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1.
Nat Neurosci ; 18(1): 17-24, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25420066

ABSTRACT

Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro.


Subject(s)
Fibroblasts , Models, Neurological , Nociceptors , Pain/physiopathology , Sensory Receptor Cells , Animals , Dysautonomia, Familial/pathology , Electrophysiological Phenomena/physiology , Humans , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Peripheral Nervous System Diseases/pathology , Transcription Factors
2.
Neuroreport ; 19(4): 393-8, 2008 Mar 05.
Article in English | MEDLINE | ID: mdl-18287934

ABSTRACT

Rett syndrome, a pervasive X-linked neurodevelopmental disorder in young girls, is caused by loss-of-function mutations in the gene that encodes the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2). Mecp2-knockout mice phenocopy the major symptoms found in human patients and have advanced our understanding of the function of MeCP2 and mechanism of Rett syndrome. To study the behavior of the MeCP2 protein in vivo, we have generated a knock-in reporter mouse model that expresses MeCP2-enhanced green fluorescent protein (EGFP) fusion protein instead of endogenous MeCP2. Here we show that expression of the fusion protein in the brain remarkably mirrors endogenous MeCP2 expression in all temporal and spatial aspects. This mouse model may be a valuable tool for studying Rett syndrome and for developing therapies.


Subject(s)
Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Methyl-CpG-Binding Protein 2/genetics , Recombinant Fusion Proteins/genetics , Rett Syndrome/genetics , Rett Syndrome/metabolism , Animals , Brain/growth & development , Brain/metabolism , Brain/physiopathology , Disease Models, Animal , Gene Expression Regulation, Developmental/genetics , Genetic Predisposition to Disease/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Biology/methods , Mutation/genetics , Rett Syndrome/physiopathology , Staining and Labeling/methods
3.
Nat Genet ; 39(3): 391-6, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17322882

ABSTRACT

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.


Subject(s)
Colorectal Neoplasms/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , Mitosis , Alleles , Cell Proliferation , Cell Survival , Colorectal Neoplasms/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/physiology , DNA Methylation , HCT116 Cells , Humans , Models, Biological , Models, Genetic
4.
J Biol Chem ; 280(18): 17986-91, 2005 May 06.
Article in English | MEDLINE | ID: mdl-15757890

ABSTRACT

DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G(1) to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism.


Subject(s)
Chromosomal Instability/physiology , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , DNA/metabolism , Fibroblasts/enzymology , Animals , Cell Line, Transformed , Cells, Cultured , DNA/genetics , DNA (Cytosine-5-)-Methyltransferases/deficiency , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Mice , Mice, Inbred C57BL , Mice, Knockout
5.
Mol Cell Biol ; 24(20): 9048-58, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15456878

ABSTRACT

Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , Heterochromatin/metabolism , Microsatellite Repeats , Animals , Cell Differentiation , DNA/metabolism , DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Heterochromatin/genetics , Humans , Mice , NIH 3T3 Cells , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stem Cells/cytology , Stem Cells/physiology , DNA Methyltransferase 3B
6.
Nature ; 429(6994): 900-3, 2004 Jun 24.
Article in English | MEDLINE | ID: mdl-15215868

ABSTRACT

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/metabolism , Genes, Essential/genetics , Genomic Imprinting/genetics , Alleles , Animals , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , DNA Methyltransferase 3A , Embryo Loss/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Gene Deletion , Gene Expression Regulation, Developmental , Male , Mice , Mice, Knockout , Mutagenesis/genetics , Phenotype , Sex Characteristics , Spermatogenesis , Spermatogonia/cytology , Spermatogonia/metabolism
7.
J Biochem ; 135(3): 413-20, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15113840

ABSTRACT

alphaB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the alphaB-crystallin gene in a head-to-head manner. In the present study, we found a highly conserved region of 220 bp approximately 2.4-kb upstream of the alphaB-crystallin gene and examined its role in expression of the alphaB-crystallin gene. Transgenic mice containing 3 kb of the upstream sequence of the alphaB-crystallin gene showed lacZ reporter gene expression in the lens as well as the myotome and heart on embryonic day 12.5. Deletion analysis revealed that the -2656/-2267 region including the conserved region with four putative Sox binding elements (E1-E4) exhibits lens enhancer activity toward the alphaB-crystallin promoter. Gel shift assays showed that the Sox1 and Sox2 proteins preferentially bound to E2 and E4. Moreover, disruption of E2 and E4 abolished the reporter gene expression in the lens. These results indicate that the newly identified enhancer with Sox elements activates the alphaB-crystallin promoter in the lens, although they are separated by the entire HSPB2 gene.


Subject(s)
DNA-Binding Proteins/metabolism , Embryo, Mammalian/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , High Mobility Group Proteins/metabolism , Lens, Crystalline/metabolism , Nuclear Proteins/metabolism , alpha-Crystallin B Chain/genetics , Animals , Base Sequence , Conserved Sequence/genetics , Genes, Reporter/genetics , HMGB Proteins , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/genetics , Lens, Crystalline/embryology , Mice , Mice, Transgenic , Molecular Sequence Data , Protein Binding , Response Elements/genetics , SOXB1 Transcription Factors , Transcription Factors
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