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(Journal Article) |
Biochimica et biophysica acta 1443 (1-2): 75-89 (1998)
Methylation in the initiation region of the first exon suppresses collagen pro-alpha2(I) gene transcription.
P K Sengupta
,
B D Smith
ABSTRACT
Our previous studies demonstrated that the collagen alpha2(I) gene is hypermethylated in the promoter/first exon region after chemical transformation and the alpha2(I) promoter/first exon is sensitive to methylation in transfection studies. In this paper, we demonstrate that a minimum collagen promoter containing the preinitiation region (-41 to +54) driving luciferase reporter gene was inactivated by DNA methylation as judged by transfection assays. All the methylation sites within the preinitiation region were located in the first exon, not in the promoter. Methylation of the promoter construct inhibited transcription as determined by an in vitro assay, only if proteins were extracted from nuclei using 500 mM NaCl. Gel mobility shift analysis suggested that methylation within the first exon decreased the formation of the largest preinitiation complex while increasing the formation of faster migrating protein-DNA complexes. Competition gel mobility shift analysis indicated that the faster migrating protein-DNA complex could be competed by a smaller initiator probe which did not contain TATA binding region. A protein-DNA complex with increased affinity to methylated sequences was detected using the initiator probe, which contained two methylation sites and no TATA sequence (-25 to 30) suggesting that a separate repressor complex binds to the methylated sequences. Mutations at the methylation sites (+7, +23) in the first exon also increased the protein-DNA complex formation in gel shift analysis and inhibited collagen alpha2(I) transcription as judged by transient transfection and in vitro transcription assays. Therefore, these methylation sites in the preinitiation region are important for transcription of alpha2(I) gene and the protein responsible for the repression of transcription is extractable using high salt nuclear extracts.