Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.
ABSTRACT Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.
Article: Activated beta-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction.[show abstract] [hide abstract]
ABSTRACT: Wnt glycoproteins are important regulators of cellular differentiation and embryonic development. Some Wnt proteins induce stabilization of beta-catenin which cooperatively regulates gene expression with LEF/Tcf transcription factors. Here we demonstrate a direct role for beta-catenin signaling in osteoblast differentiation and in BMP2-mediated signal transduction. Similar to treatment with BMP-2 protein, ectopic expression of stabilized beta-catenin in C3H10T1/2 cells or activation of endogenous beta-catenin signaling with LiCl induces expression of alkaline phosphatase mRNA and protein, a defined marker of early osteoblast differentiation. Unlike BMP2 protein, stabilized beta-catenin does not induce osteocalcin gene expression, a marker of late osteoblast differentiation. BMP2-induced differentiation also leads to activation of endogenous beta-catenin signaling thus implicating beta-catenin in early steps of BMP2-mediated osteoblast differentiation. Effects of beta-catenin and BMP2 on C3H10T1/2 differentiation are not completely overlapping, implying that some aspects of BMP2-induced differentiation may be mediated by beta-catenin signaling and that beta-catenin can also participate in non-BMP2-dependent differentiation processes.Biochemical and Biophysical Research Communications 02/2003; 301(1):84-91. · 2.48 Impact Factor
Article: Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9.[show abstract] [hide abstract]
ABSTRACT: To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate.Molecular and cellular biology 02/2011; 31(4):686-99. · 6.06 Impact Factor
Article: SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression.[show abstract] [hide abstract]
ABSTRACT: Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between these opposing modes of transcriptional control by SOX9 may be mediated by cooperation with different partners such as GLI factors.PLoS Genetics 11/2011; 7(11):e1002356. · 8.69 Impact Factor