title: Isolation of an Alu repetitive DNA binding protein and effect of CpG methylation on binding to its recognition sequence.
abstract: The structure, expression, and evolution of Alu repetitive DNA elements have been extensively studied, but the role of these sequences in the function of primate genomes has yet to be elucidated. The contribution of Alu repetitive sequences (ARS) to the structure, maintenance, or expression of the human genome is undoubtedly mediated by one or more DNA binding proteins. As part of a larger study in this laboratory to define the molecular mechanisms that result in de-repression of the glycoprotein hormone alpha-subunit (GPH alpha) gene in a variety of tumor cell types, it was found that the gene was hypermethylated in a variety of cell lines that produce alpha-subunit at high levels and significantly less methylated in cell lines where the gene is unexpressed or expressed at low levels. This is in sharp contrast to the majority of genes examined in this regard, which show an inverse correlation between methylation and expression. The analysis was extended to a group of clones isolated from a single cell line (HeLa) that were differentially methylated over the GPH alpha gene and exhibited a 400-fold range in its expression. These analyses demonstrated that methylation of a small number of CpG dinucleotides correlated with high level expression of the gene. Two of these sites are imbedded in oppositely oriented Alu repeats located in the 5'-flanking DNA and second intron. The upstream site was examined in some detail. DNase I footprint analysis demonstrated that the protein protects a region encompassing the sequence 5'-TTGAACCCGGGAG-3', and electrophoretic gel mobility shift analysis demonstrated specific binding of a protein to an oligonucleotide containing the DNase footprint sequence. Chromatography of nuclear extracts on Sephacryl S-200, heparin--agarose, and oligonucleotide--Sepharose produced an apparently homogeneous preparation of the 50-53 kDa DNA-binding protein as judged by silver staining of sodium dodecylsulfate polyacrylamide gels. The affinity-purified material was enriched 15- to 18,000-fold over crude nuclear extracts. Binding of this protein to an oligonucleotide containing the DNase-protected sequence was severely inhibited when CpG dinucleotide in the Msp I recognition site was methylated on either the sense or antisense strands. Based on its properties, this protein has been termed MeSABp50 for methylation-sensitive Alu binding protein of 50 kDa.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Cox
authors.forename: D W
authors.initials: DW
authors.lastname: Gutkin
authors.forename: M J
authors.initials: MJ
authors.lastname: Haas
authors.forename: D E
authors.initials: DE
authors.lastname: Cosgrove
mesh_terms: D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000235:genetics* / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000235:genetics* / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors
chemical_list: C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C097691:cbp1 protein, S pombe; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII