Gene Regulation And Function Of Cartilage

Dental &Craniofacial Research

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Abstract

This project covers not only cartilage but also tooth and craniofacial development. Cartilage is a highly specialized connective tissue with distinct morphological and biochemical characteristics. Cartilage contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. When cartilage formation is impaired, skeletal malformation of the limbs, craniofacial bones, and appendicular skeleton occurs. Chondrocytes are the major cell type in cartilage and produce large amounts of cartilage-specific matrix molecules, such as type XI and type II collagen, and aggrecan. Cartilage formation is initiated by mesenchymal cell condensation to form primordial cartilage followed by chondrocyte differentiation. These include resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As a final step in endochondral bone formation, hypertrophic cartilage is invaded by blood vessels and osteoprogenitor cells, and the calcified cartilage is subsequently replaced by bone. Thus, spatial and temporal regulation of chondrocyte differentiation is essential in determining the length and width of skeletal components. Hormones and vitamins affect chondrocyte differentiation and maturation by regulating the transcription of genes. Our objective is to define the mechanisms for activating and repressing chondrocyte-specific genes and to elucidate the molecular basis of cartilage development and chondrocyte differentiation. We previously described the identification of a 24 bp sequence in the alpha 2 type XI collagen gene (Col11a2) promoter, which is able to switch promoter activity from neuronal tissues to cartilage in transgenic mice. We subsequently isolated a protein factor we named NT2, which bound to the 24 bp sequence. NT2 is a C2H2-type zinc finger protein consisting of a Kruppel-associated box (KRAB), a potent DNA-binding-dependent repression module. We found that NT2 functions as a negative regulator of Col11a2. We demonstrated by chromatin immunoprecipitation assays that NT2 forms a complex of these heterochromatin- associated proteins at the 24 bp region of the Col11a2 promoter in NIH3T3 cells, in which Col11a2 is repressed but NT2 is expressed. These results suggest that repression of Col11a2 in NIH3T3 cells is caused by heterochromatin formation. The heterochromatin can be nucleated by the 24 bp sequence, which is recognized by the zinc finger domain of NT2. The KRAB domain of NT2 then recruits KAP1 via the KRAB domain followed by the association of other proteins including histone-modifying enzymes. Our results suggest the importance of both positive and negative transcriptional gene regulation for cartilage gene expression during chondrogenesis. In our the Oral and Craniofacial Genome Anatomy Project (OC-GAP), our goal is to discover and characterize previously unknown genes to help understand how tooth and craniofacial tissues develop and to define the molecular defects underlying anomalies of these tissues or oral cancer. Craniofacial birth defects with anomalies of the mouth, neck, and head are of major public concern. We identified a cDNA clone for epiprofin, which is preferentially expressed in the tooth, by differential hybridization using DNA microarrays from an E19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Kruppel-Like Factor (KLF) family containing three characteristic C2H2-type zinc-finger motifs. The SP/KLF family consists of more than 17 proteins in mice, and has unique features including a DNA-binding domain with three tandem C2H2-type (Kruppel-like) zinc-finger motifs at the C-terminus and a transcriptional regulatory domain at the N-terminus. Except for its 5? terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of KLF14/SP6, whhhich was previously identified in EST databases and GenBank by a zinc-finger DNA-binding domain search. This sequence difference is due to differences in the assignment oof the location of exon 1. Epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblasts, and also hair follicle matrix epithelium. In addition, epiprofin mRNA is transiently expressed in cells of the apical ectodermal ridge (AER) in developing limb. We found by transfection of an epiprofin expression vector that epiprofin is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein, expressed by proliferating epithelial cells of tooth, hair follicle, and limb that may function in the development of these tissues by regulating cell growth. Because epiprofin is expressed primarily in tissues of ectodermal origin, ectodermal dysplasia may be a candidate human genetic disorder caused by mutations of the epiprofin gene.