The Zinc Finger Protein CTCF Binds to the APBβ Domain of the Amyloid β-Protein Precursor Promoter

The promoter of the amyloid β-protein precursor (APP) gene directs high levels of cell type-specific transcription with 94 base pairs 5′ to the main transcriptional start site. An essential activator domain in this proximal APP promoter is a nuclear factor binding site designated as APBβ. The recognition domain for the APBβ binding factor is located between position −93 and −82 relative to the main transcriptional start site. The nuclear factor that binds to the APBβ site was partially purified by multiple steps of ion exchange and hydroxyapatite chromatography. Based on UV cross-linking results, a protein with an apparent molecular mass of 140 kDa was selected as the putative APBβ binding protein. After the final purification step consisting of preparative SDS-polyacrylamide gel electrophoresis, partial peptide sequences were obtained that completely matched the transcriptional factor CTCF. This protein is a known regulator of c- myc and lysozyme gene expression, and it binds to a variety of diverse DNA sequences. The binding of CTCF to the APBβ domain was further established by competition with CTCF binding oligonucleotides in mobility shift electrophoresis. The identity was also confirmed by the observation that the APBβ binding factor is recognized by antibodies against C- and N-terminal sequences of CTCF. In addition, oligonucleotide competition during in vitro transcription affirmed that CTCF acts as a transcriptional activator in the APP gene promoter.