Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.
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.
Rentsendorj
Otgonchimeg
O
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt 62, H-6726 Szeged, Hungary.
Nagy
Andrea
A
Sinkó
Ildikó
I
Daraba
Andreea
A
Barta
Endre
E
Kiss
Ibolya
I
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D019902:Chondrocytes / Q000378:metabolism; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D064235:Matrilin Proteins; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry; D055758:SOX9 Transcription Factor; D055755:SOXD Transcription Factors; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; C412319:ETV3 protein, mouse; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D006609:High Mobility Group Proteins; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D055758:SOX9 Transcription Factor; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse; C095939:Sox6 protein, mouse; C527933:Sox9 protein, mouse; D014157:Transcription Factors