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1.
Proc Natl Acad Sci U S A ; 110(34): 13938-43, 2013 Aug 20.
Article in English | MEDLINE | ID: mdl-23918391

ABSTRACT

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.


Subject(s)
Angelman Syndrome/genetics , Chromosomes, Human, Pair 15/genetics , Gene Expression Regulation/genetics , Prader-Willi Syndrome/genetics , RNA, Small Nucleolar/chemistry , Topotecan/pharmacology , Animals , Chromatin/drug effects , Chromatin Immunoprecipitation , Gene Silencing , Genetic Loci/genetics , Genomic Imprinting/genetics , HEK293 Cells , Humans , Immunoblotting , In Situ Hybridization, Fluorescence , Locus Control Region/genetics , Mice , Mice, Knockout , Neurons/metabolism , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Small Nucleolar/genetics , Real-Time Polymerase Chain Reaction , Statistics, Nonparametric , Ubiquitin-Protein Ligases/genetics , snRNP Core Proteins/genetics
2.
Mol Cell Biol ; 32(14): 2894-903, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22615490

ABSTRACT

Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) lead to disrupted neuronal function and can cause the neurodevelopmental disorder Rett syndrome. MeCP2 is a transcriptional regulator that binds to methylated DNA and is most abundant in neuronal nuclei. The mechanisms by which MeCP2 regulates gene expression remain ambiguous, as it has been reported to function as a transcriptional silencer or activator and to execute these activities through both gene-specific and genome-wide mechanisms. We hypothesized that posttranslational modifications of MeCP2 may be important for reconciling these apparently contradictory functions. Our results demonstrate that MeCP2 contains multiple posttranslational modifications, including phosphorylation, acetylation, and ubiquitylation. Phosphorylation of MeCP2 at S229 or S80 influenced selective in vivo interactions with the chromatin factors HP1 and SMC3 and the cofactors Sin3A and YB-1. pS229 MeCP2 was specifically enriched at the RET promoter, and phosphorylation of MeCP2 was necessary for differentiation-induced activation and repression of the MeCP2 target genes RET and EGR2. These results demonstrate that phosphorylation is one of several factors that are important for interpreting the complexities of MeCP2 transcriptional modulation.


Subject(s)
Methyl-CpG-Binding Protein 2/chemistry , Methyl-CpG-Binding Protein 2/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Brain/metabolism , Cell Differentiation , Cell Line , Cell Nucleus/metabolism , Ether-A-Go-Go Potassium Channels/genetics , Humans , Male , Methyl-CpG-Binding Protein 2/genetics , Mice , Molecular Sequence Data , Neurogenesis/genetics , Neurogenesis/physiology , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-ret/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rett Syndrome/etiology , Rett Syndrome/genetics , Rett Syndrome/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic
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