We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from ؊199 to ؉1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the ؊147 to ؊121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Ybinding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.The collagen superfamily, one of the extracellular matrix proteins, plays an important role, not only in stabilizing the tissues as structural components but also in regulating a variety of biological functions, such as development, differentiation, proliferation, and morphogenesis (1-3). Among them, types I, II, III, V, and XI collagens are included in the group of fibril-forming collagens, based on their structural and functional features, and are divided into two subgroups, major (I, II and III) and minor (V and XI) fibrillar collagen (4) on the basis of their contents in tissues.Type XI collagen is a component of the collagen fibrillar network found in cartilage (5), and consists of three genetically distinct polypeptide chains: ␣1(XI), ␣2(XI), and ␣3(XI); the last of these is thought to be overglycosylated ␣1(II) chains (6).Although type XI collagen is a relatively minor collagen and is buried within the major collagen fibrils, it is important for the regulation of fibril diameter (7). Chondrodysplasia mice (cho), which do not synthesize ␣1(XI) chains, show irregular collagen fibrils in their cartilage (8). Contrary to the previous findings, the ␣1(XI) chain is not restricted to cartilage (9 -14), and it was demonstrated that the ␣1(XI) chain could form a heterotrimer with the ␣2(V) chain in a 2:1 ratio in non-cartilaginous cells and tissues (15,16). Although the precise function of this crosstype trimer remains unclear, the ␣1(XI) collagen gene is more broadly expressed than other collagen genes, showing that the ␣1(XI) collagen gene is the sole collagen gene to be expressed in both cartilaginous and non-cartilaginous tissues. This implies that the regulation of the ␣1(XI) collagen gene might be more complex than expected.We have previously re...

To elucidate the mechanisms underlining ␣3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the ؊129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNAprotein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to ؊122 to ؊117, the ؊101 to ؊96, the ؊83 to ؊78, and the ؊68 to ؊57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies Vertebrate collagens, a large family of extracellular proteins, are critically important for the formation and function of virtually every organ system (1). Among them, fibrillar collagen, which includes five different molecular types I, II, III, V, and XI, participates in the formation of fibrils with molecules packed in quarter-staggered arrays (2, 3). The fibrillar collagens are divided into major types (I-III) and minor types (V and XI) based on their relative expression levels. Minor fibrillar collagen types V and XI are incorporated into the fibrils of the much more abundant collagen types I and II, respectively, and act as regulators of the sizes and shapes of the resultant heterotypic fibrils (4 -7). The collagen molecules are either homotrimers with ␣ chains or heterotrimers with two or three different ␣ chains. The predominant molecular form of type V is the heterotrimer [␣1(V)] 2 ␣2(V) and is expressed in most tissues (8). Other forms of type V collagen include the [␣1(V)] 3 homotrimer that is synthesized in cultures of hamster lung cells (9) and the ␣1(V)␣2(V)␣3(V) heterotrimer that can be extracted from the placenta (10 -12). Cross-type heterotrimer composed of ␣2(V) and ␣1(XI) chains is present in the rhabdomyosarcoma cell line A204 and bovine vitreous (13,14). These findings suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner. Recently, a fourth chain, ␣4(V), expressed in rat Schwann cells was reported (15). This ␣ chain, which can form molecules with ␣1(V) and ␣2(V) chains, seems to be the counterpart of the mouse and human ␣3(V) chain (16).Type V collagen, which is widely expressed spatially and temporally, is expressed in the mouse embryo as early as 11 days post-coitum (17). Altered production of type V collagen is associated with some connective tissue pathology, such as inflammation, some forms of cancer, and atherosclerosis (18 -20). Characterization of the cis-acting elements and trans-acting nuclear factors that modulate correct patterns of gene expression is necessary for understanding physiological and pathological conditions. Among the collag...