Specificity Proteins (Sp) and Cancer.

The specificity protein (Sp) transcription factors (TFs) Sp1, Sp2, Sp3 and Sp4 exhibit structural and functional similarities in cancer cells and extensive studies of Sp1 show that it is a negative prognostic factor for patients with multiple tumor types. In this review, the role of Sp1, Sp3 and Sp4 in the development of cancer and their regulation of pro-oncogenic factors and pathways is reviewed. In addition, interactions with non-coding RNAs and the development of agents that target Sp transcription factors are also discussed. Studies on normal cell transformation into cancer cell lines show that this transformation process is accompanied by increased levels of Sp1 in most cell models, and in the transformation of muscle cells into rhabdomyosarcoma, both Sp1 and Sp3, but not Sp4, are increased. The pro-oncogenic functions of Sp1, Sp3 and Sp4 in cancer cell lines were studied in knockdown studies where silencing of each individual Sp TF decreased cancer growth, invasion and induced apoptosis. Silencing of an individual Sp TF was not compensated for by the other two and it was concluded that Sp1, Sp3 and Sp4 are examples of non-oncogene addicted genes. This conclusion was strengthened by the results of Sp TF interactions with non-coding microRNAs and long non-coding RNAs where Sp1 contributed to pro-oncogenic functions of Sp/non-coding RNAs. There are now many examples of anticancer agents and pharmaceuticals that induce downregulation/degradation of Sp1, Sp3 and Sp4, yet clinical applications of drugs specifically targeting Sp TFs are not being used. The application of agents targeting Sp TFs in combination therapies should be considered for their potential to enhance treatment efficacy and decrease toxic side effects.

SP and KLF Transcription Factors in Cancer Metabolism.

Tumor development and progression depend on reprogramming of signaling pathways that regulate cell metabolism. Alterations to various metabolic pathways such as glycolysis, oxidative phosphorylation, lipid metabolism, and hexosamine biosynthesis pathway are crucial to sustain increased redox, bioenergetic, and biosynthesis demands of a tumor cell. Transcription factors (oncogenes and tumor suppressors) play crucial roles in modulating these alterations, and their functions are tethered to major metabolic pathways under homeostatic conditions and disease initiation and advancement. Specificity proteins (SPs) and Krüppel-like factors (KLFs) are closely related transcription factors characterized by three highly conserved zinc fingers domains that interact with DNA. Studies have demonstrated that SP and KLF transcription factors are expressed in various tissues and regulate diverse processes such as proliferation, differentiation, apoptosis, inflammation, and tumorigenesis. This review highlights the role of SP and KLF transcription factors in the metabolism of various cancers and their impact on tumorigenesis. A better understanding of the role and underlying mechanisms governing the metabolic changes during tumorigenesis could provide new therapeutic opportunities for cancer treatment.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer.

Specificity protein (Sp) transcription factors (TFs) Sp1, Sp3 and Sp4, and the orphan nuclear receptor 4A1 (NR4A1) are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival. Results of knockdown and overexpression of Sp1, Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members. NR4A1 is also a pro-oncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth, survival, migration and invasion. There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin, epidermal growth factor receptor, PAX3-FOXO1, α5- and α6-integrins, ß1-, ß3- and ß4-integrins; this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites. Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells, and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

Expression of the zinc finger transcription factor Sp6-9 in the velvet worm Euperipatoides kanangrensis suggests a conserved role in appendage development in Panarthropoda.

The Sp-family genes encode important transcription factors in animal development. Here we investigate the embryonic expression patterns of the complete set of Sp-genes in the velvet worm Euperipatoides kanangrensis (Onychophora), with a special focus on the Sp6-9 ortholog. In arthropods, Sp6-9, the ortholog of the Drosophila melanogaster D-Sp1 gene plays a conserved role in appendage development. Our data show that the expression of Sp6-9 during the development of the velvet worm is conserved, suggesting that the key function of the Sp6-9 gene dates back to at least the last common ancestor of arthropods and onychophorans and thus likely the last common ancestor of Panarthropoda.

Activation of COUP-TFI by a Novel Diindolylmethane Derivative.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan receptor and member of the nuclear receptor superfamily. Among a series of methylene substituted diindolylmethanes (C-DIMs) containing substituted phenyl and heteroaromatic groups, we identified 1,1-bis(3'-indolyl)-1-(4-pyridyl)-methane (DIM-C-Pyr-4) as an activator of COUP-TFI. Structure activity studies with structurally diverse heteroaromatic C-DIMs showed that the pyridyl substituted compound was active and the 4-pyridyl substituent was more potent than the 2- or 3-pyridyl analogs in transactivation assays in breast cancer cells. The DIM-C-Pyr-4 activated chimeric GAL4-COUP-TFI constructs containing full length, C- or N-terminal deletions, and transactivation was inhibited by phosphatidylinositol-3-kinase and protein kinase A inhibitors. However, DIM-C-Pyr-4 also induced transactivation and interactions of COUP-TFI and steroid receptor coactivators-1 and -2 in mammalian two-hybrid assays, and ligand-induced interactions of the C-terminal region of COUP-TFI were not affected by kinase inhibitors. We also showed that DIM-C-Pyr-4 activated COUP-TFI-dependent early growth response 1 (Egr-1) expression and this response primarily involved COUP-TFI interactions with Sp3 and to a lesser extent Sp1 bound to the proximal region of the Egr-1 promoter. Modeling studies showed interactions of DIM-C-Pyr-4 within the ligand binding domain of COUP-TFI. This report is the first to identify a COUP-TFI agonist and demonstrate activation of COUP-TFI-dependent Egr-1 expression.

Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-κB signaling pathway.

BACKGROUND AND AIMS: Human genetic studies indicated that variations near the transcription factor Krüppel-like factor 14 (KLF14) gene locus are highly associated with coronary artery disease. Activation of endothelial cells (ECs) by pro-inflammatory molecules and pathways is a primary step in atherosclerosis development. We aimed to investigate the effects and mechanism of KLF14 on inflammatory responses in ECs. METHODS: Adenovirus-mediated overexpression of human KLF14 and EC specific Klf14 knockout mice were applied to study the role of KLF14 in EC inflammation. Intravital microscopy was used to examine leukocyte-endothelial cell interactions in vivo. RESULTS: The expression of Klf14 was markedly decreased in mouse aortic ECs in both acute and chronic inflammatory conditions. Overexpression of KLF14 inhibited inflammatory activation of human ECs stimulated by interleukin 1ß and tumor necrosis factor α. Primary pulmonary ECs from Klf14 knockout mice showed increased expression of adhesion molecules under IL-1ß stimuli. Mechanistically, KLF14 inhibited NF-κB signaling pathway by transcriptionally suppressing the expression of p65, resulting in significantly decreased leukocyte adhesion to activated ECs. Using intravital microscopy, an increased leukocyte-endothelial cell interaction was observed in endothelial specific Klf14 knockout mice compared to wild type control mice. Additionally, perhexiline, a KLF14 activator, induces KLF14 expression in ECs and reduced leukocyte-endothelial cell interactions in vitro and in vivo. CONCLUSIONS: The data revealed that KLF14 inhibited the inflammatory response in ECs and the protective effects were mediated by transcriptional inhibition of NF-κB signaling pathway. Endothelial KLF14 could be a potential therapeutic target for cardiovascular diseases.

LncRNA HAND2-AS1 sponging miR-1275 suppresses colorectal cancer progression by upregulating KLF14.

Long noncoding RNAs (lncRNAs) represent a novel type of noncoding RNAs of over 200 nucleotides, characterized by no or limited protein-coding potential. Although the function of lncRNAs attracts increasing attention recently, the relationship between lncRNA and colorectal cancer (CRC) remains further investigation. In our study, we found that lncRNA HAND2-AS1 was markedly downregulated in CRC tissues. And its expression level was negatively correlated with metastasis and advanced stage in CRC patients. Furthermore, we showed that HAND2-AS1 low expression predicted poor prognosis. Functionally, we found that overexpression of HAND2-AS1 obviously attenuated the proliferation and invasion of CRC cells. Ectopic expression of HAND2-AS1 also inhibited tumor propagation in vivo. In mechanism, HAND2-AS1 served as a sponge of miR-1275 which targeted KLF14. Through facilitating KLF14 expression, HAND2-AS1 suppressed CRC progression. In conclusion, our study demonstrated that HAND2-AS1 exerts a suppressive role in CRC by sponging miR-1275 and modulating KLF14 expression.

Bortezomib Targets Sp Transcription Factors in Cancer Cells.

Bortezomib alone and in combination with other anticancer agents are extensively used for chemotherapeutic treatment of multiple myeloma (MM) patients and are being developed for treating other cancers. Bortezomib acts through multiple pathways, and in this study with ANBL-6 and RPMI 8226 MM cells we show that bortezomib inhibited growth and induced apoptosis and that this was accompanied by downregulation of specificity protein (Sp) 1, Sp3, and Sp4 transcription factors that are overexpressed in these cells. Similar results were observed in pancreatic and colon cancer cells. The functional importance of this pathway was confirmed by showing that individual knockdown of Sp1, Sp3, and Sp4 in MM cells inhibited cell growth and induced apoptosis, and that this correlates with the results of previous studies in pancreatic, colon, and other cancer cell lines. The mechanism of bortezomib-mediated downregulation of Sp transcription factors in MM was due to the induction of caspase-8 and upstream factors, including Fas-associated death domain. These results demonstrate that an important underlying mechanism of action of bortezomib was due to the activation of caspase-8-dependent downregulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

Specificity Protein Transcription Factors and Cancer: Opportunities for Drug Development.

Specificity protein (Sp) transcription factors (TFs) such as Sp1 are critical for early development but their expression decreases with age and there is evidence that transformation of normal cells to cancer cells is associated with upregulation of Sp1, Sp3, and Sp4, which are highly expressed in cancer cells and tumors. Sp1 is a negative prognostic factor for pancreatic, colon, glioma, gastric, breast, prostate, and lung cancer patients. Functional studies also demonstrate that Sp TFs regulate genes responsible for cancer cell growth, survival, migration/invasion, inflammation and drug resistance, and Sp1, Sp3 and Sp4 are also nononcogene addiction (NOA) genes and important drug targets. The mechanisms of drug-induced downregulation of Sp TFs and pro-oncogenic Sp-regulated genes are complex and include ROS-dependent epigenetic pathways that initially decrease expression of the oncogene cMyc. Many compounds such as curcumin, aspirin, and metformin that are active in cancer prevention also exhibit chemotherapeutic activity and these compounds downregulate Sp TFs in cancer cell lines and tumors. The effects of these compounds on downregulation of Sp TFs in normal cells and the contribution of this response to their chemopreventive activity have not yet been determined. Cancer Prev Res; 11(7); 371-82. ©2018 AACR.

Modified aging of elite athletes revealed by analysis of epigenetic age markers.

Recent progress in epigenomics has led to the development of prediction systems that enable accurate age estimation from DNA methylation data. Our objective was to track responses to intense physical exercise of individual age-correlated DNA methylation markers and to infer their potential impact on the aging processes. The study showed accelerated DNA hypermethylation for two CpG sites in TRIM59 and KLF14. Both markers predicted the investigated elite athletes to be several years older than controls and this effect was more substantial in subjects involved in power sports. Accordingly, the complete 5-CpG model revealed age acceleration of elite athletes (P=1.503x10-7) and the result was more significant amongst power athletes (P=1.051x10-9). The modified methylation of TRIM59 and KLF14 in top athletes may be accounted for by the biological roles played by these genes. Their known anti-tumour and anti-inflammatory activities suggests that intense physical training has a complex influence on aging and potentially launches signalling networks that contribute to the observed lower risk of elite athletes to develop cardiovascular disease and cancer.

The role of Krüppel-like factor 14 in the pathogenesis of atherosclerosis.

The Krüppel-like factor (KLF) family, as the SP/XKLF transcription factors, plays important roles in regulating the expression of genes required for the proper execution of important biological and pathological processes. Recent studies have demonstrated that KLF14, a member of the KLF family, participates in the initiation and progression of atherosclerotic cardiovascular disease (CVD). From the molecular function aspect, this review focuses on the impact of KLF14-mediated regulation in major atherosclerosis-related diseases and pathological processes, such as insulin resistance, type 2 diabetes, dyslipidemia, inflammation, obesity, metabolic syndrome, cell proliferation and differentiation. This review was designed to help understand the roles of KLF14 in the pathogenesis of atherosclerosis and define KLF14 as a potential disease biomarker and a novel therapeutic target in CVD.

SP and KLF Transcription Factors in Digestive Physiology and Diseases.

Specificity proteins (SPs) and Krüppel-like factors (KLFs) belong to the family of transcription factors that contain conserved zinc finger domains involved in binding to target DNA sequences. Many of these proteins are expressed in different tissues and have distinct tissue-specific activities and functions. Studies have shown that SPs and KLFs regulate not only physiological processes such as growth, development, differentiation, proliferation, and embryogenesis, but pathogenesis of many diseases, including cancer and inflammatory disorders. Consistently, these proteins have been shown to regulate normal functions and pathobiology in the digestive system. We review recent findings on the tissue- and organ-specific functions of SPs and KLFs in the digestive system including the oral cavity, esophagus, stomach, small and large intestines, pancreas, and liver. We provide a list of agents under development to target these proteins.

Bardoxolone Methyl and a Related Triterpenoid Downregulate cMyc Expression in Leukemia Cells.

Structurally related pentacyclic triterpenoids methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate [bardoxolone-methyl (Bar-Me)] and methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF3DODA-Me) contain 2-cyano-1-en-3-one and 2-trifluoromethyl-1-en-3-one moieties, respectively, in their A-rings and differ in the position of their en-one structures in ring C. Only Bar-Me forms a Michael addition adduct with glutathione (GSH) and inhibits IKKß phosphorylation. These differences may be due to steric hindrance by the 11-keto group in CF3DODA-Me, which prevents Michael addition by the conjugated en-one in the A-ring. In contrast, both Bar-Me and CF3DODA-Me induce reactive oxygen species in HL-60 and Jurkat leukemia cells, inhibit cell growth, induce apoptosis and differentiation, and decrease expression of specificity proteins (Sp) 1, 3, and 4, and cMyc, and these effects are significantly attenuated after cotreatment with the antioxidant GSH. In contrast to solid tumor-derived cells, cMyc and Sp transcriptions are regulated independently and cMyc plays a more predominant role than Sp transcription factors in regulating HL-60 or Jurkat cell proliferation and differentiation compared with that observed in cells derived from solid tumors.

Sp1 is a competitive endogenous RNA of Klf4 during odontoblast differentiation.

Our previous studies have demonstrated that KLF4 is a critical transcription factor that promotes the odontoblastic differentiation of dental papilla cells. Klf4 mRNA was found to be regulated by multiple microRNAs (miRNAs). Competitive endogenous RNAs (ceRNAs) are a group of transcripts post-transcriptionally regulating each other by competing for their common miRNAs. However, the regulation of Klf4 by ceRNAs in odontoblastic differentiation remains unknown. In this study, we predicted a group of potential Klf4 ceRNAs with bioinformatics approach, and examined the expression of Klf4 and five interested potential ceRNAs including Sp1 using real-time PCR during odontoblastic differentiation of mDPC6T. The expression levels of both Sp1 and Klf4 were significantly upregulated during this process. In situ hybridization verified that Sp1 was co-expressed with Klf4 in the differentiating and the mature odontoblasts in vivo. Knockdown of Sp1 using siRNA resulted in a significant reduction of Klf4 and vice visa. This interaction was further confirmed to be miRNA dependent. Common miRNAs of Klf4 and Sp1 were predicted, among which miR-7a, miR-29b and miR-135a were able to downregulate both Klf4 and Sp1 expression after their separate overexpression in the mDPC6T cells. Dual luciferase assays showed that these miRNAs separately regulated the 3'UTRs of both Klf4 and Sp1, and the down-regulation of Klf4 3 'UTR by Sp1 siRNA was abolished when these three miRNAs' binding sites were mutated in the Klf4 3 'UTR. Therefore, our results indicate that Sp1 functions as a ceRNA of Klf4 during odontoblastic differentiation through competing for miR-7a, miR-29b and miR-135a.

NF-Y and SP transcription factors - New insights in a long-standing liaison.

For long it has been recognized that CCAAT boxes and GC-rich elements co-occur in many human and murine promoters within 100bp upstream of the transcription start site. The trimeric transcription factor NF-Y is the major CCAAT box-binding factor, and members of the SP family of transcription factors are the major GC box-binding proteins. Recent chromatin immunoprecipitations coupled with high throughput sequencing (ChIP-seq) have examined binding of NF-Y and the ubiquitous SP factors SP1, SP2 and SP3 genome-wide, allowing for comprehensive comparison of NF-Y and SP factor actions in the context of chromatin. Here, I attempt a synthesis of the earlier single-promoter type of analysis with the more recent genome-wide studies. In particular, I also discuss different modes of genomic interactions between SP factors and NF-Y that have emerged recently, and identify a key technical issue, which needs to be taken into account in a critical evaluation of genome-wide studies. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Penfluridol Represses Integrin Expression in Breast Cancer through Induction of Reactive Oxygen Species and Downregulation of Sp Transcription Factors.

It was recently demonstrated the penfluridol inhibited breast tumor growth and metastasis and this was associated with downregulation of α6- and ß4-integrins. In this study, we observed the penfluridol induced reactive oxygen species (ROS) and this was the primary mechanism of action. Penfluridol-mediated growth inhibition, induction of apoptosis, and inhibition of breast cancer cell migration was attenuated after cotreatment with glutathione. Penfluridol also downregulated Sp transcription factors Sp1, Sp3, and Sp4 through epigenetic downregulation of cMyc and cMyc-regulated miRNAs (miR27a and miR20a/miR17) and induction of the miR-regulated Sp transcriptional repressors ZBTB10 and ZBTB4. α6- and ß4-integrins as well as α5- and ß1-integrins are Sp-regulated genes that are also coregulated by the orphan nuclear receptor NR4A1 and these integrins can be targeted by agents such as penfluridol that suppress Sp1, Sp3, and Sp4 and also by NR4A1 antagonists. Mol Cancer Ther; 16(1); 205-16. ©2016 AACR.

Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer.

The antineoplastic agent benzyl isothiocyanate (BITC) acts by targeting multiple pro-oncogenic pathways/genes, including signal transducer and activator of transcription 3 (STAT3); however, the mechanism of action is not well known. As reported previously, BITC induced reactive oxygen species (ROS) in Panc1, MiaPaCa2, and L3.6pL pancreatic cancer cells. This was accompanied by induction of apoptosis and inhibition of cell growth and migration, and these responses were attenuated in cells cotreated with BITC plus glutathione (GSH). BITC also decreased expression of specificity proteins (Sp) Sp1, Sp3, and Sp4 transcription factors (TFs) and several pro-oncogenic Sp-regulated genes, including STAT3 and phospho-STAT3 (pSTAT3), and GSH attenuated these responses. Knockdown of Sp TFs by RNA interference also decreased STAT3/pSTAT3 expression. BITC-induced ROS activated a cascade of events that included down-regulation of c-Myc, and it was also demonstrated that c-Myc knockdown decreased expression of Sp TFs and STAT3 These results demonstrate that in pancreatic cancer cells, STAT3 is an Sp-regulated gene that can be targeted by BITC and other ROS inducers, thereby identifying a novel therapeutic approach for targeting STAT3.

Natural Products as Mechanism-based Anticancer Agents: Sp Transcription Factors as Targets.

Naturally occurring anticancer agents and their derivatives act on multiple pathways to inhibit carcinogenesis and their inhibition of migration, invasion, growth, survival, and metastasis is associated with downregulation of genes associated with these responses. Several phytochemical-derived anticancer drugs including curcumin, betulinic acid, phenethylisothiocyanate and celastrol, and many others induce reactive oxygen species, and their effects on gene regulation show some overlap in various cancer cell lines. We hypothesize that reactive oxygen species-inducing anticancer agents and many other natural products target a common pathway in cancer cells, which initially involves downregulation of specificity protein 1 (Sp1), Sp3, and Sp4, which are highly expressed in tumors/cell lines derived from solid tumors. This hypothesis is supported by several published reports showing that a large number of phytochemical-derived anticancer agents downregulate Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes involved in cell growth (cyclin D1 and growth factor receptors), survival (bcl-2 and survivin), angiogenesis and migration (MMP-9, vascular endothelial growth factor and its receptors), and inflammation (NF-kB). The contribution of this pathway to the anticancer activity of drugs such as curcumin, celastrol, betulinic acid, and phenethylisothiocyanate must be determined in order to optimize clinical applications of drug combinations containing these compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms.

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.