MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions.

Francesco M Piccolo,Nathaniel Heintz,Robert Tjian,Ching-Lung Hsu,Peng Dong,Elitsa I Stoyanova,Zhe Liu,Anjana Rao

doi:10.7554/elife.51449

Abstract

Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.

Highlights

Methyl-CpG-binding-protein 2 (MeCP2) is an abundant nuclear protein that was initially identified as the founding member of the methyl-DNA binding proteins family (Lewis et al, 1992)
To study MeCP2 at single molecule resolution in live neurons, we transfected mouse cerebellar granule cells with a construct expressing MeCP2-HaloTag fusion protein and cultured them for 12–14 days to generate a monolayer of neuronal cells that is ideal for image acquisition (Figure 1A)
While mobile particles are blurred into the background, immobile single molecules are selectively recorded as individual bright spots that can be followed for several frames until they dissociate from their binding sites (Figure 1—figure supplement 1)

Summary

Introduction

Methyl-CpG-binding-protein 2 (MeCP2) is an abundant nuclear protein that was initially identified as the founding member of the methyl-DNA binding proteins family (Lewis et al, 1992). MeCP2 is expressed by all cell types, it is present at very high levels in neurons (Skene et al, 2010). Loss of MeCP2 is not lethal to these cells but results in alterations in gene expression and reduced cellular growth (Armstrong et al, 1995; Chahrour et al, 2008). Given the abundance of MeCP2 and its important role in the regulation of chromatin structure and gene expression, further investigation of its chromatin interactions in neurons is of interest

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Conclusion