Purification of nuclear proteins that potentially regulate transcription of the MUC1 mucin gene induced by a soluble factor.
Transcriptional regulation of the MUC1 mucin gene in KM12C human colon carcinoma cells, which is induced by a soluble stimulatory factor derived from normal colonic connective tissues, was investigated. The minimum responsive element that was sufficient for this upregulation by the soluble factor is the upstream sequence of the MUC1 mucin gene from -531 to -488. Several factors in nuclear extracts of KM12C cells bound to this sequence in gel retardation assays. Neither the quantities nor the mobilities of the retarded bands changed on treatment with the soluble factor. Mutagenesis within the region from ACAGGGAGCGGTTAGAAGGGTGGGGCTATTCCGGGAAGTGGTGG to ACAGGGAGCGGTTAGAA[TTT]TGGGGCTATTCCGGGAAGTGGTGG (bracketed letters were mutated) substantially decreased the induction of the MUC1 mucin gene by the soluble factor. Two retarded bands were observed when the unmutated sequence was used as a probe; the bands disappeared when the mutated sequence was used as a probe. These results indicate that factors corresponding to each band were responsible for the upregulation of the MUC1 mucin gene, although the quantities of these proteins and their affinity to the nucleotide sequence did not change during the induction. Purification of the protein components comprising each band by a combination of column chromatographies indicated that one band contained four proteins (111, 106, 101, and 95 kDa) and the other consisted of two proteins (66 and 64 kDa).
Shirotani
K
K
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Irimura
T
T
D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000235:genetics*; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000502:physiology*
D017077:Culture Media, Conditioned; D018396:Mucin-1; D009687:Nuclear Proteins