IN-VITRO BINDING OF H1 HISTONE SUBTYPES TO NUCLEOSOMAL ORGANIZED MOUSE MAMMARY-TUMOR VIRUS LONG TERMINAL REPEAT PROMOTOR

The binding of all known linker histones, named Hla through H1e, inclu ding H1 degrees and H1t, to a model chromatin complex based on a DNA f ragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, w e found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes v aried between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1 d/e, and H1 degrees, completely aggregate polynucleosomes (1.3 kilobas e pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome, To form agg regates with the histones H1t and H1b, however, greater amounts of pro tein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes, S phase-specific phosphorylation with one to three phosphate groups a t specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes, In contrast, h ighly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucl eosome but a low chromatin aggregation capability. These findings sugg est that specific S phase or mitotic phosphorylation sites act indepen dently and have distinct functional roles.