Assembly into chromatin and subtype-specific transcriptional effects of exogenous linker histones directly introduced into a living Physarum cell

The apparent diversity of linker histone subtypes may be related to their s pecific roles in defining functional states of chromatin in vivo. We have d eveloped a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be intro duced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three so matic linker histone subtypes in vivo, and to demonstrate the general appli cability of this method. Exogenous linker histone proteins H1 degrees, H5 a nd H1 were directly absorbed into living cell segments of the naturally syn chronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chr omatin with subtype-specific antibodies revealed that exogenous linker hist ones were efficiently transported into nuclei and were integrated into chro matin, The immunoreactivity of a preparation of anti-H1 degrees antibodies that are blocked from binding to specific H1 degrees epitopes in native chr omatin indicates that the exogenous linker histones were similarly associat ed into Physarum chromatin, Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G(2)-phase. Furthermore, we show, that exogenous linker histones incorporat ed in early G(2)-phase inhibited transcription and that the level of inhibi tion correlates with the apparent role of the linker histone subtype in reg ulating transcription in cells where it normally occurs.