An AP-1-like motif in the first intron of human Pro?1(I) collagen gene is a critical determinant of its transcriptional activity

Abstract

The first intron of the human Pro alpha 1(I) collagen gene contains an orientation-dependent enhancer composed of both positive and negative cis-acting elements involved in the transcriptional regulation of this gene. Deletion of a 360 bp Sau 3A intronic fragment spanning nucleotide +494 to +854 (S360) resulted in dramatic down-regulation of pCOL-KT (Thompson et al., J Biol Chem 266: 2549-2556, 1991). Using a DNaseI protection assay, we demonstrate a single footprint located at +590 to +615 in the S360 fragment; nuclear extracts prepared from mesenchymal and nonmesenchymal cells exhibited similar binding characteristics. A double stranded oligonucleotide representing a consensus Ap-1 binding sequence competed with S360 for binding. In contrast to what occurred in response to S360 deletion which was always accompanied by reduced expression, the deletion of the Ap-1 binding site (+598 to +off) caused either increased or decreased expression of the reporter gene depending on the target cell. Site-directed mutations in the Ap-1-like cis-element of Pro alpha 1(I) were also tested in transient expression assays. Consistent with the paradoxical results of Ap-1 deletion, we observed that the functional consequences of mutations in the Ap-1 site also varied in different cells. In A204 cells, one point mutation, which resulted in the loss of protein binding to S360, led to increased CAT activity while another point mutant, which retained binding of the Ap-1 like trans-acting factor(s), showed decreased CAT expression. The effects of these two mutations in the HFL-1 cells were exactly opposite of what was seen for A204 cells. Based on these observations, we postulate that the Ap-1 site plays a critical role in the transcriptional activity of the human Pro alpha 1(I) gene. The implications of an apparently dual mode of regulation through a single cis-regulatory element are discussed.

p38 and Activating Transcription Factor-2 Involvement in Osteoblast Osmotic Response to Elevated Extracellular Glucose

“…Thus, these parallel pathways could converge at c-jun2TRE and cause hyperglycemic induction of c-jun expression. Our studies 1 incorporating a dominant-negative AP-1 construct (71) suggest that AP-1 activity is important for hyperglycemic up-regulation of collagen I expression, which is thought to involve AP-1 binding and transactivation (25,72,73). Therefore, suppression of c-jun expression by p38 inhibition should suppress collagen I expression, as we demonstrated in Fig.…”

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

“…In addition, the deletion tested in our study was a large one which very likely includes many potential cis-acting elements. Some of these elements have been tested individually or as part of shorter sequences in cell transfection studies, and both positively and negatively acting elements have been identified (2,6,26,29,33,34,52; see reference 8 for a review). The phenotype identified thus far in homozygous Col-Int⌬ mice may therefore reflect the net result of deleting multiple elements with opposing effects on transcription.…”

A Minimal Murine Msx-1 Gene Promoter

“…For supershift assays, nuclear extracts were preincubated with polyclonal antibodies against MyoD, myogenin, c-Myc, Max, USF-1, USF-2, or Sp-1 for 2 h at 4°C prior to initiation of the binding reaction. The contents of the binding reactions were electrophoresed at 4°C on a 4% nondenaturing polyacrylamide gel in 1 ϫ TBE (135 mM Tris, 45 mM boric acid, and 2.5 mM Na 2 EDTA, pH 8.9) and fluorographed; we have described EMSA methods in detail previously (50,51). All antibodies to transcriptional factors and the oligonucleotides containing the consensus recognition motifs used in EMSA were purchased from Santa Cruz Biotechnology, Inc.…”