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DNA-protein interaction sites in differentiating cells. I. Two-dimensional mapping of modulated sites.

We have previously shown that DNA-protein attachment sites form during the induction of hematopoietic cell differentiation. Affinity phase-partitioning studies of DNA/protein complexes demonstrated that the DNA involved is not randomly distributed throughout the genome. The object of this study was to use filter binding followed by two-dimensional (2D) polyacrylamide gel electrophoresis (using a neutral 6% gel in the first dimension and a denaturing gradient gel in the second dimension) to gain insight into changes in DNA-protein interactions during induced granulocytic and monocytic differentiation of HL60 cells. Nitrocellulose filter-binding enriched samples for protein-associated DNA sufficiently to change the pattern of DNA spots on 2D gels. The patterns of spots obtained was reasonably reproducible between experiments and highly reproducible within experiments. Gels obtained from cells induced to differentiate by either phorbol ester or all-trans retinoic acid (RA) showed identical patterns for the majority of spots but changes in a small proportion of spots with respect to uninduced controls. Both intensification and reduction/disappearance of spots was observed, demonstrating the existence of both invariant and variant DNA/protein attachment sites during the early stages of hematopoietic cell differentiation. Previous studies have implicated DNA topoisomerase II in chromatin structural changes that are necessary for induction of granulocytic differentiation. We therefore examined the filter-binding DNA preparation by 5'-exonuclease digestion (since topoisomerase II is known to bind covalently to the 5'termini on either side of its cleavage sites). The filter-associated DNA exhibited increased 5' exonuclease protection (with respect to filter flow-through DNA), and the degree of protection increased significantly with exposure to phorbol ester and less markedly with retinoic acid. However, since not all filter DNA was 5' protected, it remains unresolved whether the specific differentiation-associated DNA-protein interactions revealed here involve DNA topoisomerase II or some other protein.