Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

Abstract

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.

Figure 1

FRAP analysis of mutant and wild-type (WT) H1-GFP. (a) Sequence of the globular domain of H1°. Residues are numbered from the initiator methionine to allow direct comparison to the H5 sequence. Asterisks indicate amino acids mutated in this study. Boxes and arrows below the sequence indicate α-helices and β-sheets, respectively, as determined from the crystal structure of H5 (ref. 21). (b) Quantitative FRAP analysis of stable transfectants expressing H1°-GFP constructs containing multiple mutations in putative binding sites. S1, site 1 mutations, K69A K73A K85A; S2, site 2 mutations, K40A R42A K52A R94A; S1S2, all seven of these mutations. (c,d) Quantitative FRAP analysis of stable transfectants expressing H1-GFP constructs containing single mutations in putative binding residues. (e) FRAP results upon mutation of each basic residue in the globular domain to a neutral residue. Transfectants expressing these H1°-GFP constructs were analyzed by FRAP to determine t₅₀ and t₈₀ (see Table 1). Values are averages from at least ten cells from three experiments. For clarity, error bars are omitted; typical standard deviations were below 10%.

Figure 2

Electrostatically conservative mutations have much smaller effects on binding affinity. (a,b) FRAP analysis of cell lines expressing the indicated construct. (c) Selected FRAP results (Table 1). Values are averages from at least ten cells from three experiments. Error bars show s.d.

Figure 3

Mutant and wild-type H1-GFP proteins protect chromatosome-sized DNA. (a) Nuclei from parental 3T3 cells and transfectants expressing wild-type H1°-GFP were digested with MNase. Soluble chromatin was separated into nucleoprotein particles and stained with ethidium bromide (left). Particles containing H1-GFP were visualized by fluorescence excitation (right). M3G, mononucleosomes containing H1-GFP; M3, mononucleosomes containing H1; M1, mononucleosomes lacking H1. The more slowly migrating material represents di- and higher-order chromatosomes. (b) Nucleoprotein particles from cell lines expressing mutant and wild-type H1-GFP were separated on nucleoprotein gels, then excised and electroeluted, and purified, labeled DNA was separated by PAGE. For all mutants, the pattern and mobility of mononucleosome particles as visualized by fluorescence and ethidium bromide staining was identical to that of material from cells expressing wild-type H1-GFP.

Figure 4

Map of the interaction surface of H1° based on data from Table 1. Yellow, binding residues in site 1; green, site 2; blue, nonbinding residues.

Figure 5

Molecular model of the location of H1 within the nucleosome. Blue, chromatosomal DNA; yellow, nucleosome dyad; red, the globular domain of H1.