell epigenetic regulators, appears to be disrupted in HPV-induced carcinogenesis providing the possibility of new therapeutic strategies against HPV-induced disease.


Chromosomal architecture, which is constituted by chromatin loops, plays an important role in cellular functions. Gene expression and cell identity can be regulated by the chromatin loop, which is formed by proximal or distal enhancers and promoters in linear DNA (1D). Enhancers and promoters are fundamental non-coding elements enriched with transcription factors (TFs) to form chromatin loops. However, the specific cooperation of TFs involved in forming chromatin loops is not fully understood.Here, we proposed a method for investigating the cooperation of TFs in four cell lines by the integrative analysis of DNA sequences, ChIP-Seq and ChIA-PET data. Results demonstrate that the interaction of enhancers and promoters is a hierarchical and dynamic complex process with cooperative interactions of different TFs synergistically regulating gene expression and chromatin structure. The TF cooperation involved in maintaining and regulating the chromatin loop of cells can be regulated by epigenetic factors, such as other TFs and DNA methylation.Such cooperation among TFs provides the potential features that can affect chromatin’s 3D architecture in cells. The regulation of chromatin 3D organization and gene expression is a complex process associated with the hierarchical and dynamic prosperities of TFs.The online version of this article (10.1186/s12864-019-5535-2) contains supplementary material, which is available to authorized users. Drosophila embryos is required for the sustained proximity of enhancer to its target promoters. Although the chromatin loop has an important role in gene regulation and disease [The spatial structure of the genome is important to cellular functions, such as gene expression and regulation, cell differentiation and identity, and tissue development –3. Linea disease , the speGenome-wide profiling of the TF binding sites is extensively studied across linear genomic DNA –13. For Recently, the molecular mechanisms and proteins in chromatin interactions have been studied. In situ Hi-C  can prodTo obtain a nucleotide resolution map of cells, scientists have developed the ChIA-PET  , 20 to sIn the present study, we provided the HidPET  method to study the hierarchy and dynamics of TF cooperation by integrating ChIP-Seq and ChIA-PET datasets. This method mainly focuses on enhancer–promoter interactions, which play a dominant role in chromatin interaction. The networks are constructed by using the enrichment information of the 3D chromatin data of ChIA-PET and the 1D linear genomic data of ChIP-Seq. Then, the networks are fused with the additional protein–protein interactions (PPIs). The hierarchy and local network parameters are analyzed across four cell lines. Hierarchical structure, community and clique analysis revealed the hierarchical and dynamic features of synergistic cooperative TF interactions in regulating gene expressions and chromatin 3D architecture.We developed the HidPET method to combine ChIA-PET and ChIP-Seq data with PPI data to systematically study the hierarchy and dynamics of TFs in four cell lines. The flow chart of the HidPET method is presented in Fig. In general, the genomic segments of chromatin interactions are enriched in promoters and enhancers. Enhancers extensively interact with promoters, which form loops and regulate the expressions of distant or proximal genes . In our A community of PPI is a candidate functional module , 30. TheThe directed biological network can be compared with the “chain-of-command” structures in social networks . A previHomo sapiens (phylostratum 16) [In addition, because the TFs do not arise simultaneously, they generally occur through the re-organization of pre-existing genes or de novo . De novoatum 16) . To undeAlthough TFs have a community structure and hierarchical structure to synergistically work together, the specific cooperation between TFs is not fully understood. To further analyze the cooperation between TFs, we calculate for the maximal cliques for the fused matrix of TFs in different cell types. The clique of a network is a fully connected sub-graph, which is a basic topological module of a network. In this work, we apply the MClique method in Cytoscape  to detecp-values for the GM12878, K562, HUVEC, and MCF7 cell lines are 0.00265, 2.2e-16, 2.2e-16, and 0.00125, respectively. This result means that the corresponding genes of the TFs in the maximal clique tend to be co-regulated and work synergistically.To further characterize the synergistic feature of TFs in a given clique, we calculate for the Spearman’s rank correlation coefficients of the TFs’ expression. For each cell line, the corresponding expression levels of these TFs are collected from the RNA-Seq data deposited in the ENCODE project. The Spearman‘s rank correlation coefficient is calculated for the expression of all pairs of TFs within the same maximal clique. The same procedure is applied to all pairs of TFs that are not in the same maximal clique. The distribution of Spearman’s rank correlation coefficients is shown in a violin plot  databases . The speThe chromatin states for K562, GM12878, and HUVEC are downloaded from the ENCODE project at UCSC  All datasets used in this study  with accession codes and Parameter selection. To balance the Additional file 2:Illustration of the fused network communities of TFs and the overlapping communities of GM12878 cell line. Nine groups are calculated, and the specific list of TFs in each community is listed. (XLSX 10 kb)Additional file 3:Figure S1. Hierarchical structures of the GM12878 TFs network. Figure S2. Percentage of TFs from different levels in the phylostrata. Figure S3. Violin plots of Spearman’s rank correlation coefficient for TF expressions in the same clique or not in four cell lines. Figure S4. Distribution of ZNF143-YY1-SMC3-SIX5-RAD21-CTCF-CTCFL seven TF binding sites and loops of enhancer and promoters in hierarchical structures of chromatin in chromosome 19 of GM12878 cell line. Figure S5. Parameter selection in motif scanning. (PDF 687 kb)Supporting figures. Additional file 4:Calculation for the maximal cliques for the fused matrix of TFs in different cell types, the specific TF in each maximal clique for the four cell lines is listed. (XLSX 12 kb)Additional file 5:The biclustering results of cliques and genes. We adopt the 51 cliques and 1223 regulated genes of K562 as an example and apply the biclustering method fabia to group cliques and genes into 10 classes. The specific clique and gene list for each superclique  and gene group  are listed. (XLSX 22 kb)


SBSN). Originally described as a component of the cornified envelope, the function of stratified epithelia-expressed SBSN is unknown. Both the lack of knowledge about the gene role under physiological conditions and the emerging link of SBSN to various human diseases, including cancer, attract research interest. The association of SBSN expression with poor prognosis of patients suffering from oesophageal carcinoma, glioblastoma multiforme, and myelodysplastic syndromes suggests that SBSN may play a role in human tumourigenesis. Three SBSN isoforms code for the secreted proteins with putative function as signalling molecules, yet with poorly described effects. In this first review about SBSN, we summarised the current knowledge accumulated since its original description, and we discuss the potential mechanisms and roles of SBSN in both physiology and pathology.Among the ~22,000 human genes, very few remain that have unknown functions. One such example is suprabasin ( SBSN) has been associated with multiple diseases, including cancer. SBSN isoforms are putative signalling molecules inducing cellular signalling  and various cellular processes, such as migration, proliferation, neovascularization, therapy-, apoptosis- and immune-resistance. Therefore, SBSN is considered an oncogene and is a proposed biomarker in a couple of diseases, lung carcinoma and myelodysplastic syndromes (MDS). The apparent need for a deeper understating of the nature and the function of SBSN prompted us to compile all current knowledge of SBSN together with suggestions for future research work.Since its original description in human and mouse keratinocyte differentiation , suprabaSBSN gene is located on human chromosome 19  close to other keratinocyte-differentiation associated genes dermokine-α/β and KDAP [SBSN gene is a part of a coordinately expressed new stratified epithelium-related gene cluster, tentatively named stratified epithelium secreted peptides complex, SSC [SBSN was studied mostly in human and mice; however, other mammals possess SBSN homologs. Predicted SBSN peptide encoded in the genome of Gorilla gorilla gorilla possesses 97.6% amino acid sequence similarity to the human SBSN-1. Relatively high amino acid sequence identity (58.9%) between mouse and human orthologs of the largest SBSN isoform  suggests strong gene integrity and conserved function among the species. Paralogs of SBSN were not defined, and no genes with confidently high sequence identities were identified. The Ensembl database [SBSN orthologs , SBSN-2  and SBSN-3 . Importantly, SBSN-2 represents a fully spliced isoform [SBSN-1 contains an in-frame retention of the first intron. SBSN-3 is also a fully spliced isoform but lacks exon 2. Strikingly, the mouse (Mus musculus) isoform corresponding to human SBSN-2 has not been identified. This may restrict the applicability of a mouse model for functional studies of SBSN isoforms. Interestingly, a sequence with high sequence identity (81.4%) to human exon two is present within the murine Sbsn-1 exon one, and the major difference between the human intron one exon two junction and homologous murine sequence (CACGAGGCCGGG vs. AACCAGGGTCAA) implies that in mice, the splicing site was likely not established, or was lost. The murine putative exon two is spliced together with the entire exon one in murine Sbsn-2, hence resembling human isoform three, based upon amino acids sequence identity analysis . Multiple other rodents lack homolog corresponding to the human SBSN-2 isoform, and interestingly, several primates  lack putative SBSN-2 as well. However, this may be due to the absence of its identification, rather than sequence deviation.Human  introns a. Human SBSN isoforms , glycine-(20%), and histidine-(10%) rich due to the presence of short tandem Glycine-x-Histidine-Histidine repeats  encoded by the retained intron one. Note, structural proteins often contain compositional biases of similar characteristics to SBSN-1 Ala-/Gly-/His-rich domain (aa 26—524), and based upon these observations, SBSN was proposed as a component of the cornified envelope (CE), the essential structure of corneocytes responsible for the skin protective function . NotablySBSN possesses similar features. The N-terminal signal peptide was predicted (aa 1–23), and the compositional bias of Ala-/Gly-/His-rich domain (aa 97—478) in SBSN-1  is also present in mouse homolog. The current description of other murine Sbsn isoforms is rather confusing. Originally, murine Sbsn-1 isoform was identified as a 700 aa long peptide [The mouse homolog of icted aa –23, and O-glycosylation in various human cell lines [Secreted murine Sbsn-2 was detected using overexpression experiments , showingll lines . Three pll lines ; howeverPseudomonas sp. mis38 lipase (PDB: 2ZJ6) served as a template for structure prediction with 12% sequence identity and 99.9% prediction confidence. Collagen α chain was among other possible templates with slightly lower confidence levels. The methyl-accepting chemotaxis protein of Escherichia coli (PDB: 1QU7), showing 13% sequence identity with SBSN-2, was used as a template for the structure prediction of the isoform with 98.5% confidence. Interestingly, the methyl-accepting chemotaxis protein of Thermotoga maritima (PDB: 2CH7) was suggested as a second template with 98.4% confidence. SBSN-3 isoform model was predicted upon the structure of human micelle-bound α-synuclein (PDB: 1XQ8), which showed 18% sequence identity.Three-dimensional structure of SBSN is still unknown. We performed Phyre2-based structure prediction  of all tSBSN expression is tightly associated with stratified epithelia, but other expression sites have also been defined. Mechanisms of SBSN transcriptional control are not clearly described. We utilized the JASPAR algorithm [SBSN proximal promoter (2 kbp upstream) and downstream coding region. Additionally, we verified putative binding sites. Based on these results, we established a model of human SBSN promoter , but meSBSN is physiologically expressed in mouse stratified epithelia including the suprabasal epithelial layer of epidermis, tongue, oesophagus, palate, stomach, uterus, thyroid, trachea, lung, vagina, thymus, and urinary tract. In human, SBSN expression is associated with epidermis, thymus, uterus, tonsils, vagina, and oesophagus [SBSN expression in mouse embryonic and adult brain [SBSN expression in human astrocytes, and its elevation under pathological conditions [SBSN mRNA levels in basal ganglia, but evaluation of SBSN protein presence in the human brain is needed to confirm this expression site [Sbsn mRNA; neither do several other mouse tissues and organs such as heart, kidney, or smooth muscle [SBSN expression sites seem to be conserved, except for the brain.sophagus ,2,15. Orlt brain ,16,17, bnditions . TranscrSbsn mRNA was detected on day seven of embryonic development when the expression is likely mediated by extra-embryonic tissue. Hence, the embryonic expression was detected on day 15 of the development at first, which is in coincidence with epidermal stratification. Sbsn mRNA levels then peak on day 17 [SBSN expression is associated with expression of dermokine-α/β [SBSN mRNA was elevated in skeletal muscle cells of Alaskan sled dogs during an acute response (2 h post-exercise) after a prolonged endurance training together with dermokine and keratin 5 [SBSN expression is supported by the microarray analysis of therapy-resistant cancer cells in vitro, which showed transcript elevation of innate immune response genes and SBSN following 5-aza-2′-deoxycytidine (5-AC)-treatment or γ-radiation. Notably, activation of the ERK pathway downstream of IFN signalling emerged as a direct activator of SBSN expression [SBSN expression inducing processes.Mouse kine-α/β , the compression . TherefoSBSN transcription is not specifically defined, but some are suggested. The Sbsn transcript elevates in differentiating mouse keratinocytes in vitro, whereas, several genes of the cornified envelope are downregulated upon SBSN knockdown [SBSN expression; however, the role of ERK in keratinocyte differentiation provides contradictory results [JUNB elevation, which plays an essential role in epidermal differentiation [JUNB and SBSN transcripts, together with disruption of keratinocyte granulation and development of stratum corneum in an organotypic model of the human epidermis [Srf in basal cells, showed reduced Sbsn transcript levels [SBSN proximal promoter region of both, human and mouse  and one binding site (−1777/−1760) in human with a 0.82 relative score). Changes in the actin cytoskeleton and MAL/SRF promote physical stimuli-induced keratinocyte differentiation via JUNB [JUNB transcript is elevated 2 h post-exercise, while the Fos transcript is downregulated [SBSN is further supported by increased expression of Sbsn in murine endothelial cells following treatment with Egf, but not bFgf [SBSN expression efficiently [SBSN expression during differentiation of primary epidermal keratinocytes in vitro was suppressed with PKC inhibitor [SBSN expression likely via, but not only, AP-1-enabled transcription. Indeed, SBSN promoter region contains multiple AP-1 binding sites. In total, a JASPAR search predicted 72 AP-1 binding sites, with extensive sequence overlaps, hence lesser number of regions is more likely. Five regions showed >0.9 relative scores . The composition of transcription factors and stimuli responsible for nockdown . This prnockdown . Targetsnockdown . This su results ,22. Addintiation . Inhibitpidermis . Mouse bt levels , and indvia JUNB . Note, ing site −77/−1760 not bFgf . FurtherSBSN [SBSN promoter exposed to lower temperatures. S100A4 was identified to be a cold-induced gene [S100A6, which is supported by a previous study [SBSN, and JUND transcript levels were elevated following cold-treatment [Lower temperature (33 °C) is frequently used in biotechnology for culture/propagation of Chinese hamster ovary cells. In a recent study, a list of cold-induced genes was established using RNAseq 48 h post change of condition, which included SBSN . This waced gene , togetheus study . Furtherreatment .SBSN transcript was one of 58 significantly upregulated in carotid arteries in sheep [SBSN expression. Note, the upstream pathway, ERK, and a target of SBSN signalling, AKT, showed increased activation in sheep carotid arteries accompanied long-term hypoxia [In response to hypoxic stress, high altitude acclimatization leads to lower oxygen tension and hypobaric pressure with enhanced hematopoiesis, increased blood volume, and neoangiogenesis to redistribute blood flow to vital organs, including the brain. The latter is mediated by carotid arteries. Upon acclimatization to high-altitude-associated long-term hypoxia, in sheep . Interes hypoxia .SBSN gene promoter was originally described as AT-rich, lacking CpG islands and containing a canonical TATA box [SBSN promoter region. Conversely, we observed an initiator element at +9/+15  is mediated by FOXO1 [SBSN transcript levels. We predicted four FOXO1 binding sites within 2 kbp SBSN promoter region  of primary non-small cell lung carcinoma (NSCLC) samples observed in the same study. Hypomethylation of the SBSN promoter was an effect of a dysregulated proto-oncogenic zinc finger transcription factor, CTCFL/Brother of the Regulator of Imprinted Sites (BORIS) [SBSN transcription can be induced in vitro in a salivary gland adenoid cystic carcinoma (ACC) cell line with 5-AC and trichostatin A (TSA) resulting in hypomethylation of the CpG island [SBSN transcripts are induced with 5-AC in human cancer cell lines such as DU-145, MCF-7, and HeLa [SBSN gene promoter contains two experimentally confirmed CTCFL/BORIS binding sites in the first exon close to the transcription start site (+202/+374) and in the second intron in front of a CpG island (+2678/+2840). BORIS-mediated induction of SBSN is associated with demethylation of a CpG island in SBSN second intron and changes in histone marks comprising elevation of the active H3K4me3 and H3K14Ac, and downregulation of the repressive H3K9me3 modifications. Importantly, the elevation of SBSN transcript levels mediated by BORIS is dose-dependent. Relatively low BORIS levels were responsible for significantly higher SBSN transcript levels compared to high BORIS levels associated with re-methylation of the second intron of SBSN and increased nucleosome occupancy of SBSN transcription start site. A repressive histone mark H3K9me3 mirrored the SBSN second intron methylation pattern. Interestingly, CTCF and BORIS compete for the same binding sites of the SBSN promoter region [SBSN expression. Hence, methylation of regulatory sites represents the main feature responsible for the regulation (suppression) of SBSN transcription. Multiple studies described aberrant elevation of gnancies ,29,32, bilencing . Indeed,t levels . This istely 50% /28 of prG island . The SBSand HeLa . Notablyand HeLa , indicattely 50% /28 of prSBSN expression suggested previously [SBSN expression in oesophageal squamous cell carcinoma (ESCC) [SBSN was confirmed by chromatin immunoprecipitation (ChIP), and remains the only determining region of SBSN promoter with the validated transcription binding factor. Furthermore, double knockout Klf2 and Klf4 mouse cardiac microvascular endothelial cells showed significantly reduced SBSN transcript levels compared to control mice [SBSN promoter region, two most prominent are depicted  . SOX2 birol mice . We idenSbsn knockout mice do not manifest any abnormal skin phenotype [SBSN knockdown abrogated the development of stratum granulosum, disrupted the formation of keratohyalin granules, and affected the morphology of some keratinocytes in the human living skin equivalent model [SBSN in human malignancies provided deeper insight regarding its function. Induction of SBSN activated AKT and p38MAPK kinases, and the WNT/β-catenin pathway [SBSN is depicted in As already mentioned, the physiological function of SBSN is currently unknown. henotype , but shRnt model . The secnt model ,38,39,40nt model , indicat pathway ,29. The  pathway ; thus, SSBSN is from cancer research. Several studies have suggested the role of SBSN in adaptation to stress conditions [SBSN coding sequence and its proximal promoter, is described in various cancer types, including ovarian, cervical, pancreatic, and breast carcinomas [The most evidence about nditions , activatnditions ,29, and nditions ,29, the rcinomas ,42,43,44SBSN expression in cancer was a reported increase of SBSN mRNA levels linked with the hypomethylation of the SBSN promoter in almost half of non-small cell lung carcinoma cases [SBSN in lung squamous cell lines resulted in increased anchorage-dependent growth in soft agar assay. The expression of SBSN in lung cancer correlates with BORIS [SBSN CpG island in the second intron is significantly hypomethylated in primary salivary gland adenoid cystic carcinoma (ACC) compared to the normal salivary gland tissue. Knockdown of SBSN in a salivary gland ACC cell line suppressed anchorage-dependent growth in the soft agar and invasiveness in Matrigel invasion assay. Nevertheless, no correlation of SBSN with gender, smoking history, tumour or nodal stage, or metastatic disease of ACC was found. Also, no correlation with patient survival or time to disease relapse were found [The first evidence of ma cases . The ectth BORIS ,33. BORIth BORIS ,46,47. Bth BORIS . Additiore found . SBSN was originally proposed in a study of oesophageal squamous cell carcinoma (ESCC) [SBSN mRNA and the 25 kDa protein isoform. Importantly, ESCC tissue samples also showed elevated SBSN mRNA levels compared to unaffected tissue. Immunohistochemical analysis of 170 clinical ESCC specimens revealed a positive correlation between SBSN expression and tumour size, tumour clinical stage, and patient vital status. Patients with higher SBSN expression showed shorter overall survival [SBSN isoform 2 promoted proliferation and anchorage-independent growth of ESCC cell lines in vitro  and tumour growth in vivo, whereas SBSN knockdown showed the opposite effect. SBSN-2 in ESCC cell lines increased the activity of WNT/β-catenin signalling pathway, which resulted in TCF/LEF transcriptional activity, nuclear translocation and reduced phosphorylation of WNT/β-catenin, and increased transcript levels of WNT/ β-catenin signalling-regulated genes such as AXIN2, MYC, CCND1, FRA1, MMP7 and JUN. The effect of SBSN on WNT signalling was mediated by phosphorylation of GSK3 β. WNT/ β-catenin signalling pathway regulates several cancer-associated processes such as cell growth and cell death, migration, invasiveness, stemness, and differentiation. Again, SBSN expressing ESCC cell lines formed in vivo tumours with higher microvascular density indicating the role of SBSN in angiogenesis [SBSN was suggested to mediate the angiogenic potential of early-stage ESCC via AKT signalling [The oncogenic role of a (ESCC) . In compsurvival . A subsesurvival . The oveogenesis . In lineogenesis ; howevergnalling . de novo angiogenesis in tumour tissue. In accordance with the effects of SBSN in angiogenic processes [SBSN compared to non-tumour (NEC) endothelial cells. SBSN was upregulated in mouse TEC isolated from human tumour xenografts, and its knockdown significantly suppressed VEGF-A-mediated migration and tube formation of mouse TEC compared to mouse NEC. Moreover, Sbsn knockdown reduced the phosphorylation status of Akt, but not Erk kinase, in mTEC indicates that Sbsn may take part in TEC migration, tube formation and pro-angiogenic activity via Akt activation [SBSN expression to vein cell function is further supported by an observation that knockdown of SBSN affected the capability of vein wall-derived cells to contract collagen gel in vitro [Tumour growth depends on rocesses ,29, humativation . The linin vitro .SBSN transcript levels correlated with significantly lower survival of glioblastoma multiforme (GBM) patients. SBSN transcript levels are elevated in a GBM subtype with mesenchymal signature, the phenotype associated with a strong immunosuppressive milieu, therapy resistance, and the elevation of angiogenic markers [SBSN was not determined. In another glioblastoma cell line U373, the siRNA-mediated knockdown of SBSN expression its participation in radioresistance via an unknown mechanism [In the case of malignant brain tumours, the higher  markers ,52. Util markers ) revealeechanism . SBSN in therapy resistance is supported by other findings. The expression of SBSN following radio- or chemo-therapy regimes in stem-like cells with a low-adhesive phenotype was mediated by ERK1/2 activity in breast, prostate and cervical cancer cell lines [SBSN relieved ERK1/2 activity-dependent anoikis resistance of low-adherent cells. Similarities in a gene expression pattern in differentiating mouse skin [The role of ll lines . Knockdouse skin  and irrause skin  can be oSBSN was identified among 16 genes negatively associated with lymph node metastases in head and neck cancer (HNC) patients [CNFN) represented a hub gene of the SBSN-containing gene cluster. This cluster was associated with gene set enriched for the p53 pathway and estrogen pathway genes. This suggests that the CNFN and SBSN-16-genes containing set could be functionally connected [SBSN expression were linked via elevation of aromatase transcript levels in confluent ASCs [SBSN expression following 17beta-estradiol treatment of immortalized vaginal epithelial cells [patients . In thisent ASCs  and indual cells . Importaal cells . Note, aal cells , hence sSBSN expression was shown to be aberrantly elevated by myeloid compartment, predominantly by myeloid-derived suppressor cells (MDSCs) and early-stage MDSCs (eMDSCSs), in bone marrow (BM) of MDS patients [SBSN expression was therapy-independent, and hence an intrinsic feature of the disease. Importantly, SBSN levels in peripheral blood reflected BM levels of SBSN [O-glycosylated at threonine 221 was observed specifically in peripheral blood (PB) plasma of gastric cancer patients [SBSN-expressing BM MDSCs play a crucial role in the pathogenesis of MDS [SBSN as a component of the innate immune response. MDS represent a heterogeneous group of pre-leukemic diseases. MDS are characterized by ineffective haematopoiesis affecting the myeloid lineage predominantly . Two stupatients . Importa of SBSN . Togethe of SBSN , MDS rep of SBSN . Furtherpatients  and redupatients  support patients . Importas of MDS ; hence ns of MDS . Among ms of MDS , this fuSBSN with ovarian carcinoma. Ovarian carcinoma derived SK-OV-3 cell line expresses SBSN in vitro and marked elevation of SBSN transcripts was found in patients’ biopsies of ovarian tumours [SBSN in ovarian carcinoma, but the association of SBSN with the prognosis of ovarian cancer requires further investigation. There is also limited evidence for a possible association of  tumours . These rSBSN expression with metastatic stages of malignant diseases is supported by a study employing mouse 4T1 cancer model. The blood-circulating 4T1 cells and 4T1 cells isolated from liver metastases showed higher Sbsn transcript levels compared to the primary tumour or parental 4T1 cells [Sbsn to metastatic disease is supported by another study [Tgm3, desmoglein 1beta, and corneodesmosin specifically in nude mouse. Interestingly, histopathology revealed a difference in cervical lymph node metastatic tumours of wild-type and nude mice, showing reduced keratinization potential of these tumour sites developed in nude mice. This was accompanied with markedly reduced Sbsn transcript levels in metastatic SCC in nude mice. Comparison of primary and metastatic tumours cells developed in nude mice showed even stronger reduction in Sbsn expression [Sbsn in DMBA-induced HNSCC primary tumour cells and metastatic cells compared to C57Bl6J mice [Sbsn expression is a response of cancerous cells to anti-tumour T cell activity. The relationship of T1 cells . Furtherer study . To elucpression . Since Tl6J mice , we can With the onset of mass spectrometry (MS)-based proteomics, large datasets opened new avenues in understanding human diseases. Besides cancer, a number of studies showed the presence of SBSN peptides also under other pathological conditions, but only limited peptide counts and coverage of SBSN protein have been achieved ,40,62. TSbsn transcript levels in the retinal tissue [SPRR2A, SPRR2D, SPRR2F, SPRR3, and SBSN [To investigate the pathology of Fuchs endothelial corneal dystrophy (FECD), label-free quantitative tandem mass spectrometry proteome analysis was performed revealing SBSN peptides specifically in the aqueous humor of FECD patients . FECD isl tissue , but thil tissue ,68. Simiand SBSN . In lineA follicular fluid isobaric tags for relative and absolute quantitation (iTRAQ) proteome study of polycystic ovary syndrome (PCOS) patients revealed downregulation of complement components and dysregulation of proteins functionally associated with angiogenic processes, for instance, PLG, AGT, LYVE1 (pro-angiogenic), and SERPINA1 . Importantly, the follicular fluid of PCOS patients was enriched in SBSN peptides . Note, cIn a rat model of pre-motor Parkinson’s disease (PD), the blood plasma proteome analysis of citrullinated proteins revealed a Sbsn peptide among PD-specific hits . CitrullPorphyromonas gingivalis-expressed peptidylarginine deiminase (PPAD). Besides PPAD, P. gingivalis secretes a group of arginine-/lysine-targeting proteases, gingipains. Cleavage of host proteins with subsequent PPAD-mediated citrullination may lead to autoimmune response via antibodies recognizing the modified peptides. P. gingivalis is commonly associated with chronic periodontitis and oral squamous cell carcinoma [P. gingivalis to arteries [P. gingivalis is associated with cardiovascular diseases [P. gingivalis proteins are present in the majority of Alzheimer’s disease patients’ brains and correlate with the disease progression [P. gingivalis showing elevated SBSN mRNA levels 16 h post treatment. Additionally, tumour-associated macrophages showed pro-tumorigenic M2-transition following the bacterial treatment [P. gingivalis infection could promote SBSN expression. Certainly, elucidation of the role of SBSN or presence of antibodies recognizing SBSN represent the next steps in further investigations. Another source of pathological citrullinated peptides is arcinoma . Transiearteries , and artdiseases . Additiogression , and gingression . Recentlreatment . AltogetThe analysis of MHC II immunopeptidome revealed an abundance of SBSN peptides in axillary, brachial, inguinal, and skin-draining lymph nodes of healthy mice , indicatSBSN expression in hippocampal astrocytes of NPSLE patients compared to healthy individuals where SBSN was also detected. Once again, these observations show aberrant SBSN expression under pathological conditions. Besides others, CSF of NPSLE patients is enriched in inflammatory cytokines [SBSN expression in cancer cells [Indeed, in a recent search for neuropsychiatric systemic lupus erythematosus (NPSLE)-specific autoantibodies in cerebrospinal fluid (CSF) that could potentially be used as diagnostic markers of the disease, SBSN peptide-recognizing immune complexes were identified . This inytokines , includiytokines , a recener cells . Intereser cells . SBSN transcript elevation follows P. gingivalis stimulation of oral cancer cells [SBSN implication in bacterial response seems to be more profound, but yet not clear. As described above, er cells . SBSN imTrypanosoma cruzi represents a pathogen with an ability of congenital transmission. To elucidate the response of human prenatal tissue to T. cruzi infection, human placental extract (HPE) was infected with trypomastigotes of T. cruzi. Two hours post-treatment transcripts of pro-inflammatory cytokines, TLRs, and NLRs were elevated, but, 24 h post-infection, the SBSN transcript was among top upregulated genes [Mycobacterium tuberculosis, or ssRNA  significantly elevated SBSN mRNA levels after 6 h [SBSN expression can be a direct response following TLR receptors stimuli, and a downstream target of NF-kappaB as already proposed [SBSN is component of innate immune response and/or early anti-microbial defence.ed genes . Furthered genes ). Additifter 6 h . These rproposed ; therefoSBSN and TGM3 [Escherichia coli (UPEC) colonizing the lumen and the epithelial cells of the bladder and responsible for chronic bacterial infection of the urinary tract, initiated innate immune defence and epithelial cell detachment [Sbsn, Ivl, Lcn2, and complement components B and C3 were markedly elevated by murine urothelial cells in close proximity of intracellular UPEC colonies [E. coli, or both, increased transcript levels of innate immune response, keratinization, and cell differentiation genes. LCN2, S100A8/9 , and F3 were significantly elevated following 17beta-estradiol or 17beta-estradiol plus flagellin treatments. Besides others, 17β-estradiol elevated IL-17A transcript in the vaginal biopsies of treated post-menopausal women, implicating the stimulation of anti-microbial defence. Additionally, transcriptome analysis of mammary gland parenchyma of intact and ovariectomized prepubertal heifers revealed SBSN among estrogen-regulated genes [Transcriptome analysis of vaginal epithelial cells from post-menopausal women with vaginal dryness, symptom commonly associated with vaginal microbial infection and epithelial irritation, showed downregulation of and TGM3 . Gene ontachment ,87. Sbsncolonies . These rcolonies , in whice in MDS ), SPRR2Arocesses ) were eled genes . SBSN was found to be a FOXO1-regulated gene with a capacity to promote aromatase (CYP19A1 gene) expression. Similarly, FOXO1 depletion resulted in downregulation of CYP19A1 transcripts under confluent conditions. The ability of ASCs to express SBSN and aromatase was donor-dependent, hence likely dependent upon epigenetic state and priming of the cells [SBSN transcription in vaginal epithelial cells. This implicates a positive feedback loop which could result in dysregulation of estrogen levels and SBSN-mediated response. Obesity is a comorbidity associated with multiple human diseases, including cancer. Chronic systemic inflammation underlies obesity-related symptoms . In an ahe cells . The FOXIL-4 and IL-13 genes [Flg and Ivl [SBSN mRNA following IL-4 treatments [SBSN isoform one, whereas levels of transcripts of isoform two and three are lower [SBSN isoforms were markedly elevated in non-lesional skin samples of AD patients, but transcripts of all isoforms were reduced in skin lesion samples of AD patients [SBSN knockdown induced apoptosis of fraction of keratinocytes in human living skin equivalent model, and IL-4/IL-13-treatments enhanced the observed apoptotic features, indicating SBSN-mediated resistance to IL-4/IL-13-induced apoptosis in human keratinocytes [Despite its high prevalence, especially in children, atopic dermatitis (AD) remains a poorly understood and inefficiently treated immune system-driven cutaneous disease. Dysregulated immune cell-mediated cytokine signalling affecting the differentiation of epidermal cells as one of the features of AD is detrimental and predominantly driven by Th2-expressed 13 genes ,93. In t and Ivl . Similareatments , but, hueatments . Healthyre lower . Interespatients . The proinocytes .SBSN remains a gene of unknown molecular function. SBSN proteins represent secretory molecules with the ability to activate the signalling cascades, but its receptor(s) are not yet identified. Based on current evidence, SBSN is associated with multiple human diseases, including cancer and autoimmune disorders. Various cellular compartments express SBSN in diseases, hence uncovering stimuli and conditions responsible for SBSN expression in different cell types would provide valuable knowledge. Several SBSN expression-inducing stimuli are already confirmed . Also, other stimuli lead to SBSN transcript elevation , but their SBSN-inducing mechanisms remain to be elucidated. SBSN expression is likely post-transcriptionally regulated, and SBSN undergoes multiple post-translation modifications, which suggests a complex nature of SBSN biology. Importantly, dysregulated SBSN expression and SBSN plasma level alterations mark malignant conditions, potentially promoting systemic effect and patients response to the therapy [ therapy ,32,37. T therapy  which ha therapy . SBSN expression were documented. Cancer cells expressing SBSN display stem markers [SBSN expression specifically in ESCC [SBSN in colon cancer and the effect of SBSN on endothelium is indicated in several reports [SBSN might play a role in the response of endothelial cells to aberrant conditions. Chronic inflammatory signalling accompanies the onset of tumorigenesis, promotes stemness [SBSN was negatively correlated with BM T cell abundance in MDS [SBSN expression in different cell types of the malignant landscape.Several cellular sources of  markers ,19 and i in ESCC . Tumour  reports ,25, suggstemness , and thestemness . MDSCs ae in MDS ; hence SSBSN in immune regulation is strengthened with recent observation in Sbsn-null mice [SBSN is crucial in Treg development induced with these stimuli [The role of ull mice . This an stimuli . Other pSBSN-inducing stimuli, we compiled various observations, indicating that innate immune response is a likely candidate [SBSN transcripts elevated following treatments with bacterial components or lysates in different animal models [SBSN transcripts, for example, S100A family representatives. Innate immune response accompanies early stages of tumour development associated with microenvironment remodelling and a dysregulated immune response [In search for andidate ,85. SBSNl models ,85. Otheresponse . Note, Mresponse .SBSN are not yet understood; therefore, SBSN represents an interesting target for further studies in both physiology and pathology. We predict that SBSN will emerge in other diseases as a candidate diagnostic and prognostic biomarker. Understanding its function is of great importance since SBSN likely plays a role in disease development. The fundamentals of SBSN biology that remains to be answered:(1)Is SBSN a ligand and an inducer of signalling pathway?There is a plethora of questions regarding SBSN has not been identified, current findings of SBSN secreted nature and the effects in experiments in vitro using recombinant SBSN indicate that SBSN can function as a ligand via an unknown receptor. The interacting partners of SBSN were not studied specifically. LRG1 remains to be the only shown interactor in peripheral blood of MDS patients [(2)SBSN under malignant conditions?What stimulates aberrant expression of Although the molecular function of patients . FurtherSBSN expression associated with human malignancies were reported. Some of these are already documented with a degree of certainty, such as EGF [SBSN regulation [SBSN expression [SBSN transcript elevation should be broadened by uncovering the SBSN molecular role, for instance, in innate immune response [(3)What is the functional role of individual SBSN isoforms?Several signals that could promote aberrant h as EGF , IFN-gamh as EGF ; howevergulation ,19. Addiresponse ,84,88, eresponse ,90, and response . ImportaSBSN. The oncogenic potential of SBSN is linked to human SBSN-2 [Different protein isoforms may possess diverse and contradictory functions, and this cannot be excluded in case of n SBSN-2 ,29 and sn SBSN-2 ; therefo


Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12—a yeast transcription factor regulating mating and invasion—alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the “calling-card” method, RNA sequencing, and HT-SELEX , we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions. Genetic variation in either a transcription factor or the cis-regulatory sequences to which it binds creates variation in gene expression, driving phenotypic variation, evolution, and disease. Emphasis has focused on cis-regulatory variation and its effect on gene expression for multiple reasons. First, regulatory variation likely played a large role in evolution because it resulted in less-severe pleiotropic effects on whole-organism phenotypes, allowing for subtle cell or tissue changes rather than interfering with overall body plans  medium. While STE12 deletion resulted in completely sterile strains that were complemented by a plasmid-borne wild-type STE12 gene, we did not confirm that the URA3 gene was fully deleted. Thus, in the following Ste12 variant RNA sequencing (RNA-seq), we carried out differentially expressed gene analysis after filtering out URA3.We generated yeast strains in the BY4705 STE12 locus from S. cerevisiae strain BY4705, including the intergenic regions, was introduced into the yeast vector pRS415 containing a LEU2 marker . Plasmids were transformed into yeast (BY4705 MATα) with a deleted endogenous copy of STE12 by high-efficiency lithium acetate transformation (The ormation . Cells wKanMX gene was inserted into the transposon region. We added an 8-bp (NNNNNNNN) unique molecular identifier (UMI) sequence to the transposon region of each copy of the donor plasmid to quantify unique insertion events per cell. For the helper plasmid, we replaced SP1 with a wild-type and variant full-length STE12 fragment and fused it with the PiggyBac transposase to encode Ste12-PBase, whose expression is under the control of the galactose-inducible GAL1 promoter. Gibson assembly products were introduced into Escherichia coli and the plasmid was isolated. Constructs were confirmed by Sanger sequencing.A donor plasmid carrying the PiggyBac transposon and an SP1-PBase helper plasmid were obtained from Robi Mitra (Washington University in St. Louis). To use G418 and 5-FOA selection in yeast, a MATα) by high-efficiency lithium acetate transformation . Cells were put onto selection plates with 5-FOA and G418 at varying dilutions for ∼2–3 days.Plasmids used for transformation were prepared using a plasmid miniprep kit (QIAGEN) following the manufacturer’s protocol. Paired donor plasmid and helper plasmid were transformed into yeast (BY4705 ormation . After tTaqI and RsaI (New England Biolabs) individually. Digested products were purified by DNA purification column (Zymo Research) and ligated overnight at 15° in dilute solution to encourage self-ligation. Self-ligated DNA was purified by DNA purification column (Zymo Research) and used as the template in an inverse PCR. Primers that anneal to the PiggyBac donor sequences (primer 316: 5′-GATGTCCTAAATGCACAGCGAC-3′ and primer 317: 5′-GAGGCGTGCTTGTCAATGC-3′) were used to amplify the genomic regions flanking the transposon, and then adaptor sequences that allow the PCR products to be sequenced on Illumina sequencing platforms were added by primer 367 (5′-AATGATACGGCGACCACCGAGATCTACACCTCCATCGAGACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCAATTTTACGCAGACTATC-3′) and 377 (5′-CAAGCAGAAGACGGCATACGAGATTGCTTGTCAATGCGGTAAG-3′). The PCR products were purified using a DNA purification column (Zymo Research). For each sample, the same amount of PCR product from digestion with each restriction endonuclease was pooled and submitted for Illumina sequencing.Cells were collected from selection plates and genomic DNA was extracted from each sample using the Smash and Grab method, as described by Sequencing was completed on Illumina’s NextSeq platforms. The raw data included all fastq files from the PiggyBac transposase-only control, wild-type Ste12, and the six variants. Each sample had two replicates. In read 1, the first segment was the universal primer sequence—5′-CGTCAATTTTACGCAGACTATCTTTCTAGGG-3′—followed by the flanking genome sequence of 39 bp. The first 8 bp of read 2 was the UMI sequence used to identify unique insertion events. We first filtered sequence reads with high quality and mapped them back to the yeast genome. Then we quantified independent PiggyBac insertions based on the UMIs detected in each sample. Finally, we called significant PiggyBac insertion peaks by MACS2, a peak-calling algorithm  to generate high-insertion-count sites for each replicate. We extracted the sequence from these windows and attempted to identify in vitro. Fragments of wild-type and variant Ste12 proteins were purified using a GST tag, and used for HT-SELEX . SELEX reactions with homogenous and mixed protein populations were performed identically to previous work , 100 μM EGTA, and 1 μM ZnSO4] for 2 hr. GST Sepharose  beads were added to each reaction, incubated for 30 min, and unbound ligand was removed using seven buffer washes. Output reactions were amplified by PCR after each round and these products were subsequently used to prepare high-throughput sequencing libraries. SELEX motif enrichments were analyzed using Autoseed software (We purified fragments of Ste12(1–215) expressed from pGEX-4T-2 vectors. These protein fragments have been used previously  and transcriptome data (five replicates), have been submitted to the Gene Expression Omnibus (GEO) under accession numbers: GSE141713. Supplemental material available at figshare: MATa cells with STE12 variants were mixed with MATα cells and selected using auxotrophic markers that would be present only in mated diploids. For the invasion selection, the yeast were incubated on plates until invasion had occurred and then washed from the plate surface, such that only cells embedded in the agar should remain. We identified Ste12 variants with single-amino acid changes in the DNA-binding domain that altered the preferences of yeast cells in their mating or invasion traits. To determine how the genomic targets of Ste12 might differ depending on these amino acid changes, we chose six Ste12 variants with altered mating or invasion phenotypes  and a URA3 marker. We quantified unique insertion events per cell using a random 8-bp UMI sequence in the transposon region of each copy of the donor plasmid. Second, we constructed a helper plasmid that encodes a fusion of the full-length Ste12 protein to the PiggyBac transposase (Ste12-PBase), whose expression is under the control of the galactose-inducible GAL1 promoter. We cotransformed both of these plasmids into the MATα +STE BY4705 strain .We induced the Ste12-PBase, which results in the insertion of the transposon near sites bound by a variant Ste12 protein and the conversion of cells with these insertions to G418 resistance. We then measured the chromosomal acquisition of the transposon-borne Materials and Methods), we obtained a total of 264 gene targets. Gene ontology (GO) analysis revealed that these genes are most enriched [false discovery rate (FDR) < 0.001] for cell fusion or pheromone response pathways . Thus, while Ste12-PBase was expressed under the control of the GAL1 promoter, which is much stronger than the STE12 promoter, the insertions fell into expected genes, suggesting that the high expression did not substantially affect Ste12 binding to the yeast genome.For wild-type Ste12, we obtained a total of 327 significant insertion peaks throughout the genome . A 7-mer analysis of the 100-bp region around each insertion found that highly enriched 7-mers include the common sequence TGAAAC , indicatr = 0.69–0.91, apart from A160P (r = 0.37), which resulted in the detection of only a few calling-card insertion peaks and thus had a relatively low correlation coefficient]. The fact that the variants yielded reproducible data also suggests that the high Ste12-PBase expression did not lead to adventitious nonspecific binding. However, pairwise comparisons revealed that the overlap among variants ranged only between 10 and 20%. Even for the two variants with the highest degree of overlap in target sites, K146D and K152L, 80% of the sites differed, despite their similar organismal phenotypes . For those sites that were common in all the variants, we found that their nearby genes are classical targets of Ste12, and critical to either the mating or invasion phenotype . These results indicate that single-amino acid changes in the Ste12 DNA-binding domain led to many gains and losses of binding sites throughout the genome. However, even though the Ste12 variants bound overall to highly divergent sets of genomic sites, it is the binding sites that are in common that likely explain the key phenotypic differences.The genome-wide insertion patterns for the six Ste12 variants were also characterized by the calling-card method . IndividSTE12 gene under the control of its own promoter and carried on a centromere-based plasmid, and transformed the plasmids into a ste12Δ version of the BY4705 strain, which was generated through a site-specific genomic deletion method . Cells were grown to exponential phase and total RNA was obtained. We carried out RNA-seq (five replicates) to generate the transcriptome for each variant, which yielded a combined total of 145 differentially expressed genes from the six variant strains when compared with wild-type Ste12, with an FDR of 5%. Replicability of the assay was high (r = 0.97–0.99). The numbers of differentially expressed genes in the variant strains ranged from 15 to 84, but the numbers of these genes did not correlate with the severity of the altered mating or invasion phenotypes.We sought to compare the DNA-binding sites of the Ste12 variants with genome-wide expression patterns induced by these variant proteins. We generated variant Ste12 proteins by introducing single-amino acid changes into the Unsupervised hierarchical cluster analysis of the data revealed three main differentially expressed groups of genes . We usedKanMX marker and G418 resistance. We found that the transposition efficiencies of the three mating-defective variants were only ∼10% of the wild-type and mating-competent variants  of the overlapping downregulated genes were found to be direct targets of the wild-type Ste12 based on the genome-binding profile. Of the 14, four genes  had a reduction in the DNA-binding signal  in all mating-deficient variants, and 10 genes  in at least one mating-deficient variant ; interaction between Ste12 and DNA provides the basis for the acquisition of the variants , suggestvariants , and thevariants . Eighteevariants , and the variant .In summary, we conclude that amino acid changes in the DNA-binding domain of Ste12 that disrupt its interaction with the genome can be distinguished by the many fewer calling-card insertions that were detected. This disruption results in loss-of-binding to critical gene targets and markedly reduces the expression of mating-related genes, which would be expected to dramatically decrease the mating proficiency of the mating-deficient variants .Two of the mating-defective variants (K146D and K152L) invade as well as or better than the wild-type, indicating that they are not fully defective. One possibility is that they maintain interactions with some cofactors while losing interactions with others or directly with DNA, resulting in distinct variant-specific binding and expression patterns. To test this possibility, we used the genome sequences near Ste12-PBase insertion peaks and examined the frequencies of Ste12 canonical sites across the variants. We found that the K152L and K146D variants showed a reduction in binding relative to wild-type Ste12 at canonical sites required for mating . To capte.g., SIP18, SPG4, and GRE1; SHO1 and MSB2 genes were direct targets of wild-type Ste12, suggesting a potential role for Ste12 upstream of the osmotic stress response pathway.We integrated both genome-binding and transcriptome profiles in an effort to uncover the mechanism underlying the altered invasion phenotype seen in the hyper-invasive variants . Transcriptome analysis revealed that shared upregulated genes in hyper-invasive variants are associated with the cellular response to osmotic stress  for MSB2 and K146D had a higher insertion signal for SHO1  , while they failed to efficiently bind to the Ste12 consensus site, resulting in downregulation of critical mating genes, both variants had an increased insertion signal (t level) . Due to  pathway . In the Distinct from these two mating-defective variants, the mating-competent and hyper-invasive K150A and K150I variants were the most enriched for the canonical Ste12-binding site, showing greater enrichment for this site than even the wild-type Ste12 , one that does not (K152L), and one that may involve cofactor interactions (K146D).in vivo can be influenced by cofactor interactions that affect the sequence contacted by Ste12, we asked whether direct binding specificity in vitro was altered. To characterize the in vitro DNA-binding preferences of the variants, we used the HT-SELEX method , the mating deficiency caused by a variant could be explained by a marked reduction in DNA-binding affinity, based on many fewer calling-card insertions and either a failure to enrich or reduced enrichment of the consensus site in the SELEX experiment. This failure to efficiently bind to Ste12 consensus sites led to the decreased expression of a small set of common genes, several of which function in the mating process. However, that these variants maintain, or even increase, their invasiveness suggests that they are not completely defective proteins and that separation-of-function alleles can arise readily. Moreover, the maintenance of invasion function in variants defective for DNA binding implicates cofactors in the new binding and expression patterns. Variant proteins may maintain interactions with some cofactors while losing interactions with others, resulting in distinct variant-specific expression patterns. Further analyses of DNA binding by these variants could use approaches such as gel mobility shift assays to obtain quantitative affinity data. In addition, carrying out these assays on a series of promoters from pheromone-responsive or invasion-specific genes in the presence of purified cofactors such as Tec1, Mcm1, and Matα1 might reveal protein interactions that are either enhanced or reduced by the Ste12 substitutions.The three variants that resulted in near wild-type mating  showed similar numbers of calling-card insertions and similar enrichment of the consensus site in the SELEX experiment as the wild-type protein. These results imply that DNA-binding affinity is likely to be intact in these variants. Variants that bind to the consensus site like wild-type but result in a new phenotype also suggest a change in the interaction with cofactors. The K150A and K150I variants lead to a hyper-invasive phenotype, and these two uniquely increased the expression of genes responsive to osmotic stress. The hyper-invasive variants that are mating-defective (K146D and K152L) increased the expression of additional genes, indicating that multiple paths to a hyper-invasive phenotype are possible.Although the Ste12 transcription factor variants easily gained or lost binding sites, most variant-specific binding events seemed to have little to no effect on the organismal phenotypes of mating and invasion. We attribute this phenotypic robustness to the underlying regulatory network architecture, which will amplify the effects of specific binding events and expression through feedback loops, motif degeneracy, and binding site redundancy while canceling the effect of aberrant binding and expression events. At the same time, the ease with which a single-amino acid substitution in Ste12 shifts phenotype suggests that a few novel binding events and expression changes suffice as starting points for the rewiring of regulatory programs in evolution .in vivo effects of transcription factor variants calls for the systematic large-scale functional interrogation of human transcription factor variants in vivo. Thus far, there are few options to conduct such studies at sufficient scale in human cells. In this regard, it is notable that we detected genome-wide binding patterns of Ste12 variants in the presence of the wild-type protein. Expressing libraries of transcription factor variants from a common genomic site in human cells containing their respective wild-type proteins is far more feasible than the prospect of engineering many hundreds of endogenous loci. While most transcription factor variants are unlikely to contribute to phenotypic changes, identifying those with major downstream effects remains a critical challenge.A major goal in functional genomics is the prediction of variant effects from sequence alone. However, current variant effect prediction algorithms and computational structure-based approaches (