44 research outputs found
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 âpan-neuronalâ 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
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MeCP2 recognizes cytosine methylated tri-nucleotide and di-nucleotide sequences to tune transcription in the mammalian brain
Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause several neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG) is the classical MeCP2 DNA recognition sequence, but additional methylated sequence targets have been reported. Here we show by in vitro and in vivo analyses that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCAC + mCG density and unexpectedly defines large genomic domains within which transcription is sensitive to MeCP2 occupancy. Our results suggest that MeCP2 integrates patterns of mCAC and mCG in the brain to restrain transcription of genes critical for neuronal function
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
An Orphan CpG Island Drives Expression of a let-7 miRNA Precursor with an Important Role in Mouse Development.
Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members
SALL4 controls cell fate in response to DNA base composition
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Neuronal non-CG methylation is an essential target for MeCP2 function
DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype
Tympanic Versus Rectal Thermometry in Pregnant Women
To assess the accuracy of the tympanic membrane thermometer for use with pregnant women. Design : Cross-sectional descriptive study. Setting : A major medical center in the midwestern United States. Participants : Thirty-three hospitalized, afebrile pregnant women. Main outcome measures : Tympanic membrane thermometers and glass mercury thermometers were used to measure body temperature at the ear and rectum, respectively. The results were compared using two statistical methods: the Pearson correlation coefficient and a new technique suggested by Bland and Altman (1986). Results : Auditory canal temperature measured by a tympanic membrane thermometer correlated with rectal temperature as measured by a glass mercury thermometer ( r = 0.38, p = 0.01). Thus, the tympanic membrane thermometer is acceptable for monitoring the body temperature of pregnant women. However, the device's estimation of rectal temperature is not clinically reliable. Conclusions : Tympanic membrane thermometers, when applied with direct measures, are acceptable for use with pregnant women. It is not recommended that the rectal estimate mode be used with pregnant women.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72991/1/j.1552-6909.1995.tb02556.x.pd
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