ISOLATION OF AN ALU REPETITIVE DNA-BINDING PROTEIN AND EFFECT OF CPG METHYLATION ON BINDING TO ITS RECOGNITION SEQUENCE

The structure, expression, and evolution of Alu repetitive DNA element s have been extensively studied, but the role of these sequences in th e function of primate genomes has yet to be elucidated. The contributi on 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 g lycoprotein hormone alpha-subunit (GPH alpha) gene in a variety of tum or cell types, it was found that the gene was hypermethylated in a var iety of cell lines that produce alpha-subunit at high levels and signi ficantly less methylated in cell lines where the gene is unexpressed o r expressed at low levels. This is in sharp contrast to the majority o f genes examined in this regard, which show an inverse correlation bet ween methylation and expression. The analysis was extended to a group of clones isolated from a single cell line (HeLa) that were differenti ally 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 expressio n of the gene. Two of these sites are imbedded in oppositely oriented Alu repeats located in the 5'-flanking DNA and second intron. The upst ream site was examined in some detail. DNase I footprint analysis demo nstrated that the protein protects a region encompassing the sequence 5'-TTGAACCCGGGAG-3', and electrophoretic gel mobility shift analysis d emonstrated specific binding of a protein to an oligonucleotide contai ning the DNase footprint sequence. Chromatography of nuclear extracts on Sephacryl S-200, heparin-agarose, and oligonucleotide-Sepharose pro duced an apparently homogeneous preparation of the 50-53 kDa DNA-bindi ng protein as judged by silver staining of sodium dodecylsulfate polya crylamide gels. The affinity-purified material was enriched 15- to 180 00-fold over crude nuclear extracts. Binding of this protein to an oli gonucleotide containing the DNase-protected sequence was severely inhi bited when the CpG dinucleotide in the Msp I recognition site was meth ylated on either the sense or antisense strands. Based on its properti es, this protein has been termed MeSABp50 for methylation-sensitive Al u binding protein of 50kDa. (C) 1998 Elsevier Science B.V.