Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Adaptation of the FK506 binding protein 1B to hibernation in bats.

Hibernation is an adaptive strategy used by some animals to cope with cold and food shortage. The heart rate, overall energy need, body temperature, and many other physiological functions are greatly reduced during torpor but promptly return to normal levels upon arousal. The heartbeat of torpid bats can be hundreds fold lower than that of active bats, indicating that hibernating bats have a remarkable ability to control excitation-contraction coupling in cardiac muscle. FKBP1B (calstabin 2), a peptidyl-prolyl cis-trans isomerase, is critical for the regulation of excitation-contraction coupling. Whether FKBP1B is adapted to hibernation in bats is not known. Evolutionary analyses showed that the ω values of the Fkbp1b genes of 25 mammalian species are all less than 1, and amino acid sequence alignments revealed that FKBP1B proteins are highly conserved in mammals. The expression of the Fkbp1b gene was found to be elevated at both mRNA and protein levels in two distantly related bats (Rhinolophus ferrumequinum in Yinpterochiroptera and Myotis ricketti in Yangochiroptera) during torpor. Transcription factors such as YY1 and SPs were bioinformatically determined to have a higher binding affinity to the potential regulatory regions of Fkbp1b genes in hibernating than in non-hibernating mammals. This study provides new insights into the molecular evolution of Fkbp1b in adaptation to bat hibernation.

The polyaromatic hydrocarbon ß-naphthoflavone alters binding of YY1, Sp1, and Sp3 transcription factors to the Dp71 promoter in hepatic cells.

The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, ß-naphthoflavone (ß­NF), downregulates Dp71 expression. The aim of the present study was to determine whether ß­NF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcription­quantitative polymerase chain reaction was used to measure half­life mRNA levels in ß­NF­treated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and ß­NF­treated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and ß­NF­treated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, ß­NF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that ß­NF may modulate the expression of other genes regulated by these transcription factors. In conclusion, ß­NF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.

Transcription factors YY1, Sp1 and Sp3 modulate dystrophin Dp71 gene expression in hepatic cells.

Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic ß-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5'-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter.

Specificity protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B.

N-Methyl-d-aspartate (NMDA) receptors are major glutamatergic receptors involved in most excitatory neurotransmission in the brain. The transcriptional regulation of NMDA receptors is not fully understood. Previously, we found that the GluN1 and GluN2B subunits of the NMDA receptor are regulated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1 and NRF-2 also regulate all 13 subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme, thereby coupling neuronal activity and energy metabolism at the transcriptional level. Specificity protein (Sp) is a family of transcription factors that bind to GC-rich regions, with Sp1, Sp3, and Sp4 all binding to the same cis- motifs. Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 expression is restricted to neurons and testicular cells. Recently, we found that the Sp1 factor regulates all subunits of COX. The goal of the present study was to test our hypothesis that the Sp factors also regulate specific subunits of NMDA receptors, and that they function with NRF-1 and NRF-2 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches we found that Sp4 functionally regulated GluN1, GluN2A, and GluN2B, but not GluN2C. On the other hand, Sp1 and Sp3 did not regulate these subunits as previously thought. Our data suggest that Sp4 operates in a complementary and concurrent/parallel manner with NRF-1 and NRF-2 to mediate the tight coupling between energy metabolism and neuronal activity at the molecular level.

Regulation of the varicella-zoster virus ORF3 promoter by cellular and viral factors.

The varicella zoster virus (VZV) immediate early 62 protein (IE62) activates most if not all identified promoters of VZV genes and also some minimum model promoters that contain only a TATA box element. Analysis of the DNA elements that function in IE62 activation of the VZV ORF3 promoter revealed that the 100 nucleotides before the translation start site of the ORF3 gene contains the promoter elements. This promoter lacks any functional TATA box element. Cellular transcription factors Sp1, Sp3 and YY1 bind to the promoter, and mutation of their binding sites inhibited ORF3 gene expression. VZV regulatory proteins, IE63 and ORF29, ORF61 and ORF10 proteins inhibited IE62-mediated activation of this promoter. Mutation of the Sp1/Sp3 binding site in the VZV genome did not alter VZV replication kinetics. This work suggests that Sp family proteins contribute to the activation of VZV promoters by IE62 in the absence of functional TATA box.

Regulation of the human ascorbate transporter SVCT2 exon 1b gene by zinc-finger transcription factors.

The sodium-dependent vitamin C transporter (SVCT) 2 is crucial for ascorbate uptake in metabolically active and specialized tissues. This study focused on the gene regulation of SVCT2 exon 1b, which is ubiquitously expressed in human and mouse tissues. Although the human SVCT2 exon 1b promoter does not contain a classical TATA box, we found that it does contain a functional initiator that binds Yin Yang-1 (YY1) and interacts with upstream Sp1/Sp3 elements in the proximal promoter region. These elements in turn play a critical role in regulating YY1-mediated transcription of exon 1b. Formation of YY1/Sp complexes on the promoter is required for its optional function. YY1 with Sp1 or Sp3 synergistically enhanced exon 1b promoter activity as well as the endogenous SVCT2 protein expression. Further, in addition to Sp1/Sp3, both EGR-1 and EGR-2 were detected in the protein complexes that bound the three GC boxes bearing overlapping binding sites for EGR/WT1 and Sp1/3. The EGR family factors WT1 and MAZ were found to differentially regulate exon 1b promoter activity. These results show that differential occupancy of transcription factors on the GC-rich consensus sequences in the SVCT2 exon 1b promoter contributes to the regulation of cell and tissue expression of SVCT2.

Constitutive expression of the brg1 gene requires GC-boxes near to the transcriptional start site.

We previously reported that BRG1, an ATPase subunit of SWI/SNF chromatin remodelling complexes, is constitutively expressed and that the alternative ATPase subunit (BRM) is inducibly expressed through differentiation in mammalian cells. In the present study, the regulatory elements that confer constitutive expression on brg1 were explored. First, we analysed the promoter proximal region surrounding its transcriptional start site. Using computer-aided analysis, a TATA-less, GC-rich promoter containing four putative binding sites for Sp1/3 was predicted. One of the putative Sp1/3-binding sites (from -21 to -15 bp) overlapped with a putative YY1-binding site. A gel-shift assay showed that YY1 but not Sp1/3 bound to this sequence and that Sp3 but not Sp1 bound to the other three predicted binding sites. Furthermore, chromatin immunoprecipitation analysis showed that Sp3 and YY1 bound to the promoter region together with TATA-binding protein in vivo. In vivo and in vitro binding assays showed that Sp3 and YY1 interacted with each other. Together, these results suggest that Sp3 and YY1 recruit general transcription factors and facilitate the assembly of a preinitiation complex.

Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells.

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.

Inhibition of NFkappaB and pancreatic cancer cell and tumor growth by curcumin is dependent on specificity protein down-regulation.

Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFkappaB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFkappaB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFkappaB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFkappaB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.

A role for Sp1 in transcriptional regulation of phosphatidylethanolamine N-methyltransferase in liver and 3T3-L1 adipocytes.

Phosphatidylcholine is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for phosphatidylcholine biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have now identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. We have found that PEMT mRNA and protein levels increased dramatically in 3T3-L1 cells upon differentiation to adipocytes. 5'-Deletion analysis of the PEMT promoter-luciferase constructs stably expressed in 3T3-L1 adipocytes identified a regulatory region between -471 and -371 bp (relative to the transcriptional start site). Competitive and supershift assays demonstrated binding sites for transcription factors Sp1, Sp3 (-408 to -413), and YY1 (-417 to -420). During differentiation of 3T3-L1 cells to adipocytes, the amount of Sp1 protein decreased by approximately 50% just prior to activation of PEMT. Transduction of 3T3-L1 adipocytes with retrovirus containing Sp1 cDNA demonstrated that Sp1 inhibited PEMT transcriptional activity. Similarly, short hairpin RNA directed against Sp1 in 3T3-L1 adipocytes enhanced PEMT transcriptional activation. Chromatin immunoprecipitation assays confirmed that Sp1 binds to the PEMT promoter, and this interaction decreases upon differentiation to adipocytes. These experiments directly link increased PEMT expression in adipocytes to decreased transcriptional expression of Sp1. In addition, our data established that Sp1 binding was required for tamoxifen-mediated inhibition of Pemt promoter activity.

A potent enhancer element in the 5'-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene.

Ubiquitin (Ub) plays a crucial role in almost every aspect of cellular functions. It is encoded by four genes, of which UbC is known to meet cell demand for ubiquitin in both basal and stressful conditions. To understand the molecular mechanisms regulating UbC gene expression, we performed a functional characterization of the UbC promoter. Deletion analyses on the 5' end of the -916/+878 promoter region, excluded the functional importance of nt -916/-371 in the transcriptional regulation of the gene, while 3' deletions revealed that intron removal (nt+65/+876) resulted in a marked reduction of promoter activity in all the reporter constructs, regardless of the cell types. Intron substitution with a heterologous chimeric intron failed to restore promoter activity, thus allowing to exclude that the splicing event, per se, can be responsible for the intron-mediated burst of transcription. Gel shift assays demonstrated nuclear factor binding with the +137/+766 intron region. Reporter constructs carrying partial intron deletions confirmed that this sequence is, indeed, required for maximal transcriptional activity. Computer-based analysis found potential Sp1 binding motifs within the intron sequence and electrophoretic mobility shift and supershift assays demonstrated that both Sp1 and Sp3 transcription factors interact, in vitro, with the UbC intron, at multiple binding sites. Moreover, ectopic expression of Sp1 and Sp3 revealed that both transcription factors positively regulate UbC promoter activity. Collectively, our data highlight the very new evidence that the 5'-UTR intron is crucial in regulating UbC gene expression and provide insights into the pivotal role of Sp1/Sp3 binding to the intronic enhancer in the regulation of UbC transcription.

Folate supplementation limits the aggressiveness of glioma via the remethylation of DNA repeats element and genes governing apoptosis and proliferation.

PURPOSE: We have investigated whether the folate supplementation could be used to limit the aggressiveness of glioma through the DNA remethylation because (a) the cancer genome is characterized by a low level of DNA methylation (or 5-methylcytosine, 5 mC); and (b) folate is the main generator of S-adenosyl-methionine, the methyl donor molecule in the DNA methylation reaction catalyzed by the DNA methyltranferases. EXPERIMENTAL DESIGN: The effects of folate supplementations were analyzed on the global DNA methylation status, the methylation status of DNA repeat element, the sensitivity of temozolomide-induced apoptosis, and the proliferation index of glioma cells. Finally, we analyzed whether the DNA methylation level could be used as a prognostic factor and/or a biomarker in an antiglioma therapy using folate supplementation as an adjuvant. RESULTS: Our data show that gliomagenesis is accompanied by a reduction in 5 mC levels and that this low level of 5 mC is a poor prognostic factor in Glioblastoma Multiforme patients. We also show that folate supplementation enhanced the DNA remethylation through the Sp1/Sp3-mediated transcriptional up-regulation of genes coding for Dnmt3a and Dnmt3b proteins, two de novo methyltranferases. Finally, we show that the folate-induced DNA methylation limits proliferation and increases the sensitivity to temozolomide-induced apoptosis in glioma cells through methylation of the genes implicated in these processes (PDGF-B, MGMT, survivin, and bcl-w). CONCLUSION: This study suggests that folate supplementation could be a promising adjuvant for the future design of antiglioma therapies in preclinical and/or clinical studies.

Transcriptional control of occludin expression in vascular endothelia: regulation by Sp3 and YY1.

Endothelium differentiates in response to tissue-specific signals; brain endothelium expresses tight junctions and transporters which are absent from other endothelia. The promoter of the tight junction protein occludin exhibited strong activity in a brain endothelial cell line, hCMEC/D3 but was inactive in lung endothelial cells. Expression of occludin in brain endothelium corresponded with binding of Sp3 to a minimal promoter segment close to the transcription-start site. However, in lung endothelium Sp-transcription factors did not bind to this site although they are present in the cell nucleus. In contrast, repression of occludin in lung endothelium was associated with the binding of YY1 to a remote site in the promoter region, which was functionally inactive in brain endothelium. The work identified a group of transcription factors including Sp3 and YY1, which differentially interact with the occludin promoter to induce expression of occludin in brain endothelium and repression in other endothelia. The mechanism controlling occludin expression is similar to that which controls tissue-specific expression of the transferrin receptor in brain endothelium, leading to a scheme for endothelial differentiation, in which activation or repression of tissue-specific proteins is maintained by a set of transcription factors which include Sp3 and YY1.

Allyl mercaptan, a garlic-derived organosulfur compound, inhibits histone deacetylase and enhances Sp3 binding on the P21WAF1 promoter.

Histone deacetylase (HDAC) inhibitors have the potential to derepress epigenetically silenced genes in cancer cells, leading to cell cycle arrest and apoptosis. In the present study, we screened several garlic-derived small organosulfur compounds for their ability to inhibit HDAC activity in vitro. Among the organosulfur compounds examined, allyl mercaptan (AM) was the most potent HDAC inhibitor. Molecular modeling, structure activity and enzyme kinetics studies with purified human HDAC8 provided evidence for a competitive mechanism (K(i) = 24 microM AM). In AM-treated human colon cancer cells, HDAC inhibition was accompanied by a rapid and sustained accumulation of acetylated histones in total cellular chromatin. Chromatin immunoprecipitation assays confirmed the presence of hyperacetylated histone H3 on the P21WAF1 gene promoter within 4 h of AM exposure, and there was increased binding of the transcription factor Sp3. At a later time, 24 h after AM treatment, there was enhanced binding of p53 in the distal enhancer region of the P21WAF1 gene promoter. These findings suggest a primary role for Sp3 in driving P21 gene expression after HDAC inhibition by AM, followed by the subsequent recruitment of p53. Induction of p21Waf1 protein expression was detected at time points between 3 and 72 h after AM treatment and coincided with growth arrest in G(1) of the cell cycle. The results are discussed in the context of other anticarcinogenic mechanisms ascribed to garlic organosulfur compounds and the metabolic conversion of such compounds to potential HDAC inhibitors in situ.

Ligand structure-dependent activation of estrogen receptor alpha/Sp by estrogens and xenoestrogens.

This study investigated the effects of E2, diethylstilbestrol (DES), antiestrogens, the phytoestrogen resveratrol, and the xenoestrogens octylphenol (OP), nonylphenol (NP), endosulfan, kepone, 2,3,4,5-tetrachlorobiphenyl-4-ol (HO-PCB-Cl(4)), bisphenol-A (BPA), and 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on induction of luciferase activity in breast cancer cells transfected with a construct (pSp1(3)) containing three tandem GC-rich Sp binding sites linked to luciferase and wild-type or variant ERalpha. The results showed that induction of luciferase activity was highly structure-dependent in both MCF-7 and MDA-MB-231 cells. Moreover, RNA interference assays using small inhibitory RNAs for Sp1, Sp3 and Sp4 also demonstrated structure-dependent differences in activation of ERalpha/Sp1, ERalpha/Sp3 and ERalpha/Sp4. These results demonstrate for the first time that various structural classes of ER ligands differentially activate wild-type and variant ERalpha/Sp-dependent transactivation, selectively use different Sp proteins, and exhibit selective ER modulator (SERM)-like activity.

OX40 gene expression is up-regulated by chromatin remodeling in its promoter region containing Sp1/Sp3, YY1, and NF-kappa B binding sites.

OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40 mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-kappaB (p50 and p65) also binds to the OX40 promoter region, but the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipitation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-kappaB binds to the OX40 promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-kappaB might be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4(+)CD25(+) regulatory T (Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg cell. In CD4(+)CD25(+) Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence of in vitro activation.

Action of transcription factors in the control of transferrin receptor expression in human brain endothelium.

Brain endothelium has a distinctive phenotype, including high expression of transferrin receptor, p-glycoprotein, claudin-5 and occludin. Dermal endothelium expresses lower levels of the transferrin receptor and it is absent from lung endothelium. All three endothelia were screened for transcription factors that bind the transferrin receptor promoter and show different patterns of binding between the endothelia. The transcription factor YY1 has distinct DNA-binding activities in brain endothelium and non-brain endothelium. The target-sites on the transferrin receptor promotor for YY1 lie in close proximity to those of the transcription initiation complex containing TFIID, so the two transcription factors potentially compete or interfere. Notably, the DNA-binding activity of TFIID was the converse of YY1, in different endothelia. YY1 knockdown reduced transferrin receptor expression in brain endothelium, but not in dermal endothelium, implying that YY1 is involved in tissue-specific regulation of the transferrin receptor. Moreover a distinct YY1 variant is present in brain endothelium and it associates with Sp3. A model is presented, in which expression from the transferrin receptor gene in endothelium requires the activity of both TFIID and Sp3, but whether the gene is transcribed in different endothelia, is related to the balance between activating and suppressive forms of YY1.

Characterization of human collagen XVIII promoter 2: interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes.

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides -186 and -21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position -700 (T>G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele -700G increases transcription and binding for a still unknown transcription factor. SNP -700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide -700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.