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Rett syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is primarily caused by variants in MECP2. Variants in other genes such as CDKL5 and FOXG1 are usually associated with individuals who manifest distinct phenotypes that may overlap with RTT. Individuals with phenotypes suggestive of RTT are typically screened for variants in MECP2 and then subsequently the other genes dependent on the specific phenotype. Even with this screening strategy, there are individuals in whom no causative variant can be identified, suggesting that there are other novel genes that contribute to the RTT phenotype. Here we report a de novo deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in identical twins with a RTT-like phenotype. We also demonstrate the reduced expression of Ptpn4 in a Mecp2 null mouse model of RTT, as well as the activation of the PTPN4 promoter by MeCP2. Our findings suggest that PTPN4 should be considered for addition to the growing list of genes that warrant screening in individuals with a RTT-like phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

The discovery that Rett syndrome (RTT) is caused by mutation of the methyl-CpG-binding-protein MeCP2 provided a major breakthrough in understanding the neurodevelopmental disorder and accelerated MeCP2 research. However, gene regulation by MeCP2 is complicated. The current consensus for MeCP2 remains as a classical repressor complex, with major emphasis on its role in methylation-dependent binding and repression. However, recent evidence indicates additional regulatory roles, suggesting non-classical mechanisms in gene activation. This has opened the field of MeCP2 research and suggests that the gene targets may not be the usual suspects, that is, dependent only on DNA methylation. Here we examine how chromatin binding and sequence preference may confer MeCP2 functionality, and connect relevant pathways in an active genome. Finding both genomic and proteomic evidence to indicate MeCP2 spliceosome interaction, we consequently discovered broad MeCP2 enrichment of the transcriptome while our focus toward long non-coding RNA (lncRNA) revealed MeCP2 association with RNCR3. Our data may indicate an as-yet-unappreciated role between lncRNA and MeCP2. We hypothesize that ncRNA may mediate chromatin-remodeling events by interacting with MeCP2, thereby conferring changes in gene expression. We consider that these results may suggest new mechanisms of gene regulation conferred by MeCP2 and its interactions upon chromatin structure and gene function.

Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development.

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. The pattern of X-chromosome inactivation (XCI) is thought to play a role in phenotypic severity. In the present study, patterns of XCI were assessed by lacZ staining of embryos and adult brains of mice heterozygous for a X-linked Hmgcr-nls-lacZ transgene on a mutant mouse model of RTT. We found that there was no difference between the lacZ staining patterns in the brain of wild-type and heterozygous mutant embryos at embryonic day 9.5 (E9.5) suggesting that Mecp2 has no effect on the primary pattern of XCI. At 20 weeks of age, there was no significant difference between XCI patterns in the Purkinje cells in the cerebellum of heterozygous mutant and wild-type mice when the mutant allele was inherited from the mother. However, when the mutant allele was paternally inherited, a significant difference was detected. Thus, parental origin of the mutation may have a bearing on phenotype through XCI patterns. An estimation of the Purkinje cell precursor number based on XCI mosaicism revealed that, when the mutation was paternally inherited, the precursor number was less than that in the wild-type mice. Therefore, it is likely that the number of precursor cells allocated to the Purkinje cell lineage is affected by a paternally inherited mutation in Mecp2. We also observed that the pattern of XCI in cultured fibroblasts was significantly correlated with patterns in the Purkinje cells in mutant animals but not in wild-type mice.

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increasedSlc6a2mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on theSlc6a2promoter sequence by chromatin immunopurification strategies. IncreasedSlc6a2gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on theSlc6a2promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism ofSlc6a2gene regulation.

Cell lineage analyses trace the hierarchy of cell types derived from a progenitor population. Critical to these analyses is the ability to track reliably all or defined subsets of the clonal descendants of the progenitor population. This necessitates marking the cells with a heritable and cell autonomous marker. Transgenes encoding molecules that can be visualized directly in situ without compromising cell differentiation, such as the reporter, β-galactosidase encoded by lacZ and chloramphenicol acetyltransferase, encoded by the CAT gene are the most widely used. The lacZ can be readily detected using a sensitive histo-chemical assay such that cells in which the β-galactosidase gene is transcrip-tionally active produce a blue stain in tissue sections or in whole mounts (1). Some lineage studies demand the simultaneous detection of the lacZ product and other tissue-specific proteins or transcripts. In this chapter, we discuss the experimental strategies in which the lacZ transgene can be utilized in the analysis of cell lineages, and we detail assays for detecting β-galactosidase by X-gal histochemistry or immunological localization of the enzyme in combination with mRNA in situ hybridization, immunochemistry, and histochemical procedures, such as alkaline phosphatase staining. 1.1. Choosing the Appropriate Transgenic Animals for Lineage Analysis The expression of the lacZ transgene varies according to the nature of the regulatory elements (promoter and enhancer) that drive gene expression, the number of active copies, and the chromosomal domain where the transgene is localized. The choice of transgene is dictated by the specific questions to be addressed. Several examples of transgenes give ubiquitous nonlineage biased expression. For example, in transgenic mice where the lacZ gene is regulated by the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase promoter, the transgene is expressed in all lineages at high levels during development (2). Similarly, the ROSA-β-geo transgene produced by site-directed mutagenesis (3) and the lacZ reporter driven by rat β-actin promoter (4) also provide ubiquitous tagging of multiple cell lineages. Ubiquitously expressed transgenes provide the most ideal marker for unbiased cell lineage analyses, provided the lineage progenitors or founder cells to be studied can be isolated as a pure cell population. Integration of the transgene into a specific chromosome may be useful in some cases for tracing cell lineages. Transgenes that are expressed in the same pattern as neighboring genes at the site of integration provide a ready marker for any tissue-specific pattern of expression. For example, transgenes integrated into the X chromosome may behave like the endogenous X-linked gene; however, the fact that the expression pattern is due to a transgene, not an endogenous gene, must be taken into account when interpreting the results (5, 6). In female mice that carry the lacZ transgene on only one X chromosome, X-inactivation during embryonic development generates two cell populations, one that expresses the X-linked transgene and another that does not. Since the status of X-inactivation is heritable, descendants of either population are stable for the transgene expression. Several studies on the lineage relationship of cells in tissues, such as the retina, brain, and tongue, have been performed using the mosaicism of transgene expression generated by random X-chromosome inactivation (7–10). Finally, some transgenes display lineage and stage-specific expression through use of a tissue-specific promoter or as a consequence of their unique integration sites. For example, in Wnt1-lacZ transgenic mice, the transgene is regulated by the Wnt1 3′-cis-acting enhancer element, which directs specific expression to the dorsal part of the neural tube and subsequently in the neural crest cells derived from this region (11). Another example is R197 transgenic mice, in which the lacZ gene is expressed principally in the muscle lineage from early organogenesis stages onward as a consequence of its integration site (12). Provided that the expression of the transgene is tissue specific for a defined period of development, such transgenes offer an excellent tool for tracing the differentiation of cell lineages. A prerequisite for using these mice and any other transgenic lines is to establish the pattern of transmission and ascertain the tissue and temporal specificity of transgene expression.