The zonal expression of chicken cartilage matrix protein gene in the developing skeleton of transgenic mice.

Cartilage matrix protein (CMP) is a major noncollagenous glycoprotein of hyaline cartilage with a molecular mass of about 148 kDa. It has been proposed to be involved in matrix organization by its interactions with proteoglycan and type II collagen. The 54-kDa monomers form homotrimers stabilized by disulfide bonds. The gene for chicken cartilage matrix protein was isolated, and its regulation has been studied recently in transient expression experiments. To learn more about the spatial and temporal expression of the gene during ontogenic development, we created transgenic mice via microinjection of a 21.8-kb genomic fragment, encoding the chicken cartilage matrix protein. None of the founder animals exhibited any abnormal phenotype. The developmental stage-specific expression of the transgene was examined by immunostaining with a chicken CMP specific antiserum at different stages of embryonic development in cartilage from different sources: lower and upper limb, vertebrae, ribs and nasal septum. The level of transgene expression showed marked differences in various zones of cartilage. Briefly, high levels were found in the zones of proliferating chondrocytes, while little if any transgene product was detected in the very early and hypertrophic stage of chondrogenesis. The expression pattern of the transgene correlated with the endogenous mouse CMP and did not cause any morphological changes detectable by microscopic analysis of cartilage. These data indicate that the injected CMP gene with its flanking sequences contained all the information necessary for cell type-specific expression in transgenic mice.

Identification of a nuclear factor-I family protein-binding site in the silencer region of the cartilage matrix protein gene.

Cartilage matrix protein (CMP) is synthesized by chondrocytes in a developmentally regulated manner. Here we have dissected promoter upstream elements involved in its transcriptional regulation. We show that although the 79-base pair CMP minimal promoter is promiscuous, 1137 base pairs of 5'-flanking region are capable of directing tissue- and developmental stage-specific transcription when fused to a reporter gene. This results from two positive control regions which, in proliferating chondrocytes, relieve the repression mediated by two non-tissue-specific negative control regions. Characterization of the promoter proximal silencer by DNase I footprinting and gel shifts revealed the presence of two elements, SI and SII, which bound mesenchymal cell proteins. Methylation interference analysis indicated a gapped palindromic binding site similar to nuclear factor I (NF-I) family proteins within SI, but only a half-site within SII. Gel shift assays with specific NF-I and mutated SI competitors, binding of recombinant NF-I, as well as supershift analysis with NF-I-specific antiserum verified the binding of NF-I family proteins to the SI element. Double-stranded SI and SII oligonucleotides inserted in single copy in either orientation were found to repress both homologous and heterologous promoters upon transfection into mesenchymal cells. Transcriptional repression also occurred when a consensus NF-I site itself was fused to the CMP minimal promoter. We conclude that NF-I-related protein(s) can mediate transcriptional repression in cells of mesenchymal origin.