DNA-PROTEIN INTERACTION SITES IN DIFFERENTIATING CELLS .2. 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 employe d nitrocellulose filter binding (described in the accompanying paper) to recover these protein-DNA complexes, followed by representational d ifference analysis (RDA). RDA is an extremely powerful new technique f or cloning the differences between complex DNA samples, exploiting a c ombination of kinetic enrichment during PCR amplification, and subtrac tive hybridization. In this case, we used filter-bound DNA from undiff erentiated cells for subtraction of equivalent DNA from differentiatin g cells. A second approach used a combination of sodium dodecyl sulfat e (SDS)/KCl precipitation and affinity-phase partitioning for purifica tion of DNA bound to proteins, followed by selection of clones showing differentiation-specific attachment of proteins (by differential hybr idization to protein-attached DNA from differentiating and undifferent iated HL60 cells). Both procedures yielded a high proportion of alphoi d repetitive sequences, although slightly higher in the RDA sample tha n that of the other method (similar to 50% and similar to 30%, respect ively). Plots of purine-pyrimidine composition showed that the alphoid s recovered by both techniques were unusual and remarkably similar. Al though 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 c omposition plots of archetypal alphoid monomers and this subset. The p rincipal differences observed related to two adjacent homopyrimidine t racts present in the archetypal monomers but altered in the subset. Th e possible significance of these differences is discussed with respect to the established roles of alphoid sequences and known/putative prot ein binding sites including that for the centromeric binding protein ( CENB-P box) and response elements for retinoic acid receptors. Followi ng induction of differentiation with retinoic acid, protein attachment (monitored by filter binding) was rapid, maximal at 40 minutes, and s till elevated at 165 minutes. This was specific to the alphoid subset, and induced protein binding at a nonrepetitive site in DNA had differ ent kinetics. Changing protein attachment at this subset of alphoid re petitive sequences contributes to differentiation-associated chromatin structural changes.