NF-Y organizes the γ-globin CCAAT boxes region

Abstract

The CCAAT-binding activator NF-Y is formed by three evolutionary conserved subunits, two of which contain putative histone-like domains. We investigated NF-Y binding to all CCAAT boxes of globin promoters in direct binding, competition, and supershift electrophoretic mobility shift assay; we found that the α, ζ, and proximal γ CCAAT boxes of human and the prosimian Galago bind avidly, and distal γ CCAAT boxes have intermediate affinity, whereas the ε and β sequences bind NF-Y very poorly. We developed an efficient in vitro transcription system from erythroid K562 cells and established that both the distal and the proximal CCAAT boxes are important for optimal γ-globin promoter activity. Surprisingly, NF-Y binding to a mutated distal CCAAT box (a C to T at position -114) is remarkably increased upon occupancy of the high affinity proximal element, located 27 base pairs away. Shortening the distance between the two CCAAT boxes progressively prevents simultaneous CCAAT binding, indicating that NF-Y interacts in a mutually exclusive way with CCAAT boxes closer than 24 base pairs apart. A combination of circular permutation and phasing analysis proved that (i) NFY- induced angles of the two γ-globin 3 boxes have similar amplitudes; (ii) occupancy of the two CCAAT boxes leads to compensatory distortions; (iii) the two NF-Y bends are spatially oriented with combined twisting angles of about 100°. Interestingly, such distortions are reminiscent of core histone-DNA interactions. We conclude that NF-Y binding imposes a high level of functionally important coordinate organization to the γ-globin promoter.