Research ReportUbe3a mRNA and protein expression are not decreased in Mecp2R168X mutant mice
Introduction
Rett syndrome (RS) is a severe neurodevelopmental disorder characterized by apparently normal initial development followed by slowing of development and head growth (Hagberg et al., 1983, Hagberg et al., 1985, Hagberg et al., 2002, Rett, 1966). Purposeful hand skills are lost and replaced by characteristic stereotypies. Spoken language is lost. Patients may also develop seizures, breathing irregularities, sleep disturbance, autistic symptoms and scoliois. Mutations or deletions in methyl-CpG-binding protein 2 (MECP2) located at Xq28 are detectable in 96% of patients with RS (Amir et al., 1999, Moretti and Zoghbi, 2006). A small percentage of RS patients with early onset seizures have mutations in cyclin dependent kinase-like 5 (CDKL5) (Evans et al., 2005, Scala et al., 2005, Tao et al., 2004). CDKL5 may play a role in phosphorylation and regulation of MeCP2 (Mari et al., 2005).
Because MeCP2 binds to methyl CpG dinucleotides, an early leading hypothesis was that it serves as a global repressor of transcription. However, transcriptional profiling studies have failed to reveal a global de-repression of transcription in the setting of MeCP2 deficiency (Tudor et al., 2002). An alternate hypothesis that MeCP2 instead regulates transcription in a highly specific and selective manner has thus been raised. Strong evidence for this hypothesis is the finding that MeCP2 regulates BDNF in a calcium- and phosphorylation-dependent manner (Chen et al., 2003, Zhou et al., 2006).
Angelman syndrome (AS) is an imprinting disorder caused by a decrease in or loss of function of the maternal copy of ubiquitin protein ligase E3A(UBE3A) located at 15q11q13 (Kishino et al., 1997, Magenis et al., 1987). This protein, unlike MeCP2, is not a regulator of gene transcription but is involved in the ubiquitination pathway, which targets specific proteins for degradation. AS patients exhibit profound speech deficits, gait ataxia, seizures, characteristic EEG, postnatal acquired microcephaly, sleep disturbance, and an unusually happy demeanor with propensity to paroxysms of laughter (Angelman, 1965, Clayton-Smith and Laan, 2003, Williams et al., 1995). Several of these features are in common with RS, including the speech deficits, acquired microcephaly, sleep disturbance, and seizures. These similarities suggest that UBE3A could be a target for regulation by MeCP2. However, recent studies designed to test this hypothesis yielded conflicting results (Jordan and Francke, 2006, Makedonski et al., 2005, Samaco et al., 2005).
The study by Samaco et al. looked at two lines of mutant Mecp2 adult mice, as well as postmortem brain tissue from RS patients (Samaco et al., 2005). One mouse line studied, Mecp2tm1.1Jae, was constructed with an exon 3 deletion and characterized as functionally Mecp2 null (Chen et al., 2001). The other mouse line studied was Mecp2tm1.1Bird, which is Mecp2 null (Guy et al., 2001). They reported a significant reduction in expression of both UBE3A RNA and protein products. Next, Makedonski et al. investigated newborns of one of the same mouse lines studied by Samaco et al. (Mecp2tm1.1Bird) as well as postmortem brain tissue from RS patients and a lymphoblast cell line from an RS patient (Makedonski et al., 2005). They also reported reductions in UBE3A RNA and protein expression. Jordan and Francke then reported studies on both mouse lines, Mecp2tm1.1Jae and Mecp2tm1.1Bird, at 3 and 21 days of age (Jordan and Francke, 2006). However, in direct contrast to the previous two reports, no significant changes in Ube3a RNA or protein expression were detected. This lack of consensus prompted us to investigate Ube3a expression in a novel Mecp2 mutant mouse line designed in our laboratory.
Section snippets
Mecp2R168X mice show RS features
We designed a line of Mecp2 mutant mice, which have the most common mutation associated with RS, R168X, knocked-in the mouse gene (Mecp2R168X) (Bienvenu and Chelly, 2006). This sequence change replaces a codon for arginine with a stop codon. Using site directed mutagenesis, we altered the AGA sequence coding for arginine at codon 168 to a TGA coding for stop (Fig. 1a). The mutant transcript is transcribed and is easily detectable by RT–PCR (Fig. 1b). The amplicon was confirmed to be Mecp2 by
Discussion
Conceptually, MeCP2 regulation of UBE3A is an attractive hypothesis, given the similarities between RS and AS. However, the literature is far from a clear consensus. Two groups using mouse models and human post-mortem brain tissue observed decreases in UBE3A RNA and protein in the setting of MeCP2 deficiency (Makedonski et al., 2005, Samaco et al., 2005), while a third group found no change using the same two mouse models as the previous two groups (Jordan and Francke, 2006).
We chose to probe
Mouse lines
Site directed mutagenesis was performed using the Transformer Site-Directed Mutagenesis Kit (Clontech). Sequence alteration was confirmed by sequencing (Massachusetts General Hospital DNA Core Facility). The targeting construct containing a Neomycin resistance (neo) cassette flanked by lox P sites and the mutant sequence was electroporated onto 129SvJ embryonic stem cells. The neo cassette was cloned from pLITMUS-28 (New England Biolabs). Neomycin-resistant colonies were isolated, expanded and
Note added in proof
A BspH1 restriction site was created 3′ of point mutation during the cloning of the mutant construct. The mutant sequence of codons 167 to 172 reads 5′ AGG TGA GAT TCA TGA CCA 3′, while the wildtype sequence reads 5′ AGG AGA GAG CAG AAA CCA 3′. As this is 3′ of the premature stop codon, this will not affect the mutant product.
Acknowledgments
Funding for this study was through NIH grant MH-572901 and from a grant from the American Academy of Pediatrics Section on Genetics and Birth Defects.
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