Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene

Abstract

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Toxicogenomic Responses of Zebrafish Embryos/Larvae to Tris(1,3-dichloro-2-propyl) Phosphate (TDCPP) Reveal Possible Molecular Mechanisms of Developmental Toxicity

“…Matrilin 1 is expressed in chondrocytes in a developmental stage-specific manner in vertebrates. 32,33 Treatment with TDCPP significantly increased the expression of matrilin 1 precursor, suggesting disruption in chondrogenesis. Collectedly, we speculated that larval malformation (trunk curvature) resulted from abnormal development of somites, fast muscle, and cartilage after TDCPP exposure.…”

SOX9 overexpression plays a potential role in idiopathic congenital talipes equinovarus

“…Specific in vitro and in vivo studies have revealed that the SRY (sex-determining region Y)-box 9 (SOX9) transcription factor binds to consensus sequence pairs in the upstream region of the COL2A1 and COL9A1 genes and regulates expression (10)(11)(12)(13)(14)(15)(16). SOX9 is a protein of the HMG-box DNA-binding protein class, which recognize the CCTTGAG sequence.…”

A matrilin-1 gén transzkripciós szabályozásának vizsgálata transzgenikus egerekben

“…A Sox9 az E csoport tagjaként rendelkezik transzaktivációs doménnel, transzkripciós faktorként direkt szerepe van több porc mátrix fehérjét kódoló gén, többek között a matrilin-1 gén szabályozásában is [80].…”

“…A rövid (-334/+67), illetve a teljes hosszúságú (-2011/+67) matrilin-1 promoter által irányított luciferáz riporter gént tartalmazó konstrukciók (FO15Luc és AC8Luc) már rendelkezésünkre álltak [80].…”

“…Mivel a magas aktivitású konstrukciók mindegyike tartalmazta a rövid promotert (-334/+67), valószínőnek látszott, hogy ez a szakasz tartalmazza azokat a fı szabályozó elemeket, melyek a gén szövet-és fejlıdési állapotspecifikus expresszióját irányítják, illetve a távoli elemek hatását közvetítik. Ezek azonosítására munkatársaim in silico analízist végeztek, melynek során a rövid promoteren belül több, amniotákban különbözı mértékben konzervált szekvenciát találtak [80]. Ezek közül a legnagyobb mértékő konzerváltságot egy proximális promoter elem (Pe1) mutatta, amely egy pár, egymással ellentétes irányban elhelyezkedı motívumot tartalmaz, mely jelentıs mértékő homológiát …”