Xenopus Egg Extracts Increase Dynamics of Histone H1 on Sperm Chromatin

Benjamin S Freedman,Rebecca Heald,Kelly E Miller

doi:10.1371/journal.pone.0013111

Abstract

BackgroundLinker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems.Methodology/Principal FindingsWe have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited ∼10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase.Conclusion/SignificanceRanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.

Highlights

H1 ‘‘linker’’ histones comprise a highly conserved family of lysine-rich chromatin proteins that promote the folding of beads-ona-string nucleosome arrays into thicker, 30 nm fibers [1,2,3]
We report that cytoplasmic factors including but not limited to RanBP7 and importin beta significantly inhibit the ability of H1 to bind chromatin, and that this inhibition is greater for individual domains of H1 than for the full-length protein
First we evaluated the effects of RanBP7 and histone H1 on a simple chromatin template in vitro, in extraction buffer (100 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM K-HEPES pH 7.7, 50 mM sucrose) supplemented with ATP but in the absence of cytoplasm

Summary

Introduction

H1 ‘‘linker’’ histones comprise a highly conserved family of lysine-rich chromatin proteins that promote the folding of beads-ona-string nucleosome arrays into thicker, 30 nm fibers [1,2,3]. Metazoan H1 proteins consist primarily of a winged helix globular domain near the amino terminus and a long, apparently unstructured carboxyl-terminal tail [4]. Nuclease digestion and DNA footprinting experiments suggest a structural model wherein H19s globular domain localizes near the nucleosome dyad and crosslinks incoming and outgoing DNA, while the tail binds to linker DNA and neutralizes its negative charge [5,6,7,8]. Truncated H1 proteins lacking either the globular domain or the unstructured carboxyl terminal tail can have similar effects as full-length H1 on some purified templates [7,14,15,16]. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems

Methods

Results

Conclusion