Enrichment of estradiol-receptor complexes in a transcriptionally active fraction of chromatin from MCF-7 cells.
We have examined the interaction of the estradiol receptor molecule with chromatin in MCF-7 cells, a human breast tumor cell line responsive to estradiol. Receptor was found associated with the various nucleosomal products produced by digestion with micrococcal nuclease. In order to determine whether these receptor binding sites were distributed in a random or nonrandom manner within the chromatin, we have fractionated MCF-7 cell chromatin into transcriptionally active and inactive fractions by limited micrococcal nuclease digestion followed by Mg(2+) precipitation. A comparison of the Mg(2+)-soluble and insoluble chromatin fractions showed that the Mg(2+)-soluble fraction: (i) was composed predominantly of mononucleosomes; (ii) was enriched in nonhistone proteins; (iii) apparently lacked histone H1; (iv) was enriched approximately 5-fold in transcribed sequences as measured by a cDNA probe to cytoplasmic poly(A)-RNA sequences; and (v) was depleted at least 5-fold of globin sequences, which is presumably a nontranscribed gene in these cells. When these cells were stimulated with beta-[(3)H]estradiol, the Mg(2+)-soluble fraction showed a significant enrichment in chromatin-bound estradiol receptor: the Mg(2+)-soluble mononucleosomes showed a 3- to 4-fold enrichment and the di- and trinucleosomes, a 7- to 19-fold enrichment, when compared to the corresponding subunits in the Mg(2+)-insoluble chromatin fraction. This cofractionation of chromatin enriched in transcribed sequences and bound estradiol receptor indicated that receptor binding to MCF-7 cell chromatin was not random but, rather, occurred preferentially in specific regions of the chromatin.
D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D008970:Molecular Weight; D011960:Receptors, Estrogen / Q000378:metabolism*; D014158:Transcription, Genetic
D002843:Chromatin; D006657:Histones; D011960:Receptors, Estrogen; D004958:Estradiol; D008836:Micrococcal Nuclease