MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome

Tatiana Nikitina,Rajarshi P Ghosh,Rachel A Horowitz-Scherer,Jeffrey C Hansen,Sergei A Grigoryev,Christopher L Woodcock

doi:10.1074/jbc.m704304200

Abstract

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the approximately 146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.

Highlights

Neurological symptoms that typically make their initial appearance in the first 6 –18 months of life in affected girls
MeCP2 binding contributes to the formation in nucleosomal arrays of a distinctive structural motif in which the entering and exiting linker DNA segments are brought into close juxtaposition forming a “stem.” The stem motif appears very similar to structures induced by histone H1 on mono-nucleosomes and polynucleosomes [7, 8] and is thought to initiate the zigzag conformation and compaction of H1-containing chromatin
A distinctive property of H1-containing chromatin is the protection from micrococcal nuclease (Mnase) digestion of ϳ20 bp of DNA beyond the ϳ146 bp of the nucleosome core particle (NCP)

Summary

EXPERIMENTAL PROCEDURES

601 DNA and NAs—Substrates (termed 208-12 DNA) were based on the “601” nucleosome positioning sequence and contained 12 repeats of the following 207-bp sequence (lowercase letters denote linker DNA added to the core) inserted into pUC19: agatatcggaccctatacgcgGCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACatcctgtgcatgtggatccgaattcatattaattaatact. This sequence was derived from the previously published 213-bp-long nucleosome-positioning template [29] and contained a centered nucleosome-positioning region from the 601 DNA sequence [13], flanked by EcoRV sites (see Fig. 1). The resulting DNA fragments were purified, reconstituted with core histones, and incubated with MeCP2, and gel shifts were carried out as described [5]. Digitized images of mononucleosomes were sorted and measured using software tools from EMAN [44] and SUPRIM [45]

RESULTS

RTT differ in their linker protection

DISCUSSION