After cutting open thorax and abdomen to facilitate substrate infiltration, embryos were placed in cold phosphate-buffered saline and then fixed for 45–60 min in 0.2% glutaraldehyde, 2% formalin in 0.1 m phosphate buffer, pH 7.3, containing 2 m m MgCl 2 and EGTA. Integration of the transgenes was assessed by Southern blot hybridization to a LacZ (the gene coding for β-galactosidase) probe of DNA purified from embryonic placentas. ), stage E15.5 was chosen to avoid the problem of diminished permeability due to skin keratinization. DNA purification, restriction nuclease digestion, and Southern analysis were performed according to the standard protocol ( After 15-min incubation at room temperature, the reaction was halted by adding 2 volumes of 50 m m Tris-HCl (pH 8.0), 100 m m NaCl, 100 m m EDTA, 1% SDS, and 40 μl of proteinase K (20 mg/ml). These reactions were performed in 40 m m Tris-HCl (pH 7.6) and 6 m m MgCl 2using 10 μl of DNase I (Amersham Pharmacia Biotech, Piscataway, NJ) at concentrations between 0 and 10 units/μl. Nuclear pellets were resuspended in 5 volumes of buffer C (buffer A without Triton X-100, EDTA and EGTA) and DNA concentrations were estimated by UV absorption at 260 nm 15 OD units were used for each DNase I digestion. Cells were mechanically disrupted, the resulting homogenate was diluted with an equal volume of buffer B (buffer A without Triton X-100), and the nuclei were sedimented by centrifugation. Chromatin Analysis and DNase I Footprinting AssayĬells were washed with ice-cold phosphate-buffered saline, scraped, pelleted, and resuspended in buffer A (15 m m Tris-HCl (pH 7.6), 15 m m NaCl, 60 m m KCl, 1 m m EDTA, 0.5 m m EGTA, 0.3 m m sucrose, 0.1% Triton X-100, 0.15 m m spermine, 0.5 m m spermidine, 1 m m phenylmethylsulfonyl fluoride, 1 m mdithiothreitol). Sequences were compared using the MacVector package of programs. The sequence of the mouse proximal promoter is in the GenBank™ under accession number S48747, whereas the sequence of the far-upstream enhancer has been deposited under accession number AF345994. The 1.4-kb fragment that extends from −20.2 to −18.8 was generated by Swa1 digestion of the above 2.3-kb fragment to yield construct 20.2/18.8pLAC. The 2.3-kb fragment that extends from −21.1 to −18.8 kb was generated with proofreading Pfu DNA polymerase (Stratagene, La Jolla, CA) on DNA template of the original BAC clone and using primers 5′-TTACCCCCAATTTACAGATGAAAG-3′ and 5′-GCCTCAGCAAGCAACGTGG-3′ to yield construct 21.1/18.8pLAC. The 2.0- and 3.0-kb fragments that extend from −22.8 to −20.8 kb and from −20.8 to −17.8 kb of COL1A2 were derived by internal HindIII deletion of the above clone to yield constructs 22.8/20.8pLAC and 20.8/17.5pLAC, respectively. Louis, MO GenBank™ accession number AC002074) to yield construct 22.8/17.5pLAC. The 5.2-kb fragment that extends from −22.8 to −17.5 kb was derived by double digestion with SpeI and XbaI of a larger SpeI subclone of the BAC clone GS056H18 BAC clone (Genome Systems, St. Constructs were engineered by subcloning various upstream sequences 5′ of the −378 promoter. Hence, subtle differences may characterize the regulation of mammalian α2(I) collagen genes by evolutionarily conserved sequences. Enhancer activity substantially decreased without the segment containing FU1–7 and HS5, and inclusion of AluI repeats located 3′ of HS3 augmented position-independent expression of the transgene. Characterization of the human element documented functional differences with the mouse counterpart. The region containing HS3–5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1–12) and within stretches nearly identical to the mouse sequence. HS3, HS4, and HS5 were likewise mapped ∼20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). Four strong DNase I-hypersensitive sites (HS2–5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. We have examined the chromatin structure around and upstream of the transcriptional start site of the human α2(I) collagen (COL1A2) gene. Glycobiology and Extracellular Matrices.
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