Purification and characterization of a protein from HeLa cells that binds with high affinity to the estrogen response element, GGTCAGCGTGACC.
A non-histone protein, NHP1, that binds with high affinity to the estrogen response element (ERE), GGTCAGCGTGACC, has been purified approximately 45,000-fold from HeLa cells by a combination of chromatography on Sephacryl S-300, heparin-Sepharose, Mono Q (FPLC), and sequence-specific oligonucleotide-Sepharose. The native protein has a molecular weight of 170,000 and is composed of two polypeptides of 85 and 75 kDa. The two polypeptides are different as judged by peptide mapping, and only the 85-kDa polypeptide can be cross-linked to the bromodeoxyuridine-substituted synthetic ERE by UV irradiation. The native protein binds to the ERE with an apparent KD of 1 x 10(-11) M and has a pI of 5. The contact points of the protein with individual bases of the ERE have been determined by using partially depurinated and depyrimidinated synthetic oligonucleotides. The strongest contact points of NHP1 with the ERE are 5'AGCG3' in the center of the palindrome and differ from those of the estrogen receptor. NHP1 appears to produce specific nicks around the central CpGs of the ERE, thereby suggesting that it may play a role in active demethylation of mCpGs.
Hughes
M J
MJ
Friedrich Miescher-Institut, Basel, Switzerland.
Liang
H M
HM
Jiricny
J
J
Jost
J P
JP
D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004967:Estrogens / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011960:Receptors, Estrogen / Q000378:metabolism
D004268:DNA-Binding Proteins; D004967:Estrogens; D011960:Receptors, Estrogen; D004247:DNA