Isolation of actively transcribed nucleosomes using immobilized HMG 14 and 17 and an analysis of alpha-globin chromatin

Abstract

Taking advantage of the known specificity of interaction of HMG 14 and 17 with actively transcribed chromatin, we have covalently cross-linked these proteins to agarose and used this HMG 14-17 column to purify active nucleosomes. The column has been used to map the DNA regions surrounding the chicken alpha-globin genes for their capacity to bind HMG 14-17. The results show that at a crude level the HMG binding region correlates with the major stable primary transcript and the region of DNAase I sensitivity. At a finer level of analysis, however, the correspondence between DNAase I sensitivity and HMG binding is preserved, yet this chromosomal domain is shown to extend about 1 kb beyond the known primary transcript for the two chicken alpha genes. Why HMG 14 and 17 bind specifically to active nucleosomes is still not known. Our experiments suggest that at least one additional chemical difference distinguishes them. This difference is not reflected in the DNA:protein ratio; in the electrophoretic mobility of the particles or associated DNA; in the inner histone stoichiometry; in a marked degree of histone modification as assayed on Triton-urea gels; or in the presence of nonhistone proteins. Active nucleosomes can be dissociated and reconstituted and their ability to bind to HMG 14-17 restored. The ability to reconstitute binding should make it possible to determine whether the specificity for the interaction resides in the protein or the DNA.