Jcb_201412097 1..10

During mitosis, the duplicated genome undergoes a dramatic structural reorganization, resulting in condensed, resolved chromosomes that can be segregated by the spindle during anaphase. Core histones H2A, H2B, H3, and H4 provide the first level of genome compaction, assembling into stable octameric units around which DNA is encircled to form nucleosomes. Linker histones bind nucleosomes and the linker DNA between them, stabilizing folded or oligomeric conformations of chromatin fibers, regulating transcription, and generating higher order structures required for mitotic chromosome formation (Thoma and Koller, 1977; Thoma et al., 1979; Ausió, 2006; Harshman et al., 2013). However, the precise functions of linker histones have been difficult to define, as their sequences are more divergent than those of core histones, and many isoforms are present in eukaryotic genomes, with multiple, functionally redundant variants present in most cell types (Fan et al., 2003; Happel and Doenecke, 2009). Xenopus laevis egg extracts have provided a unique opportunity to study the contribution of linker histone H1 to chromosome structure, as the cytoplasm contains a single embryonic isoform called H1M, also known as B4 (Dworkin-Rastl et al., 1994; Saeki et al., 2005). Importantly, H1M is necessary for mitotic chromosome architecture and condensation in egg extracts (Maresca et al., 2005). In contrast to core histones, all H1 isoforms including H1M are highly dynamic components of chromatin, with residence half-times in vivo on the order of seconds, a surprising characteristic for a structural protein (Misteli et al., 2000; Lever et al., 2000; Freedman et al., 2010). Factors regulating this dynamic behavior have not previously been described. A key feature of both core and linker histones is their positive charge, which neutralizes negatively charged DNA but also causes free histones to be insoluble at physiological salt concentrations in vitro. Naked DNA and free histones bind tightly and nonspecifically to one another under these conditions, forming disordered nonnucleosomal aggregates rather than chromatin (Wilhelm et al., 1978). In vivo, free histones also bind promiscuously to negatively charged cellular components other than DNA, such as RNA complexes and tubulin (Hondele and Ladurner, 2011). To avoid nonspecific interactions and maintain histone solubility in the cytoplasm, cells use a variety of chaperones, which have been shown to be particularly important in Linker histone H1 is required for mitotic chromosome architecture in Xenopus laevis egg extracts and, unlike core histones, exhibits rapid turnover on chromatin. Mechanisms regulating the recruitment, deposition, and dynamics of linker histones in mitosis are largely unknown. We found that the cytoplasmic histone chaperone nucleosome assembly protein 1 (Nap1) associates with the embryonic isoform of linker histone H1 (H1M) in egg extracts. Immunodepletion of Nap1 decreased H1M binding to mitotic chromosomes by nearly 50%, reduced H1M dynamics as measured by fluorescence recovery after photobleaching and caused chromosome decondensation similar to the effects of H1M depletion. Defects in H1M dynamics and chromosome condensation were rescued by adding back wild-type Nap1 but not a mutant lacking sites subject to posttranslational modification by glutamylation. Nap1 glutamylation increased the deposition of H1M on sperm nuclei and chromatin-coated beads, indicating that charge-shifting posttranslational modification of Nap1 contributes to H1M dynamics that are essential for higher order chromosome architecture. Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus