The Transcription Factor CCAAT-binding Factor CBF/NF-Y Regulates the Proximal Promoter Activity in the Human α1(XI) Collagen Gene (COL11A1)

Abstract

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...

Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation

“…A second transcription factor reported to interact with the Col11a1 promoter is CBF/NF-Y (Matsuo et al, 2003). This transcription factor binds to the Col11a1 promoter within the -147 to -121 region the and activates transcription of promoter (Matsuo et al, 2003).…”

“…This transcription factor binds to the Col11a1 promoter within the -147 to -121 region the and activates transcription of promoter (Matsuo et al, 2003). At this time, there is no evidence to suggest that Lef1 could be activating the Col11a1 promoter indirectly through CBF/NF-Y.…”

“…The luciferase reporter construct (pCol11a1(-1454)-luc was kindly provided by Dr. Francesco Ramirez (Kinoshita et al, 1997;Matsuo et al, 2003). Dr. Liliana Attisano provided pCMV5B-Lef1-HA and pCMV5B-Lef1∆HMG-HA.…”

The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)

“…All mutagenesis plasmids were digested with SacI and HindIII and re-cloned into the SacI/ HindIII site of pGL3-Basic vector. Construction of the dominant negative CBF-B/NF-YA was generated by RT-PCR as previously described (26,29).…”

“…Lee and Greenspan (25) suggested that the GAGA boxes in the promoter and the first exon of the COL5A1 gene affect the transcription. We have recently characterized the proximal promoter of the COL11A1 (26) and Col5a1 genes (27). In these studies, we demonstrated that the CCAAT binding factor, CBF/ NF-Y, bound to the CCAAT motif in the proximal promoter and is involved in activation of the genes.…”