The linker histones H1, H5, and H1 0 are associated with the core histone-DNA complex and with the linker DNA between adjacent nucleosomes and are thought to modulate the condensation/decondensation of the chromatin fiber, thus influencing many nuclear activities such as transcription, replication, recombination, and DNA repair (2). H1 0 was first described in 1969 by Panyim and Chalkley (3, 4) as an H1-like protein present in mammalian tissues with little or no cellular proliferation and was later shown to increase at a terminal stage of differentiation (5-10). Some cells, however, accumulate significant amounts of the protein while still actively proliferating (11, 12) or accumulate it upon proliferation arrest without concomitant differentiation (12). In addition, H1 0 seems to be the only histone undergoing changes during malignant transformation (13). Recently, it was found that transformation of NIH 3T3 fibroblasts by c-Ha-ras Val12 oncogene causes chromatin decondensation accompanied by alterations in the content of histone H1 0 (14). All these findings suggest a role for H1 0 in the regulation of either cell proliferation or cellular differentiation.In every tissue in which H1 0 has been detected, two subfractions were present (15-18). It appears that these two H1 0 proteins, up to now named H1 0 a and H1 0 b, have specific individual functions in chromatin (15). The relative proportions of the two H1 0 forms seem to differ from tissue to tissue (15) and exhibit age-dependent changes in rat brain cortical neurons (17). The two H1 0 s are resolvable by ion-exchange chromatography on Bio-Rex 70 (16, 19) or acetic acid-urea gel electrophoresis (15-18). Most recently, Lindner et al. (20) developed a high performance capillary electrophoresis method allowing separation of H1 0 and its subfractions from other histone H1 subtypes. The two H1 0 proteins run coincidentally on sodium dodecyl sulfate-polyacrylamide gels, suggesting that the difference between them is one of charge and not of size (15). Since neither treatment with alkaline phosphatase nor exposure to alkaline conditions changed the separation of the H1 0 peak into two subfractions, phosphorylation and ADP-ribosylation have been ruled out as possible post-translational modifications responsible for the different forms (16,20,21). Although some investigators speculated that the two forms of H1 0 might be coded by different genes (15, 17), Doenecke et al. (22) found that the mammalian genomes contain only one H1 0 gene.To gain insight into the nature of the two H1 0 subfractions, we took advantage of a combined reversed phase high performance liquid chromatography (RP-HPLC) 1 /hydrophilic interaction liquid chromatography (HILIC) technique recently developed in our laboratory for separating acetylated core and phosphorylated H1 histones (23,24). By applying this two-step HPLC method human placenta histone H1 0 was resolved into four components, which were treated with cyanogen bromide and chymotrypsin. HILIC analysis of the peptide fragments
