1: J Endocrinol Invest. 2005 Jul-Aug;28(7):651-6. A naturally occurring deletion in the SRY promoter region affecting the Sp1 binding site is associated with sex reversal. Assumpcao JG, Ferraz LF, Benedetti CE, Maciel-Guerra AT, Guerra G Jr, Marques-de-Faria AP, Baptista MT, de Mello MP. Centro de Biologia Molecular e Engenharia Genetica (CBMEG), Universidade Estadual de Campinas, Sao Paulo, Brasil. Male to female sex reversal results from failure of testis development. Mutations in the SRY gene or in other genes involved in the sexual differentiation pathway are considered to cause XY gonadal dysgenesis. The majority of the mutations in the SRY described so far are located within the SRY coding region, mainly in the HMG-box conserved domain. Comparison of 5' flanking SRY gene sequences among different species indicated the presence of several putative conserved consensus sequences for different transcription regulators. In this study, we investigated a 360 bp sequence encompassing the SRY putative core promoter, in 17 patients with variable degrees of 46,XY sex reversal, which have been previously shown not to bear mutations in the SRYcoding region. Sequencing analysis of the SRYpromoter in one patient with complete XY gonadal dysgenesis revealed a three base pair deletion in one of the Sp1 binding sites. The deletion abolished Sp1 binding in vitro. This is the first report on a naturally occurring mutation affecting the Sp1 regulatory element in the SRY promoter region, which is associated with sex reversal. Additionally, upon familial investigation the father, who had 18 genital surgeries due to severe hypospadia without cryptorchidism, was found to bear the same deletion and several relatives were referred to have sexual ambiguity. PMID: 16218050 [PubMed - in process] --------------------------------------------------------------- 2: Gene. 2005 Jan 3;344:287-97. Epub 2004 Dec 10. Functional characterization of the human SOX3 promoter: identification of transcription factors implicated in basal promoter activity. Kovacevic Grujicic N, Mojsin M, Krstic A, Stevanovic M. Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. BOX 23, 11010 Belgrade, Serbia and Montenegro. SRY-related HMG-box genes (Sox genes) constitute a large family of developmentally regulated genes involved in the decision of cell fates during development and implicated in the control of diverse developmental processes. Sox3, an X-linked member of the family, is expressed in the central nervous system (CNS) from the earliest stages of development. It is considered to be one of the earliest neural markers in vertebrates playing the role in specifying neuronal fate. The aim of this study has been to determine and characterize the promoter of the human SOX3 gene and to elucidate molecular mechanisms underlying the regulation of its expression. In this study, we have isolated and performed the first characterization of the human SOX3 promoter. We have identified the transcription start point (tsp) and carried out the structural and functional analysis of the regulatory region responsible for SOX3 expression in NT2/D1 cell line. Using promoter-reporter constructs, we have determined the minimal SOX3 promoter region that confers the basal promoter activity, as well as two regulatory elements which have positive effects on the promoter activity. We have investigated in detail the functional properties of three conserved motifs within the core promoter sequence that bind transcription factors specificity protein 1 (Sp1), upstream stimulatory factor (USF) and nuclear factor Y (NF-Y). By mutational analysis, we have shown that all three sites are of functional relevance for constitutive SOX3 expression in NT2/D1 cells. We have also shown that, besides the TATA motif, at least one other essential regulatory element is required for the basal transcription of the human SOX3. Taken together, data presented in this paper suggest that transcription factors such as Sp1, USF and NF-Y could function as key regulators for the basal activation of the human SOX3 gene. PMID: 15656994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Endocrinology. 2004 Mar;145(3):1113-23. Epub 2003 Nov 20. Differential utilization of the promoter of peripheral-type benzodiazepine receptor by steroidogenic versus nonsteroidogenic cell lines and the role of Sp1 and Sp3 in the regulation of basal activity. Giatzakis C, Papadopoulos V. Department of Cell Biology, Georgetown University Medical Center, Washington, DC 20057, USA. The peripheral-type benzodiazepine receptor (PBR) is involved in many cellular functions, including steroidogenesis, oxidative processes, cellular proliferation, and apoptosis. Secretory and glandular tissues, especially steroid hormone-producing cells, are particularly rich in PBR. To understand the mechanisms of PBR expression and regulation, we established an mRNA expression profile in mouse tissues and cell lines and subsequently mapped the transcription start site and characterized the promoter of the gene. Our findings indicate that PBR tissue mRNA levels are relatively high in kidney, spleen, muscle, lung, adrenal gland, thymus, and stomach; are intermediate in pancreas, uterus, prostate, heart, and testis; and are low in brain and liver. Relatively high levels of PBR mRNA were also observed in the steroid-synthesizing MA-10 mouse Leydig tumor cells compared with adrenocortical Y1 mouse cells and nonsteroidogenic NIH-3T3 mouse fibroblasts, although PBR protein levels were much higher in both steroidogenic cells compared with fibroblasts. Transcription was initiated primarily at an adenine nucleotide 61 nucleotides upstream of the translation start site, but internal initiation was also observed. A 2.7-kb fragment of the mouse PBR promoter was cloned and sequenced. Sequence analysis revealed the absence of TATA or CCAAT boxes, but the presence of many putative transcription factor-binding sites, including Sp1/Sp3, AP2, Ik2, AP1, SOX, GATA, and SRY. Functional characterization revealed that two Sp1/Sp3 sites in the proximal promoter are important for basal activity in all cell lines tested and that the steroidogenic MA-10 and Y1 cells use different areas of the promoter compared with nonsteroidogenic NIH-3T3 cells. PMID: 14630713 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Lung Cancer. 2002 Dec;38(3):229-34. The promoter region of the human BUBR1 gene and its expression analysis in lung cancer. Seike M, Gemma A, Hosoya Y, Hosomi Y, Okano T, Kurimoto F, Uematsu K, Takenaka K, Yoshimura A, Shibuya M, Ui-Tei K, Kudoh S. Fourth Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers. PMID: 12445743 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gene. 2002 May 29;291(1-2):123-33. Genomic structure, organization, and promoter region analysis of murine guanylyl cyclase/atrial natriuretic peptide receptor-A gene. Garg R, Oliver PM, Maeda N, Pandey KN. Department of Physiology, Tulane University School of Medicine, Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 710112, USA. We have determined the complete genomic nucleotide sequence and analyzed the promoter region of murine guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1,coding for NPRA). The gene spans about 17.8 kb and contains 22 exons interrupted by 21 introns. All the exon-intron boundaries possess the consensus GT/AG splice junctions. Four different types of short interspersed nuclear elements (ten mouse B1 elements, seven mouse B2-B4 elements, one ID and one MIR element) and one medium reiteration frequency repeats have been found in the non-coding regions of the gene. Eleven tandem repeats, including three in the promoter region of the gene, have been identified. The transcription start site, 362 bp upstream from the start codon, was determined by 5'- rapid amplification of cDNA ends. The 1.98 kb 5'-flanking region contains three potential SP1 binding sites and one inverted CCAAT box but lacks the TATA box. This region also contains several putative cis-acting motifs known to bind kidney specific nuclear protein HFH-3, cAMP-responsive element binding protein (CREB) and AP-4. In addition, the binding sites for a variety of transcription factors: AML-1 alpha, SRY, Nkx-2.5, LyF-1, p300, GATA-1/2, HNF-3 beta, c/EBP alpha/beta and USF have been localized in the promoter region of Npr1 gene. The analyses and characterization of the genomic structure of murine Npr1 gene should yield important insights into the species-specific regulation of this important gene family. PMID: 12095686 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Biol Reprod. 2001 Mar;64(3):775-83. Steroidogenic factor-1 contributes to the cyclic-adenosine monophosphate down-regulation of human SRY gene expression. de Santa Barbara P, Mejean C, Moniot B, Malcles MH, Berta P, Boizet-Bonhoure B. Human Molecular Genetics Group, Institut de Genetique Humaine, CNRS UPR1142, 34396 Montpellier Cedex 5, France. In mammals, male sex determination is initiated by SRY (sex-determining region of the Y chromosome) gene expression and followed by testicular development. This study describes specific down-regulation of the human SRY gene transcription by cAMP stimulation using reverse transcription-polymerase chain reaction experiments. Using transfection experiments, conserved nuclear hormone receptor (NHR1) and Sp1 consensus binding sites were identified as essential for this cAMP transcriptional response. Steroidogenic factor-1 (SF-1), a component of the sex-determination cascade, binds specifically to the NHR1 site and activates the SRY promoter. Activation of SF-1 was abolished by cAMP pretreatment of the cells, suggesting a possible effect of cAMP on the SF-1 protein itself. Indeed, human SF-1 protein contains at least two in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites, leading after phosphorylation to a modification of both DNA-binding activity and interaction with general transcription factors such as Sp1. Taken together, these data suggest that cAMP responsiveness of human SRY promoter involves both SF-1 and Sp1 sites and could act via PKA phosphorylation of the SF-1 protein itself. PMID: 11207191 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Biochim Biophys Acta. 1998 May 11;1397(3):247-52. Characterization of two Sp1 binding sites of the human sex determining SRY promoter. Desclozeaux M, Poulat F, de Santa Barbara P, Soullier S, Jay P, Berta P, Boizet-Bonhoure B. Centre de Recherche de Biochimie Macromoleculaire, ERS155 CNRS, Montpellier, France. To investigate the molecular basis of the human SRY gene regulation, we have examined the significance of two potential binding sites for the transcription factor Sp1 (Sp1A: -124 to -131 and Sp1B: -147 to -154) by DNase I footprinting and gel mobility shift assays. Cotransfection experiments in Drosophila SL2 cells implicated Sp1 protein in the transcriptional activation of the SRY promoter. PMID: 9582429 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Biochem Biophys Res Commun. 1998 Apr 17;245(2):370-7. Identification of conserved potentially regulatory sequences of the SRY gene from 10 different species of mammals. Margarit E, Guillen A, Rebordosa C, Vidal-Taboada J, Sanchez M, Ballesta F, Oliva R. Hospital Clinic, Institut de Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse and from four additional mammalian species, not previously characterized (gorilla, gazelle, rat, and guinea pig). In order to identify conserved DNA elements potentially involved in the regulation of the SRY gene, the newly determined sequences were analyzed and compared to all mammalian SRY promoter sequences available at present. Ten highly conserved potential regulatory elements have been identified in all 10 species (AP1, Barbie, GATA, Gfi1, cMyb, vMyb, NF1, Oct1, Sp1, and SRY). The known function of several of these regulatory elements fits well with the known expression of the SRY gene. However, except for the highly conserved coding HMG motif, only a short region close to the initiation of transcription in the human SRY is conserved in the exact position along the gene in all the species analyzed. This lack of sequence identity at the orthologous positions is consistent with the suggested rapid evolution of the SRY gene. This relative lack of homology contrasts with a high sequence identity of the putative regulatory sequences found within each taxonomic group of species (primates, bovids, and rodents), which supports a common mechanism of SRY expression and possibly also a similar function. PMID: 9571157 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Gene. 1998 Jan 12;206(2):237-45. Structural organization and chromosomal localization of the mouse tesk1 (testis-specific protein kinase 1) gene. Toshima J, Nakagawara K, Mori M, Noda T, Mizuno K. Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812-81, Japan. TESK1 (testis-specific protein kinase 1) is a protein serine-threonine kinase, containing characteristic structural features composed of an N-terminal kinase domain and a C-terminal proline-rich domain. Tesk1 mRNA is predominantly expressed in testicular germ cells, and developmental changes of expression in mouse testis suggest a role for this kinase in spermatogenesis. In the present study, we isolated and determined the overall sequence of the mouse Tesk1 gene, which spans 6.1 kilobases (kb) and contains 10 exons and 9 introns. The protein kinase domain is located in exons 1-9, while the proline-rich domain is in exons 9 and 10. The deduced 627 amino acid sequence of mouse TESK1 shows 97% and 94% identity with the rat and human TESK1, respectively. Sequence of the 5'-flanking and -untranslated region is devoid of a TATA box, but does contain several potential binding sites for transcription factors, including Sp1, AP-1, c-Myc, SRY and CREM (cyclic AMP-responsive element modulator). As CREM is implicated in the activation of several male germ cell-specific genes, it is suggested that the expression of the Tesk1 gene is under the control of CREM transcription activity. The Tesk1 gene was mapped to mouse chromosome 4A5-C1 by fluorescence in situ hybridization. PMID: 9469938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Biol Reprod. 1995 Mar;52(3):591-9. Bovine SRY gene locus: cloning and testicular expression. Daneau I, Houde A, Ethier JF, Lussier JG, Silversides DW. Department of Veterinary Biomedicine Faculty of Veterinary Medicine, University of Montreal, Quebec, Canada. The bovine SRY gene was cloned by a combination of anchored polymerase chain reaction (PCR) amplification of genomic restriction fragments and reverse transcription-PCR (RT-PCR) of testicular RNA. We report 1800 bp of combined genomic and cDNA sequences including 911 bp of 5' upstream sequences, an open reading frame of 687 bp, and 202 bp of sequences corresponding to the 3' end of the mRNA. The bovine SRY gene encodes a deduced (predicted on the basis of a cDNA sequence) protein product of 229 amino acids, with sequence conservation between species, notably in the region of the high-mobility group (HMG) domain or HMG box. Outside of the HMG box, the bovine SRY structure shows greater resemblance to the human SRY than to the mouse Sry. As with human SRY promoter sequences, putative binding sites for Sp1 and for SRY itself are seen in the bovine SRY promoter region. Unlike the human SRY promoter, CAAT and TATA box motifs are present in the bovine sequences. Southern analysis and PCR amplification of male and female bovine genomic DNA show that the described sequences are specific to the Y chromosome. Northern analysis of bull testicular RNA demonstrated low levels of expression of the bovine SRY gene in adult testes with a major poly(A) species at 1.9 kb. RT-PCR amplification of bull testicular RNA revealed multiple sites of polyadenylation, but sequencing showed no consensus polyadenylation signal. PMID: 7538798 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------