1: J Cell Biol. 2004 Mar 1;164(5):747-58. Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate. Smits P, Dy P, Mitra S, Lefebvre V. Dept. of Biomedical Engineering, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., ND-20, Cleveland, OH 44195, USA. Sox5 and Sox6 encode Sry-related transcription factors that redundantly promote early chondroblast differentiation. Using mouse embryos with three or four null alleles of Sox5 and Sox6, we show that they are also essential and redundant in major steps of growth plate chondrocyte differentiation. Sox5 and Sox6 promote the development of a highly proliferating pool of chondroblasts between the epiphyses and metaphyses of future long bones. This pool is the likely cellular source of growth plates. Sox5 and Sox6 permit formation of growth plate columnar zones by keeping chondroblasts proliferating and by delaying chondrocyte prehypertrophy. They allow induction of chondrocyte hypertrophy and permit formation of prehypertrophic and hypertrophic zones by delaying chondrocyte terminal differentiation induced by ossification fronts. They act, at least in part, by down-regulating Ihh signaling, Fgfr3, and Runx2 and by up-regulating Bmp6. In conclusion, Sox5 and Sox6 are needed for the establishment of multilayered growth plates, and thereby for proper and timely development of endochondral bones. PMID: 14993235 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Biochem J. 2003 Jun 15;372(Pt 3):831-9. Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells. Lambertini E, Penolazzi L, Giordano S, Del Senno L, Piva R. Dipartimento di Biochimica e Biologia Molecolare, Universita degli Studi di Ferrara, Via L. Borsari 46, Italy. (O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription activators and co-regulators. PMID: 12659635 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Development. 2003 Mar;130(6):1135-48. Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs. Smits P, Lefebvre V. Department of Biomedical Engineering and Orthopaedic Research Center, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. The notochord has major roles in vertebral column formation: indirectly by inducing sclerotome cell differentiation; and directly by forming the nucleus pulposus of intervertebral discs. Sox5 and Sox6 encode Sry-related HMG box transcription factors that act redundantly to promote chondroblast differentiation in all cartilages of the mouse embryo. We show that Sox5 and Sox6 are expressed in the notochord cell lineage and required for notochord late development. In Sox5(-/-)/Sox6(-/-) embryos, the notochord formed a typical rod-like structure. It fulfilled its inductive functions, as indicated by expression of sonic hedgehog and sclerotome specification. However, the notochord failed to become surrounded with an extracellular matrix sheath. This phenotype was associated with a downregulation of extracellular matrix genes, including the genes for collagen 2, aggrecan and perlecan in both notochord cells and surrounding chondrocytic cells of presumptive inner annuli and vertebral bodies. The mutant notochord then underwent an aberrant, fatal dismantling after sclerotome cell migration. Its cells became removed first from intervertebral spaces and then from vertebral bodies, and it progressively underwent apoptosis. Meanwhile, the development of inner annuli and vertebral bodies was dramatically impaired. Consequently, the vertebral column of Sox5(-/-)/Sox6(-/-) fetuses consisted of a very deficient cartilage and was devoid of nuclei pulposi. In Sox5(-/-)/Sox6(+/-) and more severely in Sox5(+/-)/Sox6(-/-) embryos, the notochord sheath was thinner, but cells survived. By birth, nuclei pulposi were rudimentary, and its cells poorly swelled and still expressing sonic hedgehog. Hence, Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and formation of nuclei pulposi. Through these roles and essential roles in cartilage formation, they are central transcriptional regulators of vertebral column development. PMID: 12571105 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Mol Biol Cell. 2002 Dec;13(12):4179-94. Regulation of IkappaBbeta expression in testis. Budde LM, Wu C, Tilman C, Douglas I, Ghosh S. Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520, USA. IkappaBalpha and IkappaBbeta are regulators of the nuclear factor-kappaB (NF-kappaB) transcription factor family. Both IkappaBs bind to the same NF-kappaB dimers and are widely expressed in different cells and tissues. To better understand how these two IkappaB isoforms differ biologically, we have characterized the expression of IkappaBbeta in testis, a tissue in which IkappaBalpha is only minimally expressed. We have found that IkappaBbeta expression is localized within the haploid spermatid stages of spermatogenesis and follows the expression of nuclear NF-kappaB. IkappaBbeta expression in haploid spermatids is likely regulated by Sox family proteins, members of which are also expressed within spermatids. We have shown that both SRY and Sox-5 can bind to multiple Sox binding sites found within the IkappaBbeta promoter and can enhance transcription of a reporter gene in transient transfection assays. We also demonstrate that IkappaBbeta mRNA is strongly expressed in developing male gonads. These results therefore suggest that IkappaBbeta may be a novel target for transcription factors of the HMG-box SRY/Sox family and imply a potential role for NF-kappaB/IkappaBbeta in spermatogenesis. PMID: 12475944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gene. 2002 Sep 18;298(1):59-68. Identification and characterization of the human long form of Sox5 (L-SOX5) gene. Ikeda T, Zhang J, Chano T, Mabuchi A, Fukuda A, Kawaguchi H, Nakamura K, Ikegawa S. Laboratory for Bone and Joint Diseases, SNP Research Center, RIKEN (The Institute of Physical and Chemical Research), c/o Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. The Sox (Sry-type HMG box) group of transcription factors, which is defined by a high-mobility group (HMG) DNA-binding domain, is categorized into six subfamilies. Sox5 and Sox6 belong to the group D subfamily, which is characterized by conserved N-terminal domains including a leucine-zipper, a coiled-coil domain and a Q-box. Group D Sox genes are expressed as long and short transcripts that exhibit differential expression patterns. In mouse, the long form of Sox5, L-Sox5, is co-expressed and interacts with Sox6; together, these two proteins appear to play a key role in chondrogenesis and myogenesis. In humans, however, only the short form of Sox5 has previously been identified. To gain insight into Sox5 function, we have identified and characterized human L-SOX5. The human L-SOX5 cDNA encodes a 763-amino-acid protein that is 416 residues longer than the short form and contains all of the characteristic motifs of group D Sox proteins. The predicted L-SOX5 protein shares 97% amino acid identity with its mouse counterpart and 59% identity with human SOX6. The L-SOX5 gene contains 18 exons and shows similar genomic structure to SOX6. We have identified two transcription start sites in L-SOX5 and multiple alternatively spliced mRNA variants that are distinct from the short form. Unlike the short form, which shows testis-specific expression, L-SOX5 is expressed in multiple tissues. Like SOX6, L-SOX5 shows strong expression in chondrocytes and striated muscles, indicating a likely role in human cartilage and muscle development. PMID: 12406576 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Dev Biol. 2001 Nov 15;239(2):270-80. Hypaxial muscle migration during primary myogenesis in Xenopus laevis. Martin BL, Harland RM. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA. In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores. Copyright 2001 Academic Press. PMID: 11784034 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Biochem J. 2001 Dec 1;360(Pt 2):461-70. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Stokes DG, Liu G, Dharmavaram R, Hawkins D, Piera-Velazquez S, Jimenez SA. Department of Medicine, Division of Rheumatology, 233 S. 10th Street, Thomas Jefferson University, Philadelphia, PA 19107, USA. During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata. PMID: 11716775 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Dev Cell. 2001 Aug;1(2):277-90. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B, Lefebvre V. Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. L-Sox5 and Sox6 are highly identical Sry-related transcription factors coexpressed in cartilage. Whereas Sox5 and Sox6 single null mice are born with mild skeletal abnormalities, Sox5; Sox6 double null fetuses die with a severe, generalized chondrodysplasia. In these double mutants, chondroblasts poorly differentiate. They express the genes for all essential cartilage extracellular matrix components at low or undetectable levels and initiate proliferation after a long delay. All cartilages are thus extracellular matrix deficient and remain rudimentary. While chondroblasts in the center of cartilages ultimately activate prehypertrophic chondrocyte markers, epiphyseal chondroblasts ectopically activate hypertrophic chondrocyte markers. Thick intramembranous bone collars develop, but the formation of cartilage growth plates and endochondral bones is disrupted. L-Sox5 and Sox6 are thus redundant, potent enhancers of chondroblast functions, thereby essential for endochondral skeleton formation. PMID: 11702786 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Osteoarthritis Cartilage. 2001;9 Suppl A:S69-75. L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Lefebvre V, Behringer RR, de Crombrugghe B. Department of Molecular Genetics, The University of Texas, MD Anderson Cancer Center, Houston 77030, USA. lefebvr@bme.ri.ccf.org OBJECTIVE: This work was carried out to identify transcription factors controlling the differentiation of mesenchymal cells into chondrocytes. DESIGN: We delineated a cartilage-specific enhancer in the collagen type 2 gene (Col2a1) and identified transcription factors responsible for the activity of this enhancer in chondrocytes. We then analyzed the ability of these transcription factors to activate specific genes of the chondrocyte differentiation program and control cartilage formation in vivo. RESULTS: A 48-bp sequence in the first intron of Col2a1 drove gene expression specifically in cartilage in transgenic mouse embryos. The transcription factors L-Sox5, Sox6, and Sox9 bound and cooperatively activated this enhancer in vitro. They belong to the Sry-related family of HMG box DNA-binding proteins, which includes many members implicated in cell fate determination in various lineages. L-Sox5, Sox6, and Sox9 were coexpressed in all precartilaginous condensations in mouse embryos and continued to be expressed in chondrocytes until the cells underwent final hypertrophy. Whereas L-Sox5 and Sox6 are highly homologous proteins, they are totally different from Sox9 outside the HMG box domain. The three proteins cooperatively activated the Col2a1- and aggrecan genes in cultured cells. Heterozygous mutations in SOX9 in humans lead to campomelic dysplasia, a severe and generalized skeletal malformation syndrome. Embryonic cells with a homozygous Sox9 mutation were unable to form cartilage in vivo and activate essential chondrocyte marker genes. Preliminary data indicated that the mutation of Sox5 and Sox6 in the mouse led to severe skeletal malformations. CONCLUSIONS: L-Sox5, Sox6, and Sox9 play essential roles in chondrocyte differentiation and, thereby, in cartilage formation. Their discovery will help to understand further the molecular mechanisms controlling chondrogenesis in vivo, uncover genetic mechanisms underlying cartilage diseases, and develop novel strategies for cartilage repair. PMID: 11680692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mol Pharmacol. 2001 Jun;59(6):1486-96. Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18. Im HJ, Smirnov D, Yuhi T, Raghavan S, Olsson JE, Muscat GE, Koopman P, Loh HH. Department of Pharmacology, The University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455, USA. imh@tc.umn.edu Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu-opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer. PMID: 11353810 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Gene. 2001 Jan 10;262(1-2):239-47. Cloning and functional analysis of the Sry-related HMG box gene, Sox18. Hosking BM, Wyeth JR, Pennisi DJ, Wang SC, Koopman P, Muscat GE. University of Queensland, Institute for Molecular Bioscience, Centre for Molecular and Cellular Biology, Ritchie Research Laboratories, B402A, St Lucia, 4072, Queensland, Australia. The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. PMID: 11179689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Mol Evol. 1999 May;48(5):517-27. Diversification pattern of the HMG and SOX family members during evolution. Soullier S, Jay P, Poulat F, Vanacker JM, Berta P, Laudet V. ERS155 du CNRS, Centre de Recherche en Biochimie Macromoleculaire, CNRS, BP5051, Route de Mende, 34293 Montpellier Cedex 5, France. From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. PMID: 10198118 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: J Biol Chem. 1999 Apr 2;274(14):9327-34. Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins. Blaise R, Grober J, Rouet P, Tavernier G, Daegelen D, Langin D. INSERM Unit 317, Institut Louis Bugnard, Universite Paul Sabatier, Hopital Rangueil, F-31403 Toulouse Cedex 4, France. The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL. PMID: 10092610 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Nucleic Acids Res. 1999 Mar 1;27(5):1359-64. The DNA-binding specificity of SOX9 and other SOX proteins. Mertin S, McDowall SG, Harley VR. The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. SOX (SRY-related HMG box) proteins are transcription factors that have critical roles in the regulation of numerous developmental processes. They share at least 50% homology in their HMG domains, which bind the DNA element AACAAT. How different SOX proteins achieve specific regulation of target genes is not known. We determined the DNA-binding specificity of SOX9 using a random oligonucleotide selection assay. The optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA-binding element AACAAT, flanked by 5' AG and 3' GG nucleotides. The specific interaction between SOX9 and AGAACAATGG was confirmed by mobility shift assays, DNA competition and dissociation studies. The 5' AG and 3' GG flanking nucleotides enhance binding by SOX9 HMG domain, but not by the HMG domain of another SOX factor, SRY. For SRY, different 5' and 3' flanking nucleotides are preferred. Our studies support the notion that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains. Furthermore, the related HMG domains of SOX9 and Sox17 have similar optimal binding sites that differ from those of SRY and Sox5, suggesting that SOX factors may co-evolve with their DNA targets to achieve specificity. PMID: 9973626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Genomics. 1996 Sep 1;36(2):354-8. Cloning and characterization of SOX5, a new member of the human SOX gene family. Wunderle VM, Critcher R, Ashworth A, Goodfellow PN. Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, United Kingdom. The mammalian Y-linked testis determining gene, SRY, encodes a protein with a DNA binding motif known as the HMG box. A large family of genes sharing a high similarity with the SRY HMG box and named Sox (Sry-related HMG box) in mouse and SOX in human has been identified from various organisms. We have cloned SOX5, a new member of the human SOX gene family. SOX5 cDNAs isolated from a human adult testis cDNA library show a high similarity with the mouse Sox5 transcript over a large region identical in all the human cDNAs. However, comparison of the 5' unique sequences of the cDNAs suggests that the SOX5 gene is subject to alternative splicing. Genomic analysis identified a SOX5 pseudogene located on 8q21.1, whereas the SOX5 gene itself, which contains a minimum of five introns, maps to 12p12.1. In contrast to the mouse gene, the human SOX5 gene is expressed in a variety of human tissues, and different size transcripts are observed in adult testis and fetal brain. PMID: 8812465 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Mol Med. 1996 Jul;2(4):405-12. SOX genes: architects of development. Prior HM, Walter MA. Ocular Genetics Research Group, University of Alberta, Edmonton, Canada. Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease. Publication Types: Review Review, Tutorial PMID: 8827711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Nucleic Acids Res. 1995 Sep 11;23(17):3365-72. The Sry-related HMG box-containing gene Sox6 is expressed in the adult testis and developing nervous system of the mouse. Connor F, Wright E, Denny P, Koopman P, Ashworth A. CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, London, UK. We have cloned and sequenced a full-length cDNA for the HMG box-containing, SRY-related gene Sox6 from mouse. The deduced protein sequence of Sox6 has considerable homology with that of the previously determined Sox5 sequence. It seems likely that these genes have diverged more recently than other members of the SOX gene family, although the two genes map to different chromosomes in the mouse. In common with Sox5, Sox6 is highly expressed in the adult mouse testis and the HMG domains of both proteins bind to the sequence 5'-AACAAT-3'. This suggests that the two genes may have overlapping functions in the regulation of gene expression during spermatogenesis in the adult mouse. However, Sox6 may have an additional role in the mouse embryo, where it is specifically expressed in the developing nervous system. PMID: 7567444 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Protein Eng. 1995 Jul;8(7):615-25. An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein. Adzhubei AA, Laughton CA, Neidle S. CRC Biomolecular Structure Unit, Institute of Cancer Research, Sutton, Surrey, UK. A new approach has been developed to reduce multiple protein structures obtained from NMR structure analysis to a smaller number of representative structures which still reflect the structural diversity of the data sets. The method, based on the clustering of similar structures, has been tested in the homology model building of the structure of Sox-5, a sequence-specific DNA-binding protein belonging to the high mobility group (HMG) nuclear proteins family. Sox (SRY box) genes are the autosomal genes related to the sex-determining SRY, Y chromosomal gene. The Sox-5 protein, encoded by one of the SRY-related genes, displays a 29% sequence identity with the HMG1 B-box domain whose structure, determined previously by NMR, has been used in our study to predict the structure of Sox-5. Two independent ensembles of HMG1 structures, each represented by closely related coordinate sets, were used. Nine representative structures for HMG1 were subsequently selected as starting points for the modelling of Sox-5. The model of the protein shows close similarity to the HMG1 fold, with differences at the secondary structure level located mainly in alpha-helices 1 and 3. A left-handed, three residue per turn polyproline II helix, forming a conserved polyproline II/alpha-helix supersecondary motif, was identified in the N-terminal region of Sox-5 and other HMG boxes. PMID: 8577691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Nucleic Acids Res. 1994 Aug 25;22(16):3339-46. DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5. Connor F, Cary PD, Read CM, Preston NS, Driscoll PC, Denny P, Crane-Robinson C, Ashworth A. CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London, UK. Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2. PMID: 8078769 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------