A Zinc Finger Transcription Factor, αA-Crystallin Binding Protein 1, Is a Negative Regulator of the Chondrocyte-Specific Enhancer of the α1(II) Collagen Gene

Abstract

Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, ␣A-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the mouse ␣A-crystallin gene promoter, was isolated. We herein demonstrate that CRYBP1 is involved in the negative regulation of Col2a1 enhancer activity. CRYBP1 mRNA expression was downregulated during chondrocyte differentiation in vitro. In situ hybridization analysis of developing mouse cartilage showed that CRYBP1 mRNA was also downregulated during mesenchymal condensation and that CRYBP1 mRNA was highly expressed by hypertrophic chondrocytes, but at very low levels by resting and proliferating chondrocytes. Expression of recombinant CRYBP1 in a transfected rat chondrosarcoma cell line inhibited Col2a1 enhancer activity. Electrophoretic mobility shift assays showed that CRYBP1 bound a specific sequence within the Col2a1 enhancer and inhibited the binding of Sox9, an activator for Col2a1, to the enhancer. Cotransfection of CRYBP1 with Sox9 into BALB/c 3T3 cells inhibited activation of the Col2a1 enhancer by Sox9. These results suggest a novel mechanism that negatively regulates cartilagespecific expression of Col2a1.Cartilage serves as the template for the growth and development of most bones. It contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. Cartilage development is initiated by mesenchymal cell condensation, followed by a series of chondrocyte maturation processes, including resting, proliferative, and hypertrophic chondrocytes. Type II collagen, a homotrimer of the ␣1(II) chain (Col2a1), is a major extracellular matrix protein in cartilage. It forms collagen fibrils and provides a structural framework for cartilage matrix. Type II collagen is synthesized primarily by proliferating chondrocytes but not by hypertrophic chondrocytes (9). Disruption of Col2a1 expression leads to degenerative joint disorders and a variety of chondrodysplasias (24), suggesting that fidelity of type II collagen expression is essential for maintaining normal cartilage structure and function. Transcriptional regulation of Col2a1 is mediated by tissue-specific regulatory elements located within the promoter and first intron. We have shown that a 100-bp segment within the first intron is the minimal sequence sufficient for high-level, cell type-specific expression of Col2a1 (23). Recent reports suggest that Sox9, a member of the transcription factor family with a high-mobility-group-type DNA binding domain homologous to that of SRY (16,45,46

Mouse Snail Family Transcription Repressors Regulate Chondrocyte, Extracellular Matrix, Type II Collagen, and Aggrecan

“…During hypertrophic differentiation, the extracellular matrix self-organizes prior to the formation of bone. A switch from collagen type II to collagen type X occurs, and both aggrecan and link protein disappear (1,2). Studies in stable transformants revealed that overexpression of Snail and Slug prevented the expression of major chondrocyte extracellular matrix, collagen type II, and aggrecan when transfected cells were induced to differentiate.…”

“…Type II collagen and aggrecan are major components of the cartilage extracellular matrix produced by proliferating and prehypertrophic chondrocytes, and type X collagen is a major collagen type produced by hypertrophic chondrocytes. The differentiation into hypertrophic chondrocytes is accompanied with changes in ECM; switching of collagen types from type II to type X and the disappearance of aggrecan and link protein (1,2). Dysfunction of these extracellular macromolecules in mutant mice results in striking skeletal malformation, suggesting their important roles in skeletal formation (3)(4)(5)(6)(7).…”

Inhibition of NF-κB by ZAS3, a zinc-finger protein that also binds to the κB motif

“…Members of the ZAS family have been shown to inhibit transcription. ZAS1 negatively regulates the ␣1(II) collagen gene (27), whereas ZAS2 represses c-myc transcription from the major P2 promoter (28).…”

Regulation of Human COL9A1 Gene Expression

“…The best studied gene in this regard is the COL2A1 gene, which gives rise to the main fibrillar collagen in the cartilage matrix. The expression of the COL2A1 gene is controlled through transcription factors that interact with both the promoter and the chondrocyte-specific enhancer located within the first intron (1)(2)(3)(4). Recent work has shown that both positive and negative factors interact with the COL2A1 gene to regulate its expression (2)(3)(4)(5)(6)(7)(8).…”

“…The expression of the COL2A1 gene is controlled through transcription factors that interact with both the promoter and the chondrocyte-specific enhancer located within the first intron (1)(2)(3)(4). Recent work has shown that both positive and negative factors interact with the COL2A1 gene to regulate its expression (2)(3)(4)(5)(6)(7)(8). Positive regulation of the COL2A1 gene during chondrocyte differentiation is afforded by the interaction of members of the Sry-type HMG box (SOX) 1 family of transcription factors with a specific region of the intronic enhancer (5)(6)(7)(8).…”