N- and C-terminal Domains Determine Differential Nucleosomal Binding Geometry and Affinity of Linker Histone Isotypes H10 and H1c

Payal Vyas,David T Brown

doi:10.1074/jbc.m111.312819

Abstract

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.

Highlights

Switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes
Quantification of Histone H1 Binding Dynamics Using Dual Color FRAP Analysis—A bicistronic expression vector containing an internal ribosome entry site was constructed to co-express two linker histone-fluorescent protein hybrids (Fig. 1a)
Stable cell lines expressing linker histone proteins encoded by the bicistronic vector were established in murine BALB/c 3T3 fibroblast cells

Summary

Introduction

Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H10 and H1c. Switching the C-terminal domains altered the chromatin interaction surface of the globular domain These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins

Methods

Results

Conclusion