Comparative analysis of potential broad-spectrum neuronal Cre drivers

Cre/Lox technology is a powerful tool in the mouse genetics tool-box as it enables tissue-specific and inducible mutagenesis of specific gene loci. Correct interpretation of phenotypes depends upon knowledge of the Cre expression pattern in the chosen mouse driver line to ensure that appropriate cell types are targeted. For studies of the brain and neurological disease a pan-neuronal promoter that reliably drives efficient neuron-specific transgene expression would be valuable. Here we compare a widely used “pan-neuronal” mouse Cre driver line, Syn1-cre, with a little-known alternative, Snap25-IRES2-cre. Our results show that the Syn1-cre line broadly expresses in the brain but is indetectable in more than half of all neurons and weakly active in testes. In contrast the Snap25-IRES2-cre line expressed Cre in a high proportion of neurons (~85%) and was indetectable in all non-brain tissues that were analysed, including testes. Our findings suggest that for many purposes Snap25-IRES2-cre is superior to Syn1-cre as a potential pan-neuronal cre driver

How human practices have affected vector-borne diseases in the past: a study of malaria transmission in Alpine valleys

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Incongruent restricted disjoint covering systems

We define an incongruent restricted disjoint covering system on [1,n] as a set of congruence classes such that each integer in the interval [1,n] belongs to exactly one class, and each class contains at least two members of the interval. In this paper we report some computational and structural results and present some open problems concerning such systems.7 page(s

Affinity for DNA Contributes to NLS Independent Nuclear Localization of MeCP2

MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target \beta-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease

Molecular basis of R133C Rett syndrome

Rett syndrome is a debilitating autistic spectrum disorder affecting one in ten thousand girls. Patients develop normally for up to eighteen months before a period of regression involving stagnation in head growth, loss of speech, hand use and mobility. It is almost exclusively caused by mutation in Methyl CpG binding Protein 2 (MeCP2). MeCP2 has traditionally been thought of as a transcriptional repressor, although its exact function remains unknown and it has recently been shown that the protein can also bind to hydroxymethylation and non-CpG methylation, which occurs predominantly at CAC sites in the mature nervous system. Genotype-phenotype studies of the most common Rett-causing mutations in affected patients revealed that a missense mutation, R133C results in a milder form of Rett syndrome. The reasons for this are unclear, as the mutation lies right in the heart of the methylated DNA binding domain. Previous in vitro studies of R133C showed a severe deficit in binding to methylated cytosine. A subsequent study found that R133C binding to hydroxymethylated cytosine was specifically impaired, whereas binding to methylated cytosine was indistinguishable from wildtype. Defining the DNA binding impairment of MeCP2R133C would yield important insights into Rett disease pathophysiology and provide an explanation for the phenotypic spectrum seen in patients. To shed light on these matters, a novel mouse model of the R133C mutation was created. The R133C mouse had a phenotype that was less severe than other missense mutant mice, in terms of survival, growth, Rett-like phenotypic score and some behavioural paradigms thus recapitulating the patient data. At the molecular level in adult mouse brain, MeCP2R133C protein abundance was reduced. Immunohistochemistry showed that MeCP2R133C had an abnormal pattern of localisation in the nucleus of neurons. In vitro electrophoretic mobility shift assays suggested that MeCP2R133C binding to (hydroxy)methyl-CAC may be reduced to a greater extent than binding to mCpG. Chromatin immunoprecipitation experiments confirmed the deficit in binding to methylated sites and supported a disproportionate reduction in binding to methylation in a CAC sequence context. Analysis of adult mouse cerebellar gene expression revealed a subtle upregulation of long genes and downregulation of short genes. Based on these data, it is proposed that Rett syndrome caused by the R133C mutation results from a combination of protein instability and defective binding to methylated DNA. Methyl-CAC binding is potentially abolished. The downstream biological consequence of this is a length-dependent deregulation of gene expression in the brain

DNA methylation reader MECP2:Cell type- and differentiation stage-specific protein distribution

Background: Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues. Results: Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2−/y) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2−/y and Mecp2wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2−/y mice. Conclusions: MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction

Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes

This is a pre-copyedited, author-produced version of an article accepted for publication in Human Molecular Genetics following peer review. The version of record Ross, P. D., et al. (2016). "Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes." Human Molecular Genetics 25(20): 4389-4404.] is available online at: https://doi.org/10.1093/hmg/ddw269Work in SC’s laboratory was supported by the Biotechnology and Biological Sciences Research Council (PhD studentship for PDR), a consortium grant from the Rett Syndrome Research Trust, the Chief Scientist Office (Scottish Executive Health Department) [grant ETM/334], RS Macdonald Charitable Trust, Rosetrees Trust [grant M530], and the Rett Syndrome Association Scotland. Work in AB’s laboratory was supported by a Consortium Grant from the Rett Syndrome Research Trust, by Wellcome Trust programme grant [091580] and by Wellcome Trust Centre Core Grant [092076]. Funding to pay the Open Access publication charges for this article was provided by the Scottish Executive Health Department

CpG islands influence chromatin structure via the CpG-binding protein Cfp1

CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides(1,2). Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity(3,4). In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro(5,6). Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins