PROTECTED REGIONS IN THE CHICKEN ALPHA-2(1) PROCOLLAGEN PROMOTER IN DIFFERENTIATED TISSUES

The higher ordered structure of the chicken alpha 2(I) procollagen gen e was analyzed in chromatin isolated from expressing (lung) and nonexp ressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gen e was methylated in all tissues examined and that no differences exist ed in the promoter methylation patterns between expressing and nonexpr essing tissues. DNAse 1 hypersensitive sites were located between 100- 300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single-stran d specific S-1 nuclease, implying that this region of the gene in the chromatin is either in an unfolded single-stranded conformation or und er severe conformational stress. These differences in the alpha 2(I) c hromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressi ng tissues than in the nonexpressing tissues. Digestion of chromatin w ith Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA-binding proteins in the two tissu e types. These protected sites were located as far upstream as -1,600 and downstream within the first intron at +800. (C) 1994 Wiley-Liss, I nc.