PAR1 and PAR2 play central roles in tumor biology, and PAR3 acts as a coreceptor. The significance of PAR4 in neoplasia is just beginning to emerge. PAR1 has been shown to be overexpressed in malignant epithelia, in direct correlation with tumor aggressiveness, but there is no expression in normal epithelium. In this review, the involvement of key transcription factors such as Egr1, p53, Twist, AP2, and Sp1 that control PAR1 expression levels specifically, as well as hormone transcriptional regulation by both estrogen receptors (ER) and androgen receptors (AR) are discussed. The cloning of the human protease-activated receptor 2; Par2 (hPar2) promoter region and transcriptional regulation of estrogen (E2) via binding of the E2–ER complex to estrogen response elements (ERE) are shown. In addition, evidence that TEA domain 4 (TEAD4) motifs are present within the hPar2 promoter is presented since the YAP oncogene, which plays a central part in tumor etiology, acts via the TEAD4 transcription factor. As of now, no information is available on regulation of the hPar3 promoter. With regard to hPar4, only data showing CpG methylation promoter regulation is available. Characterization of the PAR transcriptional landscape may identify powerful targets for cancer therapies.G protein-coupled receptors (GPCRs), the largest family of cell receptors, act as important regulators of diverse signaling pathways. Our understanding of the impact of GPCRs in tumors is emerging, yet there is no therapeutic platform based on GPCR driver genes. As cancer progresses, it disrupts normal epithelial organization and maintains the cells outside their normal niche. The dynamic and flexible microenvironment of a tumor contains both soluble and matrix-immobilized proteases that contribute to the process of cancer advancement. An example is the activation of cell surface protease-activated receptors (PARs). Mammalian PARs are a subgroup of GPCRs that form a family of four members, PAR The hPar1 mRNA level was shown to be high in aggressive tumors utilizing a panel of tissue biopsy specimens and cell lines accompanied by in situ hybridization and reverse transcription-PCR (RT-PCR) analyses [hPar1 gene copy number remains unchanged regardless of the hPar1 expression level, indicating that overexpression of hPar1 does not stem from gene amplification. Consequently, hPar1 transcription rates and mRNA stability were evaluated to determine the elevation rate of hPar1 mRNA levels. To analyze the stability of hPar1 mRNA, cells were treated with the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosyl benzimidazol (DRB). At various time points, mRNA was extracted and the level of hPar1 mRNA was analyzed by both Northern blotting and real-time PCR to determine levels of hPar1 mRNA. Degradation rates for hPar1 mRNA were similar regardless of whether RNA came from cells with high  or low  hPar1 expression levels [hPar1 transcription rate was observed in the highly metastatic PC3 cells, which express high hPar1 levels compared with LNCaP of low metastatic potential where there are low levels of hPar1 expression [hPar1 RNA levels in the malignant cells are primarily due to increased hPar1 transcription. This outcome guided us to center our focus and study the transcription factor (TF) landscape associated with PAR overexpression. Significantly, master TFs are conserved throughout evolution in coordinating transcriptional gene regulation networks functioning via binding to specific short sequence arrays (“motifs”) in matching promoter regions to control the transcriptional expression of a panel of target genes in a plethora of pathological and physiological functions (G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and they are involved in a wide array of physiological processes, yet their role in cancer etiology is poorly addressed ,2,3. Mam biology ,7,8,9,10unctions . 1&2 play a central role in epithelial tumor advancement and are potent inducers of the canonical Wnt/β-catenin stabilization path, a core process both in developmental and tumor progression pathways [3 is a co-receptor, PAR4 (named F2RL3) has emerged as a potent stem cell marker out of a wide panel of GPCR-induced stem-cell sphere formation candidates identified with high-throughput RNA sequencing [The idea of a rigid hierarchical stem cell niche organization in a tumor has been challenged, suggesting that within a heterogeneous cancer cell population, targeting and identifying the stem cell compartment is the main task ahead. PARpathways ,11. Whilquencing .1, and partially PAR2 and PAR4) in epithelial malignancies. In this respect, we discuss up-to-date knowledge on transcriptional controls including biochemical and structural aspects. While PAR1 is the prototype member, overexpressed directly with the tumor aggressiveness, we address also hormone regulation of PAR1 and PAR2, as also YAP regulation by TEA domain 4 (TEAD4) on PAR2. The regulation of hPar4 by CpG methylation is discussed as well. Finally, we provide data by searching online platform of patient data available at Gene Expression Profiling Interactive Analysis , on expression levels of TFs in normal and pathological epithelia.In the present review we outline and center on relevant TFs that regulate the expression levels of PAR family members  analysis of the hPar1 promoter area along with luciferase-promoter activity aided in revealing the up-to-date part of the explicit transcription factors involved in PAR-induced epithelial cancer growth and progression [hPar1 promoter genomic sequence (accession number U63331) reveals a potential Early Growth Response-1 (Egr-1) motif located between −354 and −335 bp3 [The promoter of the PARgression ,13,14,15−335 bp3 .hPar1 promoter region was shown, the possibility that Egr-1 binds initially with Sp1 to form an Sp1/Egr-1 complex, as shown in the control of hepatocyte growth factor levels [hPar1 expression in prostate carcinoma [Although a direct association between Egr-1 and the r levels , cannot arcinoma . Egr-1 iarcinoma ,19,20,21arcinoma ,23,24,25arcinoma ,27,28,29arcinoma ,31. Our current understanding is that miRNAs can function as either oncogenes or tumor suppressors ,32. BecaAlternatively, Egr-1 may also act to inhibit cancer progression by targeting, for instance, miR-203a, which functions as an anti-oncogene. It can induce expression of miR-203a and indirectly inhibit the expression of HOXD3 transcription factor in hepatocellular carcinoma (HCC) via miR-203a, causing attenuation of HCC progression . HOXD3 sHSPE) gene. This is a well-known endo-β-d-glucuronidase that plays an important role since it cleaves and degrades the basement membrane component heparan sulfate, thereby promoting tumor invasion and metastasis. HPSE eRNA associates with the heterogeneous nuclear ribonucleoprotein U (hnRNPU) to facilitate its contact with p300, consequently giving rise to chromatin looping between the super enhancer, eRNA, and the HPSE regulatory region. Recruitment of Egr-1 causes elevated expression and function of HPSE. Generally, this indicates the crucial roles of the axis: eRNA/hnRNPU/p300/EGR1/HPSE in tumor development [Aspects of chromatin architecture and structure should also be considered when discussing the likelihood of TF accessibility and association with target gene promoters. Chromatin looping and participation of histone acetyltransferases p300 and CREB binding protein (CBP) are necessary for an open chromatin design and are the required components for bridging the gap between target gene-promoters and a new regulatory element termed enhancer RNA. Enhancers are another group of important control components of the genome that contribute to appropriate instigation of gene regulatory sites via creation of chromosomal coils . An examelopment .Increasing evidence suggests that Egr-1 stimulation may act as a master alteration in many pathological processes, including cardiovascular diseases and cancers. Egr-1 has been designated in the development of a spectrum of epithelia-derived tumors such as breast, prostate, colon, and esophageal cancers ,42,43,44mt) p53 play distinctly different roles. Whereas wild-type (wt) p53 acts in controlling the expression of genes that control a selection of cell-associated procedures comprising apoptosis, cell senescence, and cell cycle checkpoints, the mt p53 primarily act as oncogenes endorsing cell survival, invasion, and metastasis. We have demonstrated that wt p53 negatively regulates the level of hPar1 via transcriptional inhibition [In the last 20 years it has become evident that the tumor suppressor p53 and its tumor-associated mutants  and those that are located at the DNA binding region. While structural features may account for the gain-of-function of mt pro tumor p53, the DNA-binding alterations may suggest that these mutants recognize a specific response element for mt p53, permitting their oncogenic function; however, there is no consensus on the sequence of such a response element. In a tumor, when unctions ,48,49,50unctions ,52. Frequnctions . As suchmt p53 relates to its ability to associate with other transcription factors and modify their target gene levels. For example, the mt p53 (p53R175H) induces EGR-1 via physical contact between the mt p53-EGR-1 promoter, forming a complex that provides considerable action related to oncogenic gain-of-function [hPar1 in prostate cancer, leading to hPar1 overexpression and enhancement of invasive properties [hPar1 levels may be stimulated indirectly by mt p53 via the induced EGR-1 level. Another option is association via the NF-Y transcription factor that binds to the CCAAT motif sequence [hPar1 regulatory area (at −2736 and −2516) to recruit mt p53. The acetylase p300 is bound to the mt p53-hPar1 promoter complex, where it helps to “open” the chromatin structure toward transcription [wt p53 is a sequence-specific transcription factor that associates with selective response elements and is negatively regulated by Mdm2, an E3 ubiquitin ligase that assigns p53 for ubiquitination and proteasomal degradation. Bioinformatic analysis for the p53 consensus sites [1 start-site coding region. ChIP analyses conclusively exhibited direct binding and physical association between p53 and the hPar1 regulatory area for a delicate fine-tuning of the PAR1 oncogenic function in prostate cancer growth and progression. Along with this line of evidence, while there is an inverse association between PAR1 and wt p53 levels, a direct association was shown between mt oncogenic p53 and hPar1 levels in a panel of prostate cancer cell lines that exhibit low-to-high aggressive properties of prostate cancer with corresponding low-to-high hPar1 levels, respectively. Both the level of expression and the functionality of PAR1 protein were evaluated as indicated by the level of phosphorylated-FAK ) and transmigration through a matrigel layer. A direct association was demonstrated between the level of hPar1 expression and mt p53, as indicated by temperature sensitive (ts) mutants (inactive at 32 °C and active at 37 °C), the level of phospho-FAK, and hPar1 levels in luciferase activity assays. This outcome provides compelling evidence for the inhibition and delicate regulation of PAR1 levels in prostate tumor in the presence of wt p53. The presence of mt p53 elicits marked levels of oncogenic PAR1, suggesting that the hPar1-mt p53 axis as a target for future therapeutic modalities. The best known transcriptional role of function . As discoperties . In genesequence , two of cription ,58. Notaus sites  showed tThe molecular description of breast tumor subtypes has led to important advancement in treatment. Tumor biopsy specimens that display an estrogen receptor (ER) or a progesterone receptor (PR) respond well to either anti-ER vehicles or aromatase inhibitors, which attenuate estrogen synthesis. HER2-expressing cancers are treated by personalized drugs directed to block HER2 ligand binding and dimerization via antibodies , or alternatively by small molecules that inhibit HER2 function such as an HER2 kinase inhibitor (Trikerb). Still, 15–20% of breast cancers fall through the flaws of this system. 2) hormone, which acts through transcriptional regulation of a panel of target genes. Transcriptional regulation of E2 includes the association of E2 to ER followed by receptor phosphorylation, receptor dimerization, and binding of the ligand-ER compound to specific motifs, namely, estrogen response elements (ERE) within the promoter of target genes.“Triple-negative” breast tumors lack all of the main three molecular signs: ER, PR, and high HER2 expression. Breast cancer expressing ER is controlled by the estrogen  and shorter overall survival (OS) compared with those that expressed ER but lacked PAR1 [hPar1 has an aggressive profile in the breast cancer gene imprint. The presence of PAR1 may certainly tilt the outcome scoring or stand on its own when making a treatment choice. PAR1 classifies a group of patients that need additional therapeutic tools, either chemotherapy or personalized anti-PAR1 biological compounds. The detailed outline of E2-ER regulation of PAR1 as evaluated by ChIP and RT-PCR. Luciferase hPar1 promoter activity and immunostaining analyses are outlined in detail in Salah Z. et al. [2-ER it may regulate breast cancer through nontranscriptional regulation recruiting signaling effectors and activating multiple pathways that lead to cellular proliferation [Ligation of Eelopment ,60, servelopment ,62,63,64ked PAR1 . A gene ked PAR1 ,66. For . et al. . It shouferation ,68.2 on cancer cells via one of three approaches: (a) preventing E2 from associating with ER with an anti-E2, for example tamoxifen [2 synthesis with an aromatase inhibitor [2, for example fulvestrant  [2 action is the selective estrogen receptor modulators (SERMs) for the inhibition and treatment of diseases as osteoporosis and breast tumors [2 inhibitor in breast tissue via competitive association to ER, thereby inhibiting an E2-stimulated increase in breast tissue cells [AIB1) [AIB1 (Amplified-in-breast cancer 1) is augmented in ER-positive human breast cancers [AIB1, also known as SRC-3 , is part of the p160 family, which also includes SRC-1 and SRC-2 [AIB1 is a transcriptional coactivator that endorses the transcriptional activity of many nuclear receptors such as ER and other transcription factors such as E2F1, AP-1, Sp1 [AIB1 [AIB1 overexpression, in combination with high levels of HER-2, was to elicit agonist activity of tamoxifen in experimental cell schemes, and to facilitate resistance to adjuvant tamoxifen treatment [2 is the second PAR family member with a major part in breast tumor growth. This is based, among other things, on the detected delay in tumor onset in a murine model for tumor growth following intercross between PAR2 knock-out mice and polyoma middle T (PyMT) mice, but not through intercross with PAR1−/− mice [2 takes a dominant role over PAR1 and drives protumor functions. These PAR2 protumor functions may be initiated by tissue factor (TF), another coagulation factor, since TF cytoplasmic domain-deleted mice were also shown to have delays in spontaneous breast tumor growth in the polyoma middle T model [1 and PAR2 are located in a close proximity and act as one functional unit when establishing heterodimers [2 plays a dominant part in PAR1–PAR2 instigated tumor activity, since shRNA silencing of hPar2 effectively inhibits PAR1-induced function but silencing hPar1 does not affect PAR2-associated signaling. Accordingly, PAR2 represents an attractive therapeutic target in cancer [Presently, the treatment approach for hormone-dependent breast tumors is to block the action of Eamoxifen ,65; (b) nhibitor ; or (c) 182,780) . The most tumors . Tamoxifue cells . Data coue cells . Tamoxifue cells ,74. Howeue cells ,76. The  cancers . AIB1, aP-1, Sp1 ,80,81. Ep1 [AIB1 ,82,83. A T model ,85. Accurodimers ,87. Consn cancer . hPar2, with an E2-like effect of tamoxifen on hPar1 and hPar2 expression  and prepared deletion constructs of varying length ; −186–167; 5’-cgcaGGTgAgtac gctgct-3′(R) and −210–192 5′-ttccGGTCccggggcgtgg-3′(R) .For the purpose of studying Eg length a. This egg-3′(R) c. In paractivity b were em0−9 M E2 d, but wahPar2 expression and ER in breast tumors, we also aimed to assess the effect of tamoxifen on hPar2 expression. A marked increase in the level of hPar2 was observed in both RNA and protein in the presence of tamoxifen. To demonstrate AIB1 involvement in the regulation of hPar2 by tamoxifen, a stable clone of MCF-7 cells was generated, and it was silenced using shRNA-aib1 following lentiviral infection. MCF-7 cells expressing shRNA for aib1 were then treated with tamoxifen or by E2 and evaluated for hPar2 level of expression by analysis of RNA levels as well as Luc-promoter activity. A marked inhibition in tamoxifen-treated cells was seen in the presence of shRNA-aib1  or control IgG to immunoprecipitate chromatin from the cell lysates (before and after E2 usage), negligible levels, which were not affected by E2, were seen (pS2 gene (a known estrogen target gene) were utilized in a similar ChIP assay, E2 stimulated occupancy of the pS2 regulatory region by both ER and AIB1, while tamoxifen, as assessed, recruited ER, but not AIB1, to the pS2 promoter. These results are in line with the data obtained for HER2 and ER-AIB1 [AIB1 to the hPar2 promoter by tamoxifen. E2-ligated ER axis might clarify the agonist function of tamoxifen on the hPar2 level.While showing a correlation between RNA-aib1 e. ChIP aere seen f,g. When ER-AIB1 . Our res1. Since the androgen hormone drives increases in prostate tumor growth, the standard therapy is androgen ablation. Ablation therapy results in tumor regression up to a point, at which the tumor reappears in an aggressive and androgen-independent form. Androgen receptor (AR) is phosphorylated and forms homodimers upon ligand binding ). Ligated AR is translocated to the nucleus, where it functions as a transcription factor through binding to ARE. These motifs are canonical half-site TGTYCT sequences that are separated by three nucleotides from the other half. Nearly no hPar1 is expressed in normal prostate tissues, whereas high and abundant levels are detected in neoplastic prostate tissue biopsy specimens. The powerful part PAR1 plays in prostate cancer progression is shown by comparing radical prostatectomy tissues after androgen ablation with samples taken several weeks prior to the ablation surgery from the same individuals [hPar1, other genes such as FGF8b and VEGF were also shown to be regulated by ligated-AR [hPar1 promoter was shown using EMSA, Luc-promoter activities, and differential levels of expression in the clinical settings [Strongly linked to the tissue context, androgen hormones may instigate the upregulation of a gene imprint, possibly including PARividuals . Apart fgated-AR ,89,90. Tsettings . 1 stimulates pro-migratory properties in epithelial malignancies, notably epithelial-mesenchyme transition (EMT), which is characterized by the loss of epithelial indicators (E-cadherin and β-catenins) [1 promoter pointed to the CAGGTG putative binding motif (unpublished data), although the resulting functionality has not yet been clarified. Twist elicits the expression of PAR1 as a downstream gene. This leads to the development of oncogenic traits via inhibition of the Hippo pathway that is induced by PAR1 activation [The Twist family of basic helix-loop-helix (bHLH) transcription factors is known to control transcriptional regulation that activates or suppresses the transcription of downstream genes via binding to DNA E-box sequences CAGGTG or CGTCTG ,92. Accutivation . Once HihPar1 in melanoma growth and advancement from the radial to the vertical growth phase has been shown [1 levels in aggressive melanoma and the activator protein-2 (AP-2), a direct correlation is seen with the expression of specificity protein 1 (Sp1). Analysis of the PAR1 regulatory region revealed specific binding motifs for both transcription factors that bind in a mutually exclusive manner for the association motifs located at bp −365 to −329 (complex 1) and bp −206 to 180 (complex 2), depending on the tissue context. It appears that AP-2 is absent in highly aggressive cells while Sp1 takes the lead. In contrast, AP-2 is expressed in the non-metastatic phase whereby Sp1 is absent. It has been proposed that a loss of AP-2 instigates metastatic capability in melanoma [1 and Maspin, a tumor suppressor [1 inhibits binding of Ets-1 and c-Jun transcription factors to the promoter of Maspin. Consequently, PAR1 acts indirectly by inhibiting the Maspin tumor suppressor-driven inhibition of melanoma progression and as a result facilitates more aggressive behavior of melanoma cells. Transcriptional regulation of en shown . Whereasmelanoma . Anotherppressor . HoweverThe TEA domain (TEAD) family of transcription factors includes four members (TEAD1-4) that control the levels of various genes linked with cell propagation, apoptosis, and differentiation. TEAD4 is the main downstream transcription factor in the Hippo signaling pathway, a major player in tumorigenesis that is often disrupted in tumors. TEAD4 was recently found to be an oncogene and a likely prognostic marker, as well as a therapeutic target in both gastric and breast cancers ,97. The Upstream kinases in the mammalian Hippo signaling pathway, including mammalian STE20-like protein kinase 1/2 (MST1/2), activate via phosphorylation of downstream kinases, including large tumor suppressor 1/2 (LATS1/2) with the assistance of the adaptor MOB kinase activator 1A/B (MOB1A/B) and Salvador family WW domain-holding protein 1 (SAV1) . ConsecuhPar2 promoters 5′-GTGGAATGT-3′ and 5′-CATTCCA-3′ . Indeed, SLIGKV activation of PAR2 markedly promoted TEAD4 LUC promoter activity  in cancer is poorly understood, yet it has emerged as a potent stem cell marker among GPCRs for cancer cell sphere formation, as shown in high-throughput RNA sequencing [4 is regulated by CpG promoter methylation. PAR4 levels are decreased upon methylation and increased upon hypomethylation in colorectal cancer tissues when compared to matched normal tissues, particularly in poorly differentiated tumors and in lymph nodes with metastases [4 levels in the colorectal cancer LoVo cell-line [The involvement of PARquencing . Recent tastases . As a reell-line . While DDNA methylation plays a major role in imprinting and is necessary for mammalian development ,110. Cyt1 and PAR2 levels are at large upregulated in the majority of epithelial malignancies, both PARs are downregulated in either kidney renal papillary cell carcinoma (for PAR1) and kidney chromophobe and kidney chromophobe and skin cutaneous melanoma (for PAR2). When we have utilized this online platform using Gene Expression Profiling Interactive Analysis   (; reviewe


Xanthomonas campestris, we demonstrate that the global transcriptional regulator HpaR1 influences many of the same genes as another global regulator Clp, including the engXCA gene that encodes extracellular endoglucanase. We demonstrate that HpaR1 facilitates the binding of RNA polymerase to the engXCA promoter. In addition, we show that HpaR1 binds directly to the engXCA promoter. Furthermore, our in vitro tests characterize two binding sites for Clp within the engXCA promoter. Interestingly, one of these sites overlaps with the HpaR1 binding site. Mobility shift assays reveal that HpaR1 has greater affinity for binding to the engXCA promoter. This observation is supported by promoter activity assays, which show that the engXCA expression level is lower when both HpaR1 and Clp are present together, rather than alone. The data also reveal that HpaR1 and Clp activate engXCA gene expression by binding directly to its promoter. This transcriptional activation is modulated as both regulators compete to bind to overlapping sites on the engXCA promoter. Bioinformatics analysis suggests that this mechanism may be used broadly in Xanthomonas campestris pv. campestris (Xcc) and is probably widespread in Xanthomonads and, potentially, other bacteria. Taken together, these data support a novel mechanism of competitive activation by two global regulators of virulence gene expression in Xcc which is probably widespread in Xanthomonads and, potentially, other bacteria.Transcriptional regulators are key players in pathways that allow bacteria to alter gene expression in response to environmental conditions. However, work to understand how such transcriptional regulatory networks interact in bacterial plant pathogens is limited. Here, in the phytopathogen  Bacterial pathogens continuously adjust their gene expression in response to the environment in order to control and deploy appropriate virulence agents. Transcription factors are one way in which bacteria can achieve control over gene expression. These proteins can sense signals and specifically activate or inhibit target genes that are involved in physiological adaptation, pathogenesis and virulence. In general, transcriptional regulator proteins consist of an N‐terminal domain which bears a helix–turn–helix (HTH) DNA‐binding motif and a C‐terminal regulatory domain which signals bind. These factors are also classed as ‘activators’ when they bind to a promoter to activate transcription initiation, and/or ‘repressors’ when they bind to a promoter to interrupt transcription , a model bacterium used over the past several decades to study plant–pathogen interactions , Zur (zinc uptake regulator), HpaR (for hrp‐associated regulator) and HpaR1 (for hrp‐associated regulator)  and in vitro transcriptional analysis. This analysis showed that the binding site of HpaR1 to the engXCA promoter overlaps with a Clp binding site, and the binding of Clp to the engXCA promoter could be outcompeted and displaced by HpaR1. Taken together, this is the first example of the activation of a single promoter by two global regulators in Xcc. Our transcriptomic data and bioinformatic analyses suggest that this regulatory interplay may occur at multiple promoters within the Xcc genome. There are very few reports of this type of regulation, but our findings suggest that this mechanism may occur more broadly than previously thought.The global transcriptional regulators characterized in hpaR1 (ΔhpaR1) on the Xcc transcriptome was established by RNA sequencing (RNA‐seq). The transcriptome analysis, false discovery rate (FDR) ≤ 0.001 and log2 of the fold change (|log2FC|) ≥ 1 were considered for differentially expressed genes. The results showed that, of the 4273 annotated genes in Xcc strain 8004, 302 genes were found to be differentially expressed, 192 and 110 of which were up‐ and down‐regulated, respectively  were observed in both mutants.These regulated genes collectively were involved in a range of biological functions, including virulence, membrane transport, multidrug resistance, amino acid biosynthesis and signal transduction. Intriguingly, changes in the expression of genes involved in the activity of endoglucanase are consistent with previous findings (Su engXCA (XC_0639), although it is clear that both HpaR1 and Clp have independent actions on gene expression.The findings suggest that HpaR1 affects the expression of a larger number of genes than originally described, rather than having other roles in the bacterial cell. These findings also highlight that a subset of genes are controlled by both HpaR1 and Clp, in particular Xcc  activities produced by the obtained transconjugant strains ΔhpaR1/pGUSengXCA and 8004/pGUSengXCA cultured in nutrient‐yeast‐glycerol (NYG) medium were then assessed. As shown in Fig. HpaR1 and Clp have been shown previously to be required for the activity of endoglucanase in engXCA in the hpaR1‐deficient mutant was investigated by quantitative RT‐PCR (qRT‐PCR) analysis. The results showed that the engXCA transcript level in the mutant was decreased by 48% compared with the wild‐type when grown for 24 h in NYG medium  the EngXCA proteins using homologous recombination . The recombinant plasmid pK18mobengXCAH6, which was constructed by fusing the 6×His‐tag encoding sequence to the 3′ end of the engXCA gene and cloning the fused fragment into the suicide plasmid pK18mobsacB, was introduced into Xcc wild‐type strain 8004 and hpaR1 deletion mutant strain ΔhpaR1, respectively. The resulting strains, 8004/EngXCA::6×His and ΔhpaR1/EngXCA::6×His, were used to test the EngXCA protein levels by western analysis. As shown in Fig. hpaR1 results in a significant reduction in the expression level of engXCA, indicating that HpaR1 positively regulates the expression of engXCA.The engXCA is achieved by the direct binding of HpaR1 to the promoter region, a 6×His‐tagged HpaR1 protein was first obtained as described previously , named P−256/+47) was determined by EMSA . The results revealed that the HpaR1 protein bound and therefore arrested the movement of the promoter sequence in the polyacrylamide gel ‐labelled primers . The binding of HpaR1 to these fragments was tested by EMSA  and incubated with 10 μm of 6×His‐tagged HpaR1 proteins. After 5 min of digestion with DNase I, the reaction was terminated and the digestion pattern was examined on a Applied Biosystems™ 3730 DNA Analyzer . By comparing the electropherograms with and without HpaR1 protein using GeneMarker software , a specific HpaR1‐protected region within the engXCA promoter was determined  assay. A wild‐type background strain expressing the HpaR1 protein fused with 3×Flag‐tag (3×Flag::HpaR1) at the N‐terminus of HpaR1 was generated  analysis. Using this method, we determined the TIS of the engXCA promoter in Xcc 8004. As described previously  upstream of the predicted translation ATG start codon  with ‘CAAGAT’ and ‘ATCACA’ shifted to ‘CAAGCG’ and ‘ATCAGT’, respectively, were created by site‐directed mutagenesis . The activity of reporter plasmid assays revealed that the promoter sequences with mutation in −10 or −35 elements produced significantly less GUS activity than the wild‐type promoter  as template . The results revealed that the mutant promoters generated low transcript levels compared with the wild‐type promoter  and ‘−35’ (ATCACA) elements in the ter Fig. B. These In general, transcription factors repress promoter activity by binding to operator sequences that overlap with the –10 or –35 elements of the promoter, thus blocking access of RNAP. Conversely, promoter activity is enhanced by the binding of the transcription factor upstream of the ‘−35’ element, which, in turn, supports the binding of RNAP.engXCA gene, an in vitro transcription assay was first carried out. For this, 317‐bp template DNA fragments, corresponding to nucleotide positions −191 to +126 relative to TIS of the engXCA promoter, were incubated with RNAP holoenzyme from Escherichia coli with increasing amounts of purified 6×His‐tagged HpaR1 protein. The results showed that, although engXCA transcripts could be generated without HpaR1 protein, the engXCA transcription level was significantly increased when HpaR1 protein was added to the reaction  within the HpaR1 binding sequence determined by footprinting assay were deleted by site‐directed mutagenesis. The labelled 175‐bp (from −141 to +47 relative to TIS) engXCA promoter fragments with a 13‐nucleotide deletion were incubated with HpaR1 and then analysed by EMSA  with a 13‐nucleotide deletion within the HpaR1 binding site was constructed . In vitro transcription assays were performed with the mutated and wild‐type promoter as templates. Compared with the wild‐type promoter, the use of the mutated promoter showed no obvious increase in the engXCA transcription level when HpaR1 protein was added to the reaction  receptor protein] of engXCA promoter suggests that they overlap, which is not a commonly reported phenomenon in bacteria. To confirm that Clp and HpaR1 truly have overlapping binding sites on the engXCA promoter, a set of EMSAs was carried out.Our assessment of the Clp and HpaR1 binding sites on the engXCA promoter, a 6×His‐tagged Clp protein was constructed and expressed in E. coli, and increasing amounts of purified Clp were incubated with the FAM‐labelled 303‐bp DNA fragment of the engXCA promoter (spanning nucleotides −256 to +47 relative to TIS). Here, it was observed that the addition of Clp at a concentration of 10 nm resulted in a slower mobility of the 303‐bp DNA fragment in the assay. However, there were further band shifts observed when high concentrations of Clp were used  was generated to test the binding of Clp. In these EMSAs, Clp protein at a concentration of 20 or 30 nm was used. As shown in Fig. First, to test Clp binding of the . et al.  for the engXCA promoter, dye primer‐based DNase I footprinting assays were carried out with a 378‐bp FAM‐labelled DNA fragment of the engXCA promoter (spanning nucleotides −191 to +187 relative to TIS) and 20 μm Clp protein. As shown in Fig. engXCA promoter was found. This protected region, consisting of 22 nucleotides spanning positions −53 to −32 relative to TIS  within CBS I were deleted by site‐directed mutagenesis . The obtained labelled 178‐bp engXCA promoter fragments (from −141 to +47 relative to TIS) with a 10‐nucleotide deletion within CBS I, and the above labelled 175‐bp engXCA promoter fragments (with 13 nucleotides of the HpaR1 binding site or 10 nucleotides of the CBS II deletion) were incubated with Clp and analysed by EMSA. The results showed that mutations within the two Clp binding sequences clearly interfered with the Clp−Clp−engXCA promoter complex, implying that the mutations prevent Clp from binding to the corresponding site, whereas Clp still binds to the innate binding site  at the N‐terminus of Clp (ΔClp/pClp‐Flag)  Table . As showengXCA promoter prompted us to investigate the effect of both HpaR1 and Clp on the transcription of the engXCA promoter. To do this, we first evaluated the role of Clp on the expression of engXCA in Xcc strain 8004. As shown in Fig. engXCA greatly, indicating that Clp is a key regulator for the expression of engXCA. In addition, the expression level of engXCA in the double mutant of HpaR1/Clp seemed to be lower than that in the Clp deletion mutant.The fact that HpaR1 and Clp overlap in the engXCA as a result of Clp binding. An in vitro transcription assay was performed with a 317‐bp DNA fragment (nucleotides spanning positions −191 to +126 relative to TIS on the engXCA promoter) as template and increasing amounts of Clp proteins. As shown in Fig. engXCA transcription level, indicating that Clp enhances engXCA transcription in vitro. In addition, in vitro transcription assays with the mutated engXCA promoter (10 nucleotides deleted within the Clp binding sequences) were also carried out . The results revealed that mutation in CBS II, rather than CBS I, reduced Clp activation  containing the HpaR1 and Clp overlapping binding sites was used for incubation with HpaR1 and/or Clp for 40 min, followed by separation using electrophoresis. As shown in Fig. m, approximately two of three DNA molecules formed the Clp−DNA complex; however, when both HpaR1 and Clp were present simultaneously in the reaction, only the HpaR1–DNA complex appeared, but not the Clp–DNA or HpaR1−Clp−DNA complexes , and on addition of 10 nm HpaR1 (lane 6), a clear HpaR1–DNA complex was observed. On addition of 20 nm HpaR1 (lane 7), most of the DNA probe was bound by HpaR1, whereas the DNA probe bound by Clp was strongly reduced compared with that on addition of 20 nm Clp only. A labelled 188‐bp engXCA promoter DNA fragment (−141 to +47) containing two Clp binding sites was further used for EMSA . The addition of 10 nm HpaR1 to this assay resulted in the appearance of an HpaR1–DNA complex and the disappearance of the Clp–Clp–DNA complex (lane 6). Interestingly, when a concentration of 30 nm HpaR1 was used, only the HpaR1–DNA complex was observed (lane 8). This suggests that Clp does not target CBS I when HpaR1 precludes Clp from binding to CBS II.We then tested the 184‐bp min Fig. A‐b. At 2aR1 lane , most ofengXCA promoter, the quantification of EMSA was performed with a 184‐bp DNA fragment (−64 to +120) containing CBS II and the HpaR1 binding site, and a range of HpaR1 and Clp concentrations. As shown in Fig. engXCA promoter with dissociation constants (Kd) of 25.08 and 36.75 nm, respectively, indicating strong affinity binding of HpaR1 compared with Clp.To further compare the binding affinities of HpaR1 and Clp to the engXCA promoter. Furthermore, under the conditions tested, it appears that HpaR1 has a higher affinity for binding the engXCA promoter as it outcompetes Clp binding to its target site. However, in this study, although Clp acting alone in the engXCA promoter generates a higher promoter activity than that when both Clp and HpaR1 proteins act together, HpaR1 was observed to dislodge Clp binding in the engXCA promoter.Overall, these data indicate that both HpaR1 and Clp proteins specifically bind to the Xcc. The work reveals an interesting mechanism in which HpaR1 and Clp both bind specifically to the promoter of engXCA to positively control its expression. Interestingly, the binding sites for both proteins overlap, suggesting that they compete for binding and control of engXCA expression. Further analysis shows that, under the conditions tested, HpaR1 has a higher affinity for binding to the engXCA promoter.During infection, bacteria control gene expression in a multitude of ways in response to alterations in their surroundings. One of the most deeply studied mechanisms is a bacterium’s use of transcriptional regulators. Although bacteria encode large numbers of transcription factors, there has been limited investigation into how combinations of transcriptional regulators control gene expression coordinately, given that many appear to co‐regulate genes involved in microbial pathogenesis. The data generated here describe how two global transcriptional regulators, HpaR1 and Clp, co‐regulate a subset of virulence genes in E. coli, and AfsR and PhoP from Streptomyces species, which have been shown to act as competitive activators by binding to sites that overlap on their respective promoter targets  of Clp binding. Although they proposed two sites of binding (CBS I and CBS II), they discounted the first, as they could not provide evidence of Clp binding to this region of the engXCA promoter. In the current study, we have shown that Clp binds to both sites of the engXCA promoter, which is dependent on the concentration of Clp. The CBS I site is a low‐affinity site that requires high concentrations of Clp to observe binding, whereas CBS II is a high‐affinity site where binding is seen at low concentrations of Clp protein. Furthermore, the presented data suggest that the CBS I site spans nucleotides −80 to −59 relative to TIS, and the CBS II site spans nucleotides −53 to −32 relative to TIS.Previously, Hsiao et al.  have desengXCA gene expression observed when Xcc is grown in laboratory medium  medium . The total RNAs were extracted from cultures of the Xcc strains using a Total‐RNA Extraction Kit (Promega) according to the manufacturer’s instructions. RNA was reverse transcribed by Superscript II reverse transcriptase , according to the manufacturer’s protocol. For semi‐quantitative RT‐PCR, the resulting cDNA was diluted and used with specific primers  green‐labelled PCR fragments were amplified using the primer set engXCA‐F/R . The expression level of the 16S rRNA gene was used as an internal standard. The qRT‐PCR tests were performed in triplicate.The DNA manipulations followed the procedures described by Sambrook et al. . Conjugar et al. . The res/R Table , which wengXCA was constructed by fusing a 303‐bp DNA fragment upstream of the engXCA open reading frame (ORF) (including the translation start codon ATG) with the promoterless GUS‐encoding ORF (excluding the translation start codon ATG). The engXCA promoter of the Xcc wild‐type strain 8004 was amplified with the primer set PengXCA‐1F/R (Table EcoRI‐ or BamHI‐compatible ends (underlined)  Table . The DNACA Table .engXCA‐F/R. This sequence was cloned into the suicide vector pK18mobsacB  of 1.0. cDNA fragments were obtained using the 5′‐RACE Kit (Invitrogen), and the PCR products were cloned into the pMD19‐T vector and sequenced.To determine TIS of the ‐3 Table . The asset al. ]. The diluted 1 : 2000 horseradish peroxidase (HRP)‐conjugated goat anti‐mouse immunoglobulin G (IgG) was used as the secondary antibody. After washing the membrane four times, the luminescence signal was detected according to the manufacturer’s instructions. For a loading control, proteins were probed with the anti‐RNAP β‐antibody  at 1 : 2000 dilution as primary antibody, and the HRP‐conjugated goat anti‐rabbit IgG H&L  at 1 : 5000 dilution as secondary antibody.Western blotting followed the procedure described by Sambrook et al. . BacteriBamHI‐ or HindIII‐compatible ends. After confirmation by sequencing, the amplified DNA fragment was cloned into the expression vector pQE‐30  to generate the recombinant plasmid pQE‐30‐Clp. The recombinant plasmid pQE‐30‐Clp was then transformed into E. coli JM109, resulting in strain JM109/pQE‐30‐Clp. This strain was cultured to an OD600 of 0.6, and 1.0 mm of isopropyl‐thiogalactopyranoside (IPTG) was added. After the culture had been grown for a further 4 h, the cells were harvested and the fused protein was purified using Ni‐NTA resin (Qiagen). To obtain the HpaR1 protein, E. coli strain JM109/pQE‐30‐2736 expressing HpaR1 with a 6×His tag on its N‐terminus was grown and induced by IPTG  and 0.1 U of E. coli RNAP holoenzyme (sigma‐saturated) to start the transcription. After incubation at 28 °C for 30 min, the reactions were stopped and the transcription products were analysed by electrophoresis./R Table . After pm) containing the engXCA promoter, amplified by PCR using the FAM‐labelled primer sets  and 1 mm dithiothreitol, pH 8.0] containing 1 μg of sonicated salmon sperm DNA and 3 μg of bovine serum albumin, and incubated at 30 °C for 20 or 40 min. Samples were then loaded onto a 6% polyacrylamide‐Tris‐borate‐EDTA (TBE) gel, and visualized after electrophoresis.DNA fragments  were mixed with various amounts of HpaR1 or Clp and incubated at 30 °C for 20 min. After electrophoresis, the gel was scanned with a Bio‐Rad  Pharos FXTM Plus scanner, and the data were analysed with Image J software (https://imagej.nih.gov/ij/). The dissociation constant (Kd) was calculated using OriginPro8 software .To quantify the binding affinity of HpaR1 (or Clp) with the et al., engXCA promoter were obtained by PCR from the total DNA of Xcc strain 8004 using the FAM‐labelled primer sets (Table m of FAM‐labelled DNA fragments was used for incubation with HpaR1 (10 μm) or Clp (20 or 40 μm) protein. After digestion with DNase I (New England BioLabs), DNA fragments were purified and the digested DNA was added to HiDi formamide (Applied Biosystems) and GeneScan‐500 LIZ size standards (Applied Biosystems). The DNA samples were then analysed with a 3730 DNA Analyzer. The results were analysed with GeneMarker software (Softgenetics).Dye primer‐based DNase I footprinting assays were performed as described previously  with the recombinant plasmid pKengXCA as the template and the appropriate primer sets , we first cloned a 303‐bp region of the /R Table  to amplits Table . The pla/R Table . To deteus Table . The resengXCA promoter with nucleotide substitution in the ‘−10’ or ‘−35’ element (or 10/13‐nucleotide deletions in the binding sequence) used for in vitro transcription assay, a 317‐bp DNA fragment (spanning nucleotides −191 to +126 relative to TIS of the engXCA promoter) was PCR amplified with the primer set PengXCA‐2F/R. The DNA fragment obtained was cloned into the suicide plasmid pK18mob  . The plasmid pLAFR3 is a low‐copy‐number vector with an E. coli lac promoter flanking the multiple cloning sites, which expresses constitutively in Xcc  by triparental conjugation, resulting in strain ΔHpaR1/pHpa‐Flag (or ΔClp/pClp‐Flag)  at the N‐terminus of HpaR1, or a Clp protein fused with 3×Flag‐tag (3×Flag::Clp) at the N‐terminus of Clp, was first constructed. A DNA fragment encoding HpaR1 (or Clp) fused with 3×Flag peptide with R) Table . The frag) Table . As a neXcc strains were grown in NYG medium for 24 h and cross‐linked by the addition of formaldehyde to a final concentration of 1%. After incubation for 20 min at room temperature with slow shaking, glycine was added at a final concentration of 0.125 m to quench the cross‐linking reaction. Bacterial cells were collected by centrifugation at 8000 g at 4 °C for 5 min and washed twice in phosphate‐buffered saline (PBS). To lyse the cells, 10 mL of RIPA buffer  was added and thoroughly mixed by vortexing, and then disrupted by sonication. For each ChIP sample, 50 μL of anti‐Flag (agarose conjugated) were added to the bacterial lysates, and incubated with gentle shaking at 4 °C overnight. Unbound DNA fragments were washed using RIPA buffer, and the bound DNA fragments and proteins were eluted by 0.25 m glycine (pH 2.5).A415 using p‐nitrophenyl‐β‐d‐glucuronide as the substrate, as described by Henderson et al.  according to the manufacturer’s protocol. Contaminating genomic DNA was removed using RNase‐free DNase I and verified by PCR. RNA quantity was initially determined by a Nanodrop spectrophotometer ND‐8000 , and RNA quality was assessed using an Agilent 2100 bioanalyzer . Total RNA was sent to Novogene  for library construction and strand‐specific RNA sequencing. Sequencing libraries were generated using a NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (New England BioLabs) following the manufacturer’s recommendations, and sequenced on an Illumina  HiSeq 2000 platform. Clean reads were mapped to the reference genome. To eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression, the RPKM (reads per kilobase per million mapped reads) method was used to calculate the gene expression levels.To prepare RNA for transcriptome analysis, single bacterial colonies were picked and grown in 5 mL of NYG medium at 28 °C for 24 h at 200 rpm. These cells were transferred into 50 mL of NYG medium again at 28 °C for 24 h at 200 rpm. RNA was harvested once the cell turbidity had reached an ODFig. S1 Electrophoretic mobility shift assays (EMSAs) for narrowing down the binding sites of HpaR1 and Clp proteins on the engXCA promoter of Xanthomonas campestris pv. campestris (Xcc). (A) Polymerase chain reaction (PCR) fragments in series length used as probes in EMSAs. Horizontal lines represent the DNA fragments corresponding to the region within the engXCA promoter, the names of the probes are indicated on the left and the numbers after P are the nucleotide positions relative to the transcription initiation site (TIS) of engXCA. Primers used for PCR amplification are listed in Table S5. (B) EMSAs to test the binding of HpaR1 with a variety of probes; 1.0 nm DNA fragments were incubated with 0, 10 and 20 nm of HpaR1 proteins at 30 ºC for 20 min. (C) EMSAs to test the binding of Clp with a variety of probes; 1.0 nm DNA fragments were incubated with 0, 20 and 30 nm of Clp proteins at 30 ºC for 20 min.Click here for additional data file.Fig. S2 Nucleotide substitution in −35 and −10 elements reduces engXCA promoter activity. (A) The β‐glucuronidase (GUS) activity of engXCA promoter‐gusA reporters in the wild‐type strain 8004. Two engXCA mutant promoters [spanning nucleotides −256 to +47 relative to the transcription initiation site (TIS)] with ‘CAAGAT’ and ‘ATCACA’ shifted to ‘CAAGCG’ and ‘ATCAGT’, respectively, were created by site‐directed mutagenesis . These two mutant promoters were fused to the promoterless gusA gene, resulting in reporter plasmids pGUSengXCAAT and pGUSengXCACA, respectively. GUS activity was measured after the bacterial cells had been cultured in NYG medium containing 2% glucose for 24 h. The values given are the means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. (B) In vitro transcription assay. Two engXCA mutant promoters (spanning nucleotides −191 to +126 relative to TIS) with nucleotide substitution in the −10 and −35 elements were first created ; 2 nm DNA of the 317‐bp fragments of the wild‐type engXCA promoter, or mutants of the engXCA promoter, were incubated with 0.1 U of RNA polymerase (RNAP). Transcription products were then run on a 5% denatured polyacrylamide gel containing 7 m urea in 1 × Tris‐borate‐EDTA (TBE) electrophoresis buffer.Click here for additional data file.Fig. S3 Mutation analysis of the HpaR1 and Clp binding sites on the engXCA promoter. (A) Electrophoretic mobility shift assays (EMSAs) of the mutated promoter; 13 and 10 nucleotides within HBS/CBS II and CBS I were chosen for deletion by site‐directed mutagenesis . The resulting mutant fragments were designated as HBS‐13 and CBS I‐10. 6‐Carboxyfluorescein (FAM)‐labelled engXCA promoter DNA fragments [spanning nucleotides −141 to +47 relative to the transcription initiation site (TIS) of the engXCA promoter] with or without (wild‐type) mutations were incubated with increasing amounts of HpaR1 (i) or Clp  protein for 40 min at 30 ºC before EMSA. (B) In vitro transcription assays of the mutated promoter. DNA fragments (spanning nucleotides −191 to +126 relative to TIS of the engXCA promoter) with 13‐ and 10‐nucleotide deletions within HBS/CBS II and CBS I, respectively, were constructed . The resulting DNA fragments PengXCA/HBS or PengXCA/CBS I, and the wild‐type fragment PengXCA, were subjected to in vitro transcription assays with amounts of HpaR1 (i) or Clp  protein.Click here for additional data file.Table S1 Gene expression profile of the ΔhpaR1 strain when grown in NYG. Note: false discovery rate (FDR) = 0.05 and absolute value of log2 of the fold change (log2FC) = 1  were used as the cut‐off values. ‘+’ represents genes up‐regulated in the mutant ΔhpaR1 and ‘−’ represents genes down‐regulated.Click here for additional data file.Table S2 Confirmation of RNA sequencing (RNA‐seq) gene expression data by semi‐quantitative real‐time polymerase chain reaction (RT‐PCR).Click here for additional data file.Table S3 Genes regulated by both global transcriptional regulators HpaR1 and Clp.Click here for additional data file.Table S4 Bacterial strains and plasmids used in this work. aRifr, Kanr, Tetr and Spcr indicate resistance to rifampicin, kanamycin, tetracycline and spectinomycin, respectively.Click here for additional data file.Table S5 Primers used in this study.Click here for additional data file.


Transient expression of the estrogen receptor (ER) in HIT-T15 cells revealed that estrogen receptor α (ERα) repressed transcription of the rat insulin II promoter in both ligand-dependent and ligand-independent manners. The N-terminal A/B domain of ERα was not required for either activity. However, the repression was absent with mutated ER lacking the DNA-binding domain. Moreover, introducing mutations in the D-box and P-box of the zinc finger of ER  also abolished the repression. Deletion of the insulin promoter region revealed that nucleotide positions − 238 to − 144  were needed for ER repression of the rat insulin II gene. PDX1- and BETA2-binding sites were required for the repression, but an estrogen response element-like sequence or an AP1 site in the promoter was not involved. In conclusion, we found that estrogen repressed insulin mRNA expression in a beta cell line. In addition, the ER suppressed insulin gene transcription in a ligand-independent matter. These observations suggest ER may regulate insulin transcription by indirect genomic signaling.The mechanism whereby 17β-estradiol (E2) mediates insulin gene transcription has not been fully elucidated. In this study, exposure of hamster insulinoma (HIT-T15) cells to 5 × 10The online version of this article (10.1007/s11626-019-00328-5) contains supplementary material, which is available to authorized users. ERα and ERβ are members of a large family of nuclear receptors that activate or repress the transcription of hormone-regulated genes upon ligand binding Evans . Both ERWith indirect genomic signaling, a direct interaction between NFκB and ER has been demonstrated and requires the DBD (C) and D regions of the ER .The human ERα expression plasmid, pHEGO, was kindly provided by Dr. P. Chambon . Oligomers containing a BamHI site and 18 base pairs (bp) of coding strand sequence of the N-terminus of the human ERα (hERα) hinge domain or an XbaI site and 18 bp of non-coding strand sequence of the hERα C-terminus were synthesized and used as PCR primers. Aliquots of each primer (100 pmol) were hybridized to 5 ng of ERα complementary DNA (cDNA) and amplified by PCR for 30 cycles  using a PerkinElmer Gene Amp PCR System 2400 . After digestion with BamHI and XbaI, the PCR products were ligated into the corresponding sites of the pM vector. Correct insertion was confirmed by dideoxy nucleotide sequencing. Mammalian expression vectors for peroxisome proliferator activated receptor alpha (PPARα), PPARγ, retinoid X receptor alpha (RXRα), retinoic acid receptor (RAR), glucocorticoid receptor (GR), and vitamin D3 receptor (VDR) were described previously  supplemented with 10% charcoal-stripped fetal calf serum (Gibco), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. HIT-T15 cells at passages 72–82 were used in the experiments. The rat INS-1 insulinoma cells were obtained from Dr. Hideo Mogami (Hamamatsu University School of Medicine). INS-1 cells were maintained in phenol red-free RPMI 1640 (11.1 mM glucose) supplemented with 100 units/ml penicillin–streptomycin and 10% dextran charcoal-stripped bovine calf serum. Cultures were maintained at 37°C and 7% CO2.Hamster HIT-T15 insulinoma cells were obtained from the American Type Culture Collection  and cultured at 37°C in 5% CORT-PCR mixtures were prepared as described previously . After hybridization, membranes were washed at 65°C in 0.1× SSC buffer containing 0.1% SDS. The results were visualized using a Phosphor Imager . Northern blots were stripped and re-probed with GAPDH cDNA to control for RNA loading.Total RNA was isolated using an RNeasy Kit . Fifteen-microgram aliquots of total RNA were size-fractionated in a 1% denaturing agarose-formaldehyde gel, transferred onto a Hybond-N+ nylon membrane , and cross-linked with ultraviolet radiation . Hybridizations were performed in ExpressHyb solution (Clontech) at 65°C for 2 h with full-length rat preproinsulin cDNA labeled with Western blotting analysis was performed as described previously  were used for the analyses. All A, RT-PCR analysis indicated that ERα mRNA was abundantly expressed in HIT-T15, INS-1 cells, and rat pancreatic β islet cells, whereas little if any ERβ mRNA expression was observed. The predicted sizes of the PCR products for ERα and ERβ were 273 and 285 bp, respectively. Furthermore, western blotting analysis demonstrated the presence of the ERα protein in the nuclear extracts of HIT-T15 and INS-1 cells . ER mRNA- and protein-expression levels in both HIT-T15 insulinoma cells and normal β islet cells were comparable to those seen in MCF-7 breast cancer cells (data not shown). The specificity of the antibody was verified using a recombinant ERα protein translated in vitro using an unprogrammed reticulocyte lysate.We first examined the expression of ERα and ERβ in clonal HIT-T15 pancreatic β islet cells. As shown in Fig. 1C, the expression of preproinsulin mRNA in HIT-T15 cells decreased significantly following treatment with 10−7 M E2.We hypothesized that nuclear ER signaling may be involved in regulating insulin production in HIT-T15 cells. Firstly, to analyze the effects of E2 (an ER agonist) on insulin mRNA expression, we performed northern blotting analysis of total RNA from HIT-T15 insulinoma cells incubated for 48 h with E2. As shown in Fig. 1A). As shown in Fig. 2A, the addition of 10−7 M E2 suppressed activity of the transfected reporter gene by approximately 20% without transfection of the ERα-expression vector, pHEGO (P < 0.05). This repression was likely mediated by endogenous ERα. The repression caused by overexpression of ERα was further suppressed by the presence of 10−7 M E2. In contrast, E2 did not alter the T3-dependent transcriptional activity in controls (data not shown), suggesting that the repression was specific for the insulin promoter and was not due to a toxic effect of E2 in the cells. Cell viability was unaffected by the addition of E2.Transient-expression experiments were performed in HIT-T15 cells using luciferase reporter plasmids containing the promoter region of the rat insulin II gene, extending from − 695 to + 1 bp relative to the transcriptional start site. Forced overexpression of ERα repressed the promoter further: the repression was dose-dependent with the maximum suppression being 20% with 100 ng ERα . These results suggest that (1) estrogen reduced insulin promoter transcription in an ER-dependent manner and (2) transcriptional suppression by ER occurred even in the absence of estrogen.E2 exposure in the range of 10C).Next, we compared the titration curves for other receptors and ligand in terms of inhibiting the insulin promoter, relative to the activation of the luciferase reporter plasmid (ERE-TK-Luc) by ERα. The concentration range over which E2 inhibited the insulin promoter and that required for ERE activation was comparable , glucocorticoid-response element (GRE), or peroxisome proliferator-response element (PPRE) was evaluated to determine whether transcriptional repression is a general phenomenon induced by E2/ERα in HIT-T15 cells. The transcriptional activities of the TRE-, GRE-, and PPRE-TK promoters were unaffected by E2 treatment Fig. , excludi−7 E2 . Deletion of nucleotides − 695 to − 188 muted the repression by E2 (30% decrease). E2 did not significantly repress the activities of the insulin promoter variants containing additional progressive 5′ deletions (to − 144). The data indicated that the estrogen-responsive region of the insulin promoter was located between nucleotides − 238 and − 144, that nucleotides − 238 to − 188 contained sites for E2-dependent and independent repression, and that nucleotides − 188 to − 144 contained an E2-dependent repression site.To identify the insulin promoter region mediating estrogen-dependent downregulation of insulin gene transcription in HIT-T15 cells, progressive 5′ promoter deletion constructs were cotransfected with the pHEGO vector , suggesting that the N-terminus (A/B domain) of ER was not required for repression. These data indicate that an intact DBD of ER was required for E2-dependent repression of insulin transcription . To further localize the domains required for the inhibition of insulin gene transcription by ERα, mutated receptors were generated . The repression was lost upon introducing mutations in the P-box of the first zinc finger and the D-box of the second zinc finger in the DBD . These data indicate that the DBD (C domain) was necessary for the repression.To evaluate the requirement of the DBD in repression of the insulin promoter by E2, cotransfection experiments were conducted using vectors encoding an N-terminal truncation mutant of ER (ΔN ERα). In the presence of ΔN ER, E2 repressed insulin promoter activity by up to 80%, similar to that shown by the wild-type ER Fig. C, suggesion Fig. C. To furted Fig. B. The reDBD Fig. C. These A, ERα repressed the activities of PDX1 and BETA2 in an E2-dependent manner. Moreover, the repression was lost upon mutation of the first and second zinc finger domains of the ERα DBD . These observations raised the possibility that ERα interacts with PDX1 and BETA2.Next, we examined whether ER alters the activity of transcription factors such as PDX1 and E47/BETA2, which bind to the repressive region and its vicinity on the insulin promoter Fig. . Plasmid4S-transferase and ERα (GST-ERα) was used for in vitro pull-down assays. As shown in Fig. 5A, 35S-methionine-labeled, in vitro-translated BETA2 and PDX1 interacted with GST-ERα in the presence of E2. In addition, 35S-methionine-labeled ERα interacted with GST-BETA2  and GST-PDX1 . These data indicated that direct protein–protein interactions occurred between ERα and PDX1 or BETA2.As the results of the transient-expression assays suggested that interactions between ERα and PDX1 or BETA2 occur, we performed GST pull-down assays to examine whether ERα could directly interact with PDX1 or BETA2. The matrix-bound fusion protein between glutathione Recent findings have shown that estrogen is important for pancreatic β cells in mammals -mediated induction of promoters regulated by AP-1 sites . These findings were consistent with those from several previous reports  altered transcription in the same manner as the wild-type ER Fig. C. These We also demonstrated that the ER repressed insulin promoter activity in a ligand-dependent manner through a mechanism involving protein–protein interactions. We identified a region within the gene promoter that mediates transcriptional repression of insulin gene expression in an ER- and E2-dependent manner in HIT-T15 cells. By transiently expressing a series of deletion promoter–reporter constructs, we localized the optimal repressive activity between nucleotides − 238 to − 144 region in the promoter. Moreover, we demonstrated that pancreatic β cell-specific transcription factors (PDX1 and BETA2/E47), which interact with this element and its vicinity and regulate insulin gene transcription, are involved in the repression by ER.The region of E2 responsiveness in the insulin promoter was localized to nucleotides − 238 to − 144 by deletion analysis. Within this region, there is a PDX1-binding site, A3 (nucleotides − 206 to − 197), that was shown previously to regulate basal insulin transcription . Deletion of the region from − 695 to − 238 had no effect on E2-dependent repression. Therefore, the segment from − 238 to − 144 appears to play a role in E2-dependent repression of the insulin promoter.cis-acting elements


Glycine-N-methyl transferase (GNMT) a tumor suppressor for hepatocellular carcinoma (HCC) plays a crucial role in liver homeostasis. Its expression is downregulated in almost all the tumor tissues of HCC while the mechanism of this downregulation is not yet fully understood. Recently, we identified 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) as a GNMT promoter enhancer compound in HCC. In this study, we aimed to delineate the mechanism by which PGG enhances GNMT expression and to investigate its effect on GNMT suppression in HCC. Microarray and pathway enrichment analysis revealed that MYC was a major target of PGG. PGG suppressed MYC mRNA and protein expression in Huh7 and Hep G2 cells in a dose- and time-dependent fashion. Furthermore, MYC expression was also reduced in xenograft tumors in PGG treated mice. Moreover, shRNA-mediated knocked-down or pharmacological inhibition of MYC resulted in a significant induction of GNMT promoter activity and endogenous GNMT mRNA expression in Huh7 cells. In contrast, overexpression of MYC significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. In addition, antibodies against MYC effectively precipitated the human GNMT promoter in a chromatin immunoprecipitation assay. Lastly, GNMT expression was negatively correlated with MYC expression in human HCC samples. Interestingly, PGG not only inhibited MYC gene expression but also promoted MYC protein degradation through proteasome-independent pathways. This work reveals a novel anticancer mechanism of PGG via downregulation of MYC expression and establishes a therapeutic rationale for treatment of MYC overexpressing cancers using PGG. Our data also provide a novel mechanistic understanding of GNMT regulation through MYC in the pathogenesis of HCC. Despite the new advances in HCC management, the incidence rate is still rising and nearly equals to the mortality rate4. Therefore, gaining a further understanding of the molecular mechanisms underlying the development of HCC is important to identify novel targets and more effective methods for treatment of HCC.Hepatocellular carcinoma (HCC) remains sixth most prevalent and third most common cause of cancer-related deaths in the globe6. GNMT has protective effects against exposure to various carcinogens including aflatoxins and polycyclic aromatic hydrocarbons8. It has been postulated that GNMT is involved in hepatic detoxification pathways9. Recent research has shown that GNMT is involved in cellular signaling cascades that coordinate various cellular processes such as proliferation, differentiation, migration and cell survival by interacting with DEPTOR, NPC2, and PREX2 proteins11. GNMT is highly expressed in the normal liver and plays a tumor-suppressive function in HCC5. The reduced expression of GNMT in human HCC cell lines and tumor tissues of HCC patients was first reported by Chen et al. in 199812. Furthermore, other studies demonstrated that GNMT is also downregulated in livers of cirrhotic patients with diverse etiologies, livers from patients with chronic cholestatic liver disease and liver tissues of nonalcoholic fatty liver (NFLD) patients14. In accordance, we described that GNMT expression was downregulated in dietary and genetic mouse models of NFLD10. Thus, it is reasonable to propose that GNMT downregulation is an early event in hepatocarcinogenesis. Although the relationship between GNMT downregulation and HCC is well established, the molecular mechanism underlying GNMT downregulation are poorly understood. Only 3 patients with a missense mutation of GNMT gene in the coding region has been reported and shown to affect catalytic activity16. Importantly, no sequence variations (mutations or deletion) were observed in the coding region of GNMT in sequence analysis suggested that the down-regulation of GNMT may not be caused by gene mutations17. In addition, hypermethylation studies showed that 3′ region of the TSS (transcription start site) of GNMT was hypermethylated to some extent in 3 HCC cell lines and seven out of the 35 primary tumors. However, demethylating drug treatment did not show significant induction of GNMT mRNA in HCC cells. Moreover, no significant association between DNA methylation and GNMT mRNA expressions was found in HCC18. Collectively, promoter hypermethylation seems not a crucial contributor in transcriptional silencing of GNMT.GNMT a multifunctional protein has a central role in the regulation of one-carbon metabolism in the liverin vitro and in vivo19. PGG is a natural polyphenolic compound well known for its wide range of biological activities. It’s has been extracted from several medicinal herbs used in traditional treatments of human diseases. PGG possesses potent anticancer activity against numerous cancers and shown to affect various signaling pathways21. Numerous proteins, including p53, Stat3, Cox-2, VEGFR1, AP-1, SP-1, Nrf-2, NF-κB and MMP-9 have been reported to be involved in the anticancer activity of PGG22. However, the precise mechanisms underlying PGG mediated GNMT induction remains elusive. In this study, we investigated the GNMT induction mechanism of PGG to improve the understanding of GNMT downregulation in HCC.Previously, we identified PGG as a GNMT inducer and showed that PGG treatment inhibits HCC cell growth both Here we demonstrated that PGG is a potent MYC inhibitor and this inhibition is responsible for GNMT induction by PGG in HCC. Our data also revealed MYC participation in downregulation of GNMT expression in HCC.19. To delineate the mechanism by which PGG enhances GNMT expression, microarray analyses were performed on Huh7 cells treated with either vehicle or PGG. We identified 168 differentially expressed genes that were persistently affected by PGG from 6 hours to 48 hours and were subjected to pathway enrichment analysis . qRT-PCR result analysis showed that the expression levels of MYC mRNA in HCC tissues were significantly negatively correlated with GNMT mRNA levels as determined by Pearson’s correlation . Finally, using the aflatoxin B1 (AFB1) -induced mouse model of HCC we confirmed that MYC mRNA expression is associated with GNMT in vivo. As shown in  was identified at distal region (−1775/−1780). In addition, MYC could also act as transcriptional repressor via binding to transcriptional initiator (Inr) elements or SP1 transcription factor in core promoters, which can both be found in GNMT core promoter; SP1 binding site: (−97/−91); CCAAT box: (−71/−67)26. Hence, to determine whether the distal E-box or the core promoter plays a crucial role in MYC-mediated GNMT suppression, three GNMT promoter-luciferase reporters-the 1.8Kb-Luc, 1.4Kb-Luc  and 147b-Luc  were used  program was used to predict MYC binding sites in the 1.8Kb GNMT promoter fragment (−1812/+14)sed Fig. . Overexpsed Fig. . Furthersed Fig. . These rion Fig. . These rion Fig. . These dTo confirm that the GNMT promoter induced by the PGG was indeed caused by suppression of MYC expression, we studied whether knocking down of MYC would impair the induction of GNMT promoter activity by PGG treatment. As shown in Fig. 27. Therefore, we examined whether PGG induced destabilization of MYC results from increased proteasomal degradation. Immunoblot results showed that proteasome inhibitor (MG132) and velcade did not prevent PGG-induced MYC protein depletion in Huh7 cells  and liver homolog receptor 1 (LHR-1) transcription factors binds to GNMT promoter and activate GNMT29. However, the contribution of these factors in GNMT downregulation is not studied in HCC. Huidobro et al. suggested that DNA hypermethylation is partly responsible for the transcriptional repression of GNMT in HCC. In contrast, demethylating agent (AdC) did not induce GNMT expression in HCC cells significantly (less than 1.5-fold induction)18. In our previous study, we identified PGG as a GNMT inducer using GNMT promoter based assay19. In this study, we attempted to explore the mechanism of action by which PGG induces GNMT expression in HCC. We found that MYC was a molecular target of PGG. Furthermore, we showed that MYC transcriptionally regulates GNMT by interacting with the core promoter of GNMT. A negative correlation between GNMT and MYC expression was also observed in human HCC specimen and TCGA liver cancer dataset. Moreover, we demonstrated that PGG enhances GNMT promoter activity via inhibition of MYC expression in HCC  induced GNMT promoter activity and mRNA expression in Huh7 cells19. Interestingly, the combination of PGG with TSA resulted in higher induction of GNMT mRNA expression compared to any of single treatments alone in Huh7 cells , while MYC acts as a transcriptional repressor through two mechanisms: by binding to the transcriptional initiator (Inr) element in the promoter; or by binding and inhibiting Sp1 transcriptional activityin vitro and in vivo models without host toxicity. PGG has been described to exert its anticancer activity by antioxidation, anti-inflammation, anti-angiogenesis, inhibiting DNA replicative synthesis, arresting cells in G1 and S phase, autophagy-mediated senescence, and inducing apoptosis22. In the present study, we found that PGG is a novel MYC inhibitor that inhibits MYC gene expression and enhances the degradation of the MYC protein. It is important to note that MYC is a pivotal regulator of many cellular processes such as cell cycle control, metabolic homeostasis and autophagy, and cell survival36. Thus, PGG induced G1 cell cycle arrest and apoptosis in cancer cells can also be explained by MYC inhibition. Therefore, it is reasonable to propose that the suppression of MYC expression, as well as its downstream targets by PGG, plays an important role in its anticancer activity. Moreover, using SYBR green real-time RT–PCR we verified the effect of PGG on the expression of genes  identified in microarray involved in cell cycle and apoptosis  have been identified. Direct MYC inhibitors, such as 10058-F4, disrupt Myc-Max dimerization. Direct MYC inhibition has proven to be a challenging task due to lack of obvious binding pockets for small molecules and more importantly, the direct targeting compound has shown very little success for clinical use41. Indirect inhibitors, such as JQ1, inhibit MYC transcription without affecting Myc-Max heterodimerization. However, recent studies have shown that various cancers have acquired resistance to JQ1 treatment42. The other indirect inhibitors, such as dihydroartemisinin, work by modulating the MYC protein stability43. It is interesting to note that PGG suppresses MYC expression at both transcriptional and post-translational level. Ubiquitin-dependent proteasome pathways using several E3 ligases is the most frequent pathway responsible for MYC protein degradation27. Li et al. showed that proteasome activator REGγ also regulates protein stability of MYC by ubiquitin-independent pathways44. In addition, calcium-activated calpain-dependent cytoplasmic cleavage of MYC has been reported to be responsible for MYC degradation as well45. Thus it is important to know that PGG accelerated MYC protein degradation through a proteasome- as well as calpain-independent mechanisms. Interestingly, pretreatment with chloroquine and EDTA showed significant rescue in PGG-mediated MYC repression in MYC overexpressed cells providing a direction for further studies. Chloroquine is known as a lysosomal inhibitor, however, the addition of other lysosomal inhibitors (3-MA and NH4Cl) do not show significant rescue in PGG mediated MYC repression, suggesting that the other unknown mechanism of chloroquine might be responsible for this rescue46. The possible mechanisms underlying MYC inhibition by PGG is under investigation.The MYC is a transcription factor crucial for normal cell functions including cell growth, proliferation, metabolism, protein biosynthesis, and microRNA regulations47. Interestingly, Hofmann et al. demonstrated that reduction of MYC expression in MYC haploinsufficient (Myc+/−) mice promotes longevity48. Even more interesting is that PGG has been shown to extend the life span of Caenorhabditis elegans49. Together with our finding, it is reasonable to hypothesize that induction of GNMT and alteration of the SAM (S-adenosdyl-methionine) metabolism contributes to the longevity of the Myc+/− mice. In addition, it is also possible that PGG increases lifespan through MYC down-regulation and subsequent alteration of the SAM metabolism. Thus, further studies of PGG and its suppressive effect on MYC is warranted and maybe beneficial to human medicine.It is interesting to note that overexpression of GNMT extends the lifespan in DrosophilaIn summary, this study demonstrated the usefulness of gene enhancer or repressor compounds as a tool for identifying the gene regulation mechanisms in cancer. Interestingly, the next questions that arise here are how does PGG induce MYC protein degradation? Is MYC stability is regulated by proteasome-independent pathways or is there a novel MYC protein degradation mechanism? Do these mechanisms exist in normal cells or only in cancer cells? Answers to these questions will contribute to better understanding of cancer biology and drug discovery.19, Huh7-shLacZ, Huh7-shMYC were grown in DMEM supplemented with 1 μg/mL puromycin. The cyclohexmide, 3-methyladenine, MG132, chloroquine diphosphate, E-64, cycloheximide (CHX) pepstatin A and (+/−) JQ-1 were purchased from Sigma-Aldrich , Z-AVD-FMK from Selleckchem.com, calpeptin from Millipore  and PGG obtained from The One Biopharmaceutical Co., Ltd.  as described previously19.Huh7 cells and Hep G2 cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) . DMEM supplemented with 10% heat-inactivated fetal bovine serum , penicillin (100 U/mL), streptomycin (100 μg/mL), nonessential amino acids (0.1 mM/L), and L-glutamine (2 mM/L)  at 37 °C in 5% CO2. Stable cell established by lentiviral-system including H7GPL26. Plasmids for lentivirus production (pCMV-ΔR8.91 and pMD.G), shRNAs for MYC  and control plasmid for the RNA interference (pLKO.1-shLacZ) were obtained from the National RNAi Core Facility . The pcDNA-MYC plasmid and vector control plasmid for overexpression experiments were provided by Dr Shih-Ping Liu . The Renilla luciferase control reporter vector , was used for normalizing transfection efficiency. Plasmid DNAs were transfected by using TurboFect Reagent  and Lipofectamine 3000  according to the manufacturer recommendations. Lentivirus production, transduction and generation of stable cell lines were performed as described previously50.The pGL3-luciferase constructs containing the GNMT promoter region with the deletions  and 147b-Luc  were constructed and described previouslyhttp://david.abcc.ncifcrf.gov/)48. The microarray data have been submitted to the Gene Expression Omnibus (GEO) public database at NCBI (Accession No. GSE75024). http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=axkzwoyojlqbhel&acc=GSE75024.Huh7 cells were treated with PGG (0.1 mg/mL) for 6 hours, 48 hours and solvent 48 hours in triplicates. Total RNA were extracted as described above and were used for microarray gene expression analysis . Array hybridization were performed according to the Affymetrix Gene chip expression analysis technical manual. Raw data was normalized by Exprssion ConsoleTM . Significant changed probe sets between treatment and control were detected by t-test statistic . Differentially expressed genes presented by selected probe sets were annotated and pathway enrichment analysis was performed by using DAVID Bioinformatics online tools  Huh7 cells was harvested and immunoprecipitated. Immunoprecipitations was performed using ChIP grade anti-MYC  antibody and rabbit non-immune serum IgG . The immunoprecipitate was PCR amplified using promoter-specific primers. Sequences for primers are listed in Supplementary Table ChIP was performed according to the protocol described beforeTotal RNA was isolated by using Tri Reagent (Sigma-Aldrich) and cDNA was synthesized using a Super Script II Reverse Transcriptase Kit . PCR was performed on an ABI StepOne Plus System  with the LightCycler® First Start DNA Master SYBR Green I reagent . The mRNA level was normalized using the TBP as an internal control to calculate relative expression. The primers used in this study shown in Supplementary Table 50. The anti-GNMT , anti-MYC  and anti-β-actin  antibodies were used in this study.Cells or xenograft tumors were lysed by RIPA lysis buffer  and phosphatase inhibitors . Immunoblotting was carried out as described previously19. Briefly, cells were lysed in Passive Lysis Buffer (Promega) and luciferase activity was assayed using the Luciferase Assay System (Promega) and Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s recommendations. AlamarBlue® assay  and MultiTox-Fluor multiplex cytotoxicity assay (Promega) were used to evaluate the cytotoxicity according to the manufacturer’s instructions.The detailed methods of luciferase assay have been described previouslyhttp://tlcn.nhri.org.tw/TLCN/index.jsp). The liver tumor tissues removed from the patients and there is no chemotherapy treatment before surgery; thus, the pathology stage can represent the status of tumor progression. Human HCC specimen study was approved by the Institutional Review Board of Kaohsiung Medical University and the user committee of TLCN. The detailed information was shown in Table For qRT-PCR, Sixty liver cancer tissue samples were obtained from the Taiwan Liver Cancer Network . Huh7 tumor-bearing mice with the size of approximately 100 mm3 were divided into two groups, with five mice per group and treated with PGG 300 mg/kg body weight (mpk) and drug vehicle. The tumor growth was monitored by using a Vernier caliper throughout the experiment. Tumors were lysed and used for immunoblot analysis as described above in immunoblot section. Data are presented as the mean of tumor volume ± SEM.The xenograft assay was performed and described previously52. Mice were divided into two groups AFB1  treatment group or control group . Mice were intraperitoneally injected with AFB 1 (10 mg per kg of body weight) or solvent at 7 days of age and given equal booster dosages at 2 months. The liver tissues were collected separately from 3-, 6-, 12-, 24-month-old mice in both control group and AFB1 treatment group (n = 3~6). Total RNA was isolated from mouse liver using Trizol Reagent (Invitrogen). C57BL/6JNarl BALB/c and NOD.CB17-Prkdcscid/JNarl mice were purchased from the National Laboratory Animal Center (NLAC), Taipei, Taiwan. Animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University and performed in compliance with the guidelines.For aflatoxin B1 (AFB1)-induced HCC mouse model, wild-type male mice  were generated as described previouslyData were analyzed using MS excel and Graph Pad Prism 5.0 software. The unpaired two-sided student’s t test was performed to compare groups. The correlation between MYC and GNMT were calculated using Pearson’s correlation coefficient. P < 0.05 considered statistically significant and marked in the figure legends.SUPPLEMENTARY INFORMATIONSupplementary table S1


TRPC6) gene promoter could increase its activation in steroid-resistant nephrotic syndrome children of China. Plasmids containing the TRPC6 promoter region  were constructed and then transfected into human embryonic kidney (HEK) 293T cells and human podocytes. Luciferase assays were used to test the promoter activity in both cell lines with or without tumor necrosis factor-α (TNF-α) treatment, and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) analysis was used to verify the transcription factor that could bind to this mutant sequence. Luciferase results indicate that the activity of the mutant promoter was greater than that of the normal promoter of the TRPC6 gene in both cell lines. We further predicted and verified that this variation was mediated by the nuclear factor kappa B (NF-κB) subunit RELA, and TNF-α significantly enhanced the transcription activity of TRPC6 with the −254G allele. In conclusion, the −254C>G SNP is a gain-of-function variation of the TRPC6 gene, and it is also an early and effective factor for predicting steroid-resistant nephrotic syndrome (SRNS) in Chinese children.This study is aimed at exploring the mechanism by which the −254C>G single nucleotide polymorphism (SNP) on the transient receptor potential cation channel 6 ( Podocytes are the key components of the glomerular filtration barrier in the kidneys; their foot processes act as the final protection against protein loss into the urine , 2. As tα-actinin-4, and some other proteins critical for podocyte function  ion overload, which can be activated in response to G protein signaling specifically via a diacylglycerol-dependent pathway, and then induces foot process effacement, podocyte detachment, and glomerulosclerosis [TRPC6 transcription or expression, thereby causing podocyte injury, has rarely been reported.TRPC6 is a recently discovered, heterotetrameric, nonselective cation channel  variation. Further analysis of the gene frequency showed that the frequency of the −254G allele locus in SRNS children was significantly higher than that in SSNS children; the luciferase assays verified that this variant significantly upregulated TRPC6 promoter activity, suggesting that TRPC6 is not only involved in the development of kidney disease but may also be closely related to steroid resistance, and the −254C>G SNP may be significantly correlated with high TRPC6 expression [TRPC6 core promoter regions with or without the −254C>G SNP and then transiently transfected them into human podocytes and HEK 293T cells; the luciferase assays reconfirmed that this variant could significantly enhance the promoter activity of the TRPC6 gene in both cell types. The HEK 293T cell line is a specific cell line originally derived from human kidney cells grown in tissue culture, and it does not express endogenous Ca2+ channels, which is an important tool cell line for multiple calcium channel studies. Therefore, we also use it as another cell line to illustrate our results [According to our previous study, we found that the expression of TRPC6 in renal tissue of children with SRNS was significantly higher than that of steroid-sensitive nephrotic syndrome (SSNS) for the first time. Furthermore, the gene sequence analysis then revealed that the pression . In this results , 20.TRPC6 gene [The promoter is the DNA sequence located at the 5′ end of a gene, acting like the gene “switch”; it controls the start time of gene transcription and the level of gene expression. However, the promoter itself cannot control gene activity and must further activate the RNA polymerase by binding with transcription factors to guide RNA transcription. Activation of the TRPC6 channel also requires the involvement of transcription factors. It has been shown that the expression of gain-of-function mutations of TRPC6 channels triggered a constitutive activation of the nuclear factor of activated T cell- (NFAT-) regulated gene transcription and has been associated with FSGS . FurtherPC6 gene . In thesκB heterodimer formation, nuclear translocation, and activation. The NF-κB family of transcription factors regulates the induction and resolution of inflammation. Additionally, it also can regulate the expression of numerous genes that play a key role in the inflammatory response during human and experimental kidney injury; most of these genes are regulated by RELA/P50 [α and found that the nuclear translocation of NF-κB upregulates TRPC6 promoter activity. Thus, it can be seen that the TNF-α-mediated activation of NF-κB significantly enhanced the promoter activity of the TRPC6 gene with the −254C>G SNP, thereby upregulating its expression; eventually, it might lead to podocyte damage. Clinically, frequent recurrence after repeated infection in children with NS is an important cause of steroid resistance, and we also found that the frequency of the −254G locus in children with SRNS was significantly higher than that in children with SSNS; thus, the −254C>G SNP maybe an important factor involved in the occurrence and development of SRNS.RELA, also known as p65, is a REL-associated protein involved in NF-RELA/P50 , 24. TheTRPC6 without TNF-α induction; the reason for this may be the stimulation by endogenous NF-κB in cells or the involvement of other transcription factors in the reaction. NF-κB-responsive promoters contain consensus-binding sites for other transcription factors often clustered into enhancers. These factors include the constitutive transcription factor SP-1 and the inducible factors IRFs, STATs, ATFs, CEBPs, CREB, and AP-1, many of which interact directly with NF-κB [TRPC6 gene, but the result did not show any differential binding between SP1 and the TRPC6 promoter (In our experiment, the −254G allele increased the luciferase activity of promoter .TRPC6, which enhanced the transcriptional regulation of TRPC6 in an NF-κB-mediated manner, may predispose individuals who have this mutation to an increased risk of developing SRNS.In conclusion, the −254C>G SNP in