The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatin in Vivo

Abstract

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 ...

Translocation of histone H1 subtypes between chromatin and cytoplasm during mitosis in normal human fibroblasts

“…There can be at least two explanations for release of histone H1 from chromatin during mitosis; either a decreased H1 affinity for chromatin or an active exchange of H1 for other chromatin binding factors. Phosphorylation of H1, especially in its C terminal, is supposed to decrease its affinity for chromatin and increase its mobility in vivo (28) and, interestingly, recent experiments in vitro have shown that linker histones with fully phosphorylated C-terminal domains showed reduced affinity for DNA in combination with increased aggregation capacity of DNA fragments (29). In interphase cells, H1.2, H1.3, H1.4, and H1.5 are phosphorylated on serine residues (19,30).…”

Independent Biological and Biochemical Functions for Individual Structural Domains of Drosophila Linker Histone H1

“…Fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that the GD plays a dominant role in H1 binding to chromatin in cultured cells (14). FRAP experiments also indicate that the CTD plays an important role in the residence time of H1 in chromatin (15). In addition, experiments with oligonucleosome arrays assembled in vitro showed that the H1 CTD is required to condense the arrays into higher order structures (16 -18).…”

Global Changes in and Characterization of Specific Sites of Phosphorylation in Mouse and Human Histone H1 Isoforms upon CDK Inhibitor Treatment Using Mass Spectrometry

“…38,39 Purified H1 from the human HeLa cells and H5 from chicken erythrocytes have been shown to inhibit ATP-dependent chromatin remodeling activities in vitro. 37,38 In the case of H5, inhibition of remodeling was abrogated by saturating phosphorylation of H5 using the cdc2 kinase. 41 Furthermore, using Xenopus extract model systems, only nonphosphorylated recombinant mammalian H1 represses chromatin remodeling during DNA replication and repair.…”

“…Phosphorylation of CDK consensus S/T residues in mammalian H1s have been shown to alter mobility of H1 in chromatin, suggesting phosphorylation assists in release of H1 from chromatin. [35][36][37] Phosphorylation of the Tetrahymena H1 on S/T residues has been shown to influence transcriptional activity by creating an electrostatic charge patch that repels contact with DNA. 38,39 Purified H1 from the human HeLa cells and H5 from chicken erythrocytes have been shown to inhibit ATP-dependent chromatin remodeling activities in vitro.…”