295 Regulation of the expression and activity by progestins of a member of the SOX gene family of transcriptional modulators J D Graham,SMNHunt, N Tran and C L Clarke Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia (Requests for offprints should be addressed to C Clarke, Department of Medical Oncology, Westmead Hospital, Westmead, New South Wales 2145, Australia) (J D Graham is now at Department of Medicine, University of Colorado Health Sciences Centre, 4200 E. 9th Avenue, Campus Box B151, Denver, Colorado, USA) ABSTRACT The mammalian testis-determining gene Sry and progestin to increase SOX mRNA expression was the related Sox genes define a family of transcrip- confined to SOX4. The increase in SOX4 transcrip- tional regulators widely expressed during embryo- tion was reflected in increased SOX4 protein genesis. Tightly controlled temporal profiles of expression, as progestin treatment of T-47D cells expression are a feature of the Sox gene family and transfected with a SOX-responsive reporter may be required for initiation of a cascade of gene resulted in a marked increase in reporter gene expression, yet the molecular mechanisms that expression. Progesterone is essential for normal control Sox gene expression are unknown. We now development and differentiation of the female show that human SOX4 is expressed in the normal reproductive system, plays an essential role in breast and in breast cancer cells. In these cells regulating growth and differentiation of the mam- SOX4 is a progesterone-regulated gene, the mary gland and is required for opposing the expression of which is increased by progestins, proliferative effects of estrogen in specific cell types. leading to a marked increase in SOX-mediated The detection of SOX4 expression in the normal transcriptional activity. Treatment of T-47D breast and malignant breast and the demonstration that cancer cells with the synthetic progestin ORG 2058 SOX4 expression is under progesterone control directly increased SOX4 transcription, resulting in suggests that changes in SOX4 gene expression may a 4-fold increase in SOX4 mRNA levels within 4 h play a role in commitment to the differentiated of treatment. No effect of ORG 2058 was noted on phenotype in the normal and malignant mammary other SOX genes measured, nor were other gland. hormone-regulated HMG box proteins detected in Journal of Molecular Endocrinology (1999) 22, 295–304 this system, suggesting that the observed ability of INTRODUCTION et al. 1992, van de Wetering & Clevers 1992, Laudet et al. 1993, van de Wetering et al. 1993, Connor The SOX4 gene is a member of a family of et al. 1995, Hosking et al. 1995). Interaction of transcription regulators which all contain an HMG box proteins with this motif results in HMG box DNA-binding domain, and interact with bending of the DNA, suggesting that transacti- DNA in a sequence-specific fashion (Pevny & vation by these factors may occur by facilitating Lovell-Badge 1997). Many SRY-related HMG box protein–protein or protein–DNA interactions, (SOX) genes have now been identified in the bringing normally distal components into proximity human, as well as other species, on the basis of their with each other (Ferrari et al. 1992). Although it is homology in this region. Where analysed, all have still not possible to ascribe functions to all members been shown to bind specifically to the double- of the Sox gene family, there is increasing evidence stranded DNA motif, A/TA/TCAAA/TG (Denny that Sox proteins are likely to be involved in many Journal of Molecular Endocrinology (1999) 22, 295–304 Online version via http://www.endocrinology.org 0952–5041/99/022–295 1999 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 02:05:22PM via free access 296 and others · Progestin regulation of SOX4 expression and activity aspects of transcriptional regulation (Pevny & processes occurring both in embryogenesis and in Lovell-Badge 1997). Their role as transcriptional the mature animal. regulators, combined with their limited tissue Although murine Sox4 is expressed in the ovary distribution and diverse patterns of distribution and testis, which are steroid hormone-responsive during embryogenesis, suggest that members of the tissues, there is no information on steroidal Sox gene family play a pivotal role in cell fate regulation of SOX4 gene expression. SOX4 was determination in a wide range of developmental identified in searching for genes that were early processes (Pevny & Lovell-Badge 1997). More targets of progestin action: human SOX4 was recent evidence has implicated aberrant expression detected in the progesterone receptor (PR)-positive of Sox genes in malignancy: SOX21 expression has T-47D breast cancer cell line as a progestin- been associated with metastatic potential in regulated gene. Progestin regulation of SOX4 may melanoma cell lines (Tani et al. 1997), and aberrant represent an intermediate step in downstream expression of SOX4 in colon carcinoma has been progestin effects in the normal and malignant implicated in regulation of the expression of p56lck, breast. a member of the src family of tyrosine kinases (McCracken et al. 1997). The Sox4 gene, first isolated from a mouse MATERIALS AND METHODS embryo cDNA library as the Sry-related gene a4 (Gubbay et al. 1990), is specifically expressed in the Materials ovary, testis and thymus of adult mice and in mouse The general chemicals used were of molecular T and pre-B lymphocytic cell lines (van de biological or analytical reagent grade, as required by Wetering et al. 1993). Sox4 has transactivating specific methodology, and were from the same capacity when cotransfected into Sox4-negative T sources as previously listed (Clarke et al. 1990). cells with a reporter plasmid containing seven Radiochemicals, ORG 2058, Moloney murine copies of the AACAAAG motif. It was found that leukaemia virus reverse transcriptase, Taq DNA binding of at least two Sox4 transactivation domains polymerase and Megaprime DNA labelling system was required to see this effect in the absence of other were obtained from Amersham Australia (North enhancer elements, suggesting that Sox4 may Ryde, Sydney, Australia). The pSG5-hPR1 plasmid cooperate with other transcription factors to exert was a gift from Dr P Chambon (Strasbourg, France) tissue-specific control of gene expression (van de and has been described previously (Kastner et al. Wetering et al. 1993). Evidence is now emerging 1990). Oestradiol-17â was a Sigma (St Louis, MO, that cooperative function between Sox proteins USA) product. ICI 182 780 was a gift from Dr Alan and other transcription factors may be a general Wakeling, Zeneca (Macclesfield, UK) and RU feature: Sox2 cooperates with one of the POU 38486 was a gift from Dr J P Raynaud, domain factors, Oct-4, in the regulation of Roussel-Uclaf (Romainville, France). The osteopontin expression in preimplantation develop- pBL2CAT-[SOX4]4 plasmid containing four ment (Botquin et al. 1998). Sox2 is also involved SOX4-binding motifs (AACAAAG) upstream of in transcriptional regulation of the FGF-4 gene, in the thymidine kinase promoter and chlorampheni- cooperation with Oct-3 and Sp1/Sp3 (Lamb col acetyltransferase (CAT) sequences was a gift & Rizzino 1998). Sox11 and Sox4 cooperate from Dr G E O Muscat, University of Queensland with another POU protein, Brn-1, in glial cells (Brisbane, Australia). The following oligonucleotide (Kuhlbrodt et al. 1998). Sox4 cooperates also with sequences were identified from the GenBank DNA the transcription factor Ets-1 in regulating the sequence database and were obtained from expression of p56lck (McCracken et al. 1997). Bresatec (Adelaide, SA, Australia) or from Pacific During embryogenesis Sox4 is essential for Oligos (Lismore, NSW, Australia) and used as normal cardiac development. Transgenic mice probes or PCR primers as described in the text: harbouring a homozygous mutation in Sox4 die SOX4F1, 5*-AGGAGTTGAGGGGCCAGTTC-3*; during embryogenesis because of a failure in SOX4R1, 3*-CACTGCCACCGACCTTGTCTC-5*; endocardial ridge development which prevents SOX2R, 3*-CACATGTGTGAGAGGGGCAGT proper formation of semilunar valves, resulting in GTG-5* (nucleotides 942–965); SOX3R, 3*-CAGT circulatory failure (Schilham et al. 1996). Further- CTCCAGAAGGCTGTACATTG-5* (nucleotides more, introduction of bone marrow cells from these 665–688); SOX11R, 3*-GTTGCTCTCCGCTTC animals into an irradiated host results in a block in CAAGCTCTC-5* (nucleotides 91–114); mSox21F1, B-cell lineage progression at the stage of pro-B- 5*-CTCAGGCTTGGCAGTGGA-3* (nucleotides lymphocyte expansion (Schilham et al. 1996). This 2363–2380); mSox21R1, 3*-AGGCTGGGTGGA indicates that Sox4 is involved in developmental TTGCCT-5* (nucleotides 2982–2999); SOX22F1, Journal of Molecular Endocrinology (1999) 22, 295–304 Downloaded from Bioscientifica.com at 09/27/2021 02:05:22PM via free access Progestin regulation of SOX4 expression and activity · and others 297 5*-TGTAGAGCGATCCTGGGA-3* (nucleotides Adifferentially displayed band of 99 bp was 1894–1911); SOX22R1, 3*-CAGTCTCACTGGC excised and reamplified using the same PCR CAAGT-5* (nucleotides 2486–2503). The plasmid conditions as above except that the dNTP concen- pSox9·5a (Wright et al. 1995) containing a 500 bp tration in the reaction was increased 10-fold to fragment of Sox9 cDNA was a gift from Dr P 20 µM. The reamplified PCR products were Koopman, University of Queensland. A partial inserted into the pGEM-T vector