HIGH-AFFINITY YY1 BINDING MOTIFS - IDENTIFICATION OF 2 CORE TYPES (ACAT AND CCAT) AND DISTRIBUTION OF POTENTIAL BINDING-SITES WITHIN THE HUMAN BETA-GLOBIN CLUSTER

PCR-assisted binding site selection was used to define the sequence ch aracteristics of high affinity YY1 binding sites. Compilation of the s equences of 189 selected oligonucleotides containing high affinity YY1 binding sites revealed two types of core sequence: ACAT and CCAT. ACA T cores were surrounded by other invariant nucleotides, forming the co nsensus GACATNTT. A search of the 73 kb human beta-like globin cluster with this consensus revealed eight matching motifs, six of which were located within 1-3 kb upstream of the gamma and beta genes. CCAT-type cores were more variable in surrounding sequence context; the consens us VDCCATNWY was found to fit 89% of the selected CCAT-containing olig onucleotides. A search of the human beta globin cluster with CCAT cons ensus sequences revealed 171 potential YY1 binding sites. Several of t hese were tested directly in gel shift assays and confirmed as high af finity YY1 binding sites. Finally, a strategy called motif-based phylo genetic analysis was employed to determine which of the 179 total site s are evolutionarily conserved. This analysis permits the detection of functionally conserved binding sites despite sequence differences pre sent between the two species. The 21 conserved sites identified will s erve as important starting points in further dissection of the possibl e role of YY1 in globin gene regulation.