DNA-protein interaction sites in differentiating cells. II. A subset of alphoid repetitive sequences with retinoic acid induced protein attachment and an unusual purine-pyrimidine 'signature'.
Two independent methods were used to recover the tightly or covalently attached DNA-protein complexes which form during the differentiation induction of HL60 cells by all-trans retinoic acid. One method employed nitrocellulose filter binding (described in the accompanying paper) to recover these protein-DNA complexes, following by representational difference analysis (RDA). RDA is an extremely powerful new technique for cloning the difference between complex DNA samples, exploiting a combination of kinetic enrichment during PCR amplification, and subtractive hybridization. In this case, we used filter-bound DNA from undifferentiated cells for subtraction of equivalent DNA from differentiating cells. A second approach used a combination of sodium dodecyl sulfate (SDS)/KCI precipitation and affinity phase partitioning for purification of DNA bound to proteins, followed by selection of clones showing differentiation-specific attachment of proteins (by differential hybridization to protein-attached DNA from differentiating and undifferentiated HL60 cells). Both procedures yielded a high proportion of alphoid repetitive sequences, although slightly higher in the RDA sample than that of the other method (approximately 50% and approximately 30%, respectively). Plots of purine-pyrimidine composition showed that the alphoids recovered by both techniques were unusual and remarkably similar. Although the clones were related to each other, they differed more from the alphoid consensus than examples of alphoid sequences selected from data banks. There was also a contrast between the purine-pyrimidine composition plots of archetypal alphoid monomers and this subset. The principal difference observed related to two adjacent homopyrimidine tracts present in the archetypal monomers but altered in the subset. The possible significance of these differences is discussed with respect to the established roles of alphoid sequences and known/putative protein binding sites including that for the centromeric binding protein (CENB-P box) and response elements for retinoic acid receptors. Following induction of differentiation with retinoic acid, protein attachment (monitored by filter binding) was rapid, maximal at 40 minutes, and still elevated at 165 minutes. This was specific to the alphoid subset, and induced protein binding at a nonrepetitive site in DNA had different kinetics. Changing protein attachment at this subset of alphoid repetitive sequences contributes to differentiation-associated chromatin structural changes.