The considerable length of DNA in eukaryotic genomes requires packaging into chromatin to fit inside the small dimensions of the cell nucleus. Histone H1 functions in the compaction of chromatin into higher order structures derived from the repeating 'beads on a string' nucleosome polymer. Modulation of H1 binding activity is thought to be an important step in the potentiation/depotentiation of chromatin structure for transcription. It is generally accepted that H1 binds less tightly than other histones to DNA in chromatin and can readily exchange in living cells. Fusion proteins of Histone H1 and green fluorescent protein (GFP) have been shown to associate with chromatin in an apparently identical fashion to native histone H1. This provides a means by which to study histone H1-chromatin interactions in living cells. Here we have used human cells with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching in living cells. We find that exchange is rapid in both condensed and decondensed chromatin, occurs throughout the cell cycle, and does not require fibre-fibre interactions. Treatment with drugs that alter protein phosphorylation significantly reduces exchange rates. Our results show that histone H1 exchange in vivo is rapid, occurs through a soluble intermediate, and is modulated by the phosphorylation of a protein or proteins as yet to be determined.

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photobleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.Histone H1 is the fifth histone subtype and is not one of the histones that form the histone octamer of the nucleosome. Rather, histone H1 binds to the surface of the nucleosome and interacts with nucleosomal DNA at the entry and exit points (1). In doing so, histone H1 is critical in determining the higher-order folding states of chromatin. Because of this property, histone H1 has traditionally been considered a general repressor of transcription (3). Consistent with this hypothesis, histone H1 was found to be modestly depleted in transcriptionally active genes (4 -6). More recently, genetic studies have revealed contributions of H1 histones to the establishment of epigenetic silencing (7-10). In addition to a structural role, histone H1 also functions in gene-specific regulation. A large number of studies have demonstrated that H1 histones or specific variants are directly involved in the regulation of specific genes (3,(11)(12)(13)(14), consistent with the observation of differential gene expression when the sole histone H1 gene was knocked out in Tetrahymena thermophila (15).The structure of H1 histones is typically considered to consist of three separate domains (16). A short stretch of amino acids on the N terminus and a much larger stretch that comprises the C terminus show significant variability between individual subtypes as well as between species. The amino and carboxyl termini have diverged considerably throughout the evolution of metazoans (17). If we restrict the analysis to mammals, the C termini diverge between individual histone H1 variants, but the sequences of the individual C termini are well conserved between species. When histone H1 sequences are examined in a broader range of ...

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF–GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF–GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

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