Restriction enzyme digestions identify discrete domains in the chromatin around the promoter of the mouse alpha 2(I) collagen gene.
We have examined the chromatin structure around the +1 transcriptional start site of the mouse alpha 2(I) collagen gene by studying the accessibility of DNA to several restriction enzymes as well as to DNase I. In NIH 3T3 cells, which express high levels of alpha 2(I) collagen mRNA, we detect a DNase I-hypersensitive site from -240 to +110 relative to the start site of transcription at +1. By digesting chromatin with restriction enzymes, which cleave naked DNA at multiple sites within the -2000 to +1000 region, a considerably more complex picture was revealed. DNA sequences upstream of around -550 and downstream of +150 are much less accessible to restriction enzymes than the region between these sites and are, therefore, probably packaged in a more compact conformation. The region from around -550 to -240 although not within the DNase I-hypersensitive domain is nevertheless accessible to restriction enzymes and, therefore, presumably in a relatively "open" conformation. In addition, beginning 5' to -100 there is a gradual decrease in restriction enzyme accessibility as one approaches +150. Of particular interest is the finding that although sites at +65 and +126 are relatively accessible, a HinfI site at +113 is resistant in chromatin. In v-mos transformed NIH 3T3 cells which express alpha 2(I) collagen at much lower levels than untransformed NIH 3T3 cells, the DNase I-hypersensitive site as well as the majority of the chromatin restriction enzyme accessibility patterns are similar to those found in untransformed NIH 3T3 cells. However, a SphI site at +58 appears less accessible in the transformed cells. We also examined the chromatin of a myeloma cell line which does not synthesize alpha 2(I) collagen at detectable levels. In the nuclei of these cells the DNA of the alpha 2(I) collagen promoter is inaccessible to DNase I and to all restriction enzymes.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002843:Chromatin / Q000032:analysis*; D003094:Collagen / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005347:Fibroblasts / Q000032:analysis; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D002843:Chromatin; D012333:RNA, Messenger; D003094:Collagen; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I