DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases.
Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heatshock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allelespecific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.
BackgroundTo date, 43 types of Spinocerebellar Ataxias (SCAs) have been identified. A subset of the SCAs are caused by the pathogenic expansion of a CAG repeat tract within the corresponding gene. Ethnic and geographic differences are evident in the prevalence of the autosomal dominant SCAs. Few descriptions of the clinical phenotype and molecular genetics of the SCAs are available from the African continent. Established studies mostly concern the South African populations, where there is a high frequency of SCA1, SCA2 and SCA7. The SCA7 mutation in South Africa (SA) has been found almost exclusively in families of indigenous Black African ethnic origin.ObjectiveTo present the results of the first clinical description of seven Zambian families presenting with autosomal dominant SCA, as well as the downstream molecular genetic analysis of a subset of these families.MethodsThe study was undertaken at the University Teaching Hospital in Lusaka, Zambia. Ataxia was quantified with the Brief Ataxia Rating Scale derived from the modified international ataxia rating scale. Molecular genetic testing for 5 types of SCA (SCA1, SCA2, SCA3, SCA6 and SCA7) was performed at the National Health Laboratory Service at Groote Schuur Hospital and the Division of Human Genetics, University of Cape Town, SA. The clinical and radiological features were evaluated in seven families with autosomal dominant cerebellar ataxia. Molecular genetic analysis was completed on individuals representing three of the seven families.ResultsAll affected families were ethnic Zambians from various tribes, originating from three different regions of the country (Eastern, Western and Central province). Thirty-four individuals from four families had phenotypic features of SCA7. SCA7 was confirmed by molecular testing in 10 individuals from 3 of these families. The age of onset of the disease varied from 12 to 59 years. The most prominent phenotypic features in these families were gait and limb ataxia, dysarthria, visual loss, ptosis, ophthalmoparesis/ophthalmoplegia, pyramidal tract signs, and dementia. Affected members of the SCA7 families had progressive macular degeneration and cerebellar atrophy. All families displayed marked anticipation of age at onset and rate of symptom progression. The pathogenic SCA7 CAG repeat ranges varied from 47 to 56 repeats. Three additional families were found to have clinical phenotypes associated with autosomal dominant SCA, however, DNA was not available for molecular confirmation. The age of onset of the disease in these families varied from 19 to 53 years. The most common clinical picture in these families included a combination of cerebellar symptoms with slow saccadic eye movements, peripheral neuropathy, dementia and tremor.ConclusionSCA is prevalent in ethnic Zambian families. The SCA7 families in this report had similar clinical presentations to families described in other African countries. In all families, the disease had an autosomal dominant pattern of inheritance across multiple generations. All families disp...
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