CpG-binding Protein Is a Nuclear Matrix- and Euchromatin-associated Protein Localized to Nuclear Speckles Containing Human Trithorax: IDENTIFICATION OF NUCLEAR MATRIX TARGETING SIGNALS

Jeong-Heon Lee,David G Skalnik

doi:10.1074/jbc.m205054200

Jeong-Heon Lee, David G Skalnik

Open Access

https://doi.org/10.1074/jbc.m205054200

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Abstract

CpG-binding protein (CGBP) binds unmethylated CpG dinucleotides and is essential for mammalian development. CGBP exhibits a punctate nuclear localization correlated with 4,6-diamidino-2-phenylindole light regions and is excluded from metaphase chromosomes. The distribution of CGBP is distinct from the heterochromatin-associated proteins MBD1, methyl-CpG-binding protein 2, and HP1alpha. Some CGBP-containing nuclear speckles co-localize with splicing factor SC-35 and actively transcribed regions of the genome, whereas most CGBP co-localizes with acetylated histones, indicating that CGBP is localized to active chromatin. CGBP contains two nuclear localization signals that are insufficient to direct punctate subnuclear distribution. Instead, localization of CGBP to nuclear speckles requires signals within the acidic, basic, and coiled-coil domains. CGBP associates with the nuclear matrix, and fragments of CGBP that fail to associate with the nuclear matrix fail to localize to nuclear speckles and exhibit reduced transcriptional activation activity. Mutated versions of CGBP that lack DNA binding activity exhibit a normal nuclear distribution, suggesting that CGBP accumulates at nuclear speckles as a result of protein/protein interactions. Importantly, the subcellular distribution of CGBP is identical to human trithorax, suggesting that these proteins may be components of a multimeric complex analogous to the histone-methylating Set1 complex of Saccharomyces cerevisiae that contains CGBP and trithorax homologues.

Highlights

CpG-binding protein (CGBP) binds unmethylated CpG dinucleotides and is essential for mammalian development
The set of nuclear speckles that contain CGBP is identical to that detected as containing the amino terminus of HRX, which has previously been reported to localize within a novel class of nuclear speckles and to associate with the nuclear matrix [35,36,37]
The distribution of CGBP is distinct from that exhibited by the heterochromatin-associated proteins MBD1, methyl-CpGbinding protein 2 (MeCP2), and HP1␣

Summary

The abbreviations used are

MeCP2, methyl CpG-binding protein 2; CGBP, CpG-binding protein; MBD, methyl CpG-binding domain; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; CMV, cytomegalovirus; HRX, human trithorax; PHD, plant homeodomain; Pipes, 1,4-piperazinediethanesulfonic acid. CGBP contains two copies of the plant homeodomain (PHD), a motif found in several dozen proteins implicated in modulation of chromatin structure and gene regulation [11, 19]. Given the DNA binding specificity of CGBP and its structural similarity to other proteins involved in the regulation and function of cytosine methylation and chromatin structure, it is tempting to speculate that CGBP-null embryos die from dysregulated gene expression, at least partially as a consequence of altered chromatin structure. In an effort to better understand the global function of CGBP, and its possible role in modulating chromatin structure, experiments were performed to determine the subcellular localization of CGBP and define the protein domains required to direct this distribution

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION