Role of the promoter in maintaining transcriptionally active chromatin structure and DNA methylation patterns in vivo.
Establishment and maintenance of differential chromatin structure between transcriptionally competent and repressed genes are critical aspects of transcriptional regulation. The elements and mechanisms that mediate formation and maintenance of these chromatin states in vivo are not well understood. To examine the role of the promoter in maintaining chromatin structure and DNA methylation patterns of the transcriptionally active X-linked HPRT locus, 323 bp of the endogenous human HPRT promoter (from position -222 to +102 relative to the translation start site) was replaced by plasmid sequences by homologous recombination in cultured HT-1080 male fibrosarcoma cells. The targeted cells, which showed no detectable HPRT transcription, were then assayed for effects on DNase I hypersensitivity, general DNase I sensitivity, and DNA methylation patterns across the HPRT locus. In cells carrying the deletion, significantly diminished DNase I hypersensitivity in the 5' flanking region was observed compared to that in parental HT-1080 cells. However, general DNase I sensitivity and DNA methylation patterns were found to be very similar in the mutated cells and in the parental cells. These findings suggest that the promoter and active transcription play a relatively limited role in maintaining transcriptionally potentiated epigenetic states.
Kang
Sung-Hae Lee
SH
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Kiefer
Christine Mione
CM
Yang
Thomas P
TP
D001483:Base Sequence; D015139:Blotting, Southern; D002843:Chromatin / Q000378:metabolism*; D041321:Chromosomes, Human, X / Q000235:genetics; D018899:CpG Islands; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D003850:Deoxyribonuclease I / Q000378:metabolism; D018274:Electroporation; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020161:Physical Chromosome Mapping; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000494:pharmacology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D013447:Sulfites; D012313:RNA; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D003850:Deoxyribonuclease I; C009279:sodium bisulfite