Mitotic phosphorylation prevents the binding of HMGN proteins to chromatin

Condensation of the chromatin fiber and transcriptional inhibition during m itosis is associated,vith the redistribution of many DNA- and chromatin-bin ding proteins, including members of the high-mobility-group N (HMGN) family . Here we study the mechanism governing the organization of HMGN proteins i n mitosis. Using site-specific antibodies and quantitative gel analysis wit h proteins extracted from synchronized HeLa cells, we demonstrate that, dur ing mitosis, the conserved serine residues in the nucleosomal binding domai n (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate tha t the negative charge abolishes the ability of the proteins to bind to nucl eosomes. Fluorescence loss of photobleaching demonstrates that, in living c ells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1(-/-) ce lls indicates that the negatively charged protein is not bound to chromosom es. We conclude that during mitosis the NBD of HMGN proteins is highly phos phorylated and that this modification regulates the interaction of the prot eins with chromatin.