Purification and characterization of a protein from HeLa cells that binds with high affinity to the estrogen response element, GGTCAGCGTGACC

Abstract

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.

Non‐histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

“…NHPI was purified from HeLa cells as described previously [2]. The purified NHPI subunits were subjected to preparative SDS-polyacrylamide gel electrophoresis and transferred to PCGM glass paper in 50 mM sodium borate containing 0.02°,/o Nonidet P-40 at 60 V and 0.2 A.…”

“…Deletion of the CpG in the center of the dyad symmetry sequence of the ERE decreased the binding of NHP1 by 90% and a conversion of any GC pair to an AT pair diminished the affinity of the binding site for NHP1 [1]. In the purified form, but not in the pure form, NHP1 was shown to produce nicks around the central CpG of the ERE [2] indicating that NHP1 might play a role in the active demethylation of mCpGs. Furthermore, at the onset of active demethylation *Corresponding author.…”

“…Non-histone protein 1 (NHP1) was first detected as a protein with a very high affinity to the estrogen response element (ERE) of the avian vitellogenin gene [1][2][3]. The strongest contact points of NHP1 with the ERE were with 5' AGCG 3' in the center of the palindrome differing from those of the estradiol receptor (ER) [2].…”

“…The strongest contact points of NHP1 with the ERE were with 5' AGCG 3' in the center of the palindrome differing from those of the estradiol receptor (ER) [2]. Of the two polypeptides p75 and p85 forming NHP1 only the p85 subunit can be crosslinked to the bromodeoxyuridine-substituted synthetic ERE by UV irradiation [2].…”

“…Fax: (41) of the avian vitellogenin gene in the liver there was a transient increase in NHP1 binding activity [4]. It has also been suggested that NHP1 could tag the DNA for the subsequent binding of the estradiol receptor [1][2][3]. A possible involvement of NHP1 in a general mechanism of active demethylation could account for its apparent ubiquity.…”