RESUMEN
At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Inestabilidad Genómica , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Ataxias Espinocerebelosas/genética , Expansión de Repetición de Trinucleótido , Animales , Ataxina-7 , Sitios de Unión , Factor de Unión a CCCTC , Metilación de ADN , Proteínas de Unión al ADN/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/genéticaRESUMEN
Myotonic dystrophy (DM) is a complex multisystemic disorder linked to two different genetic loci. Myotonic dystrophy type 1 (DM1) is caused by an expansion of a CTG repeat located in the 3' untranslated region (UTR) of DMPK (myotonic dystrophy protein kinase) on chromosome 19q13.3. Myotonic dystrophy type 2 (DM2) is caused by an unstable CCTG repeat in intron 1 of ZNF9 (zinc finger protein 9) on chromosome 3q21. Therefore, both DM1 and DM2 are caused by a repeat expansion in a region transcribed into RNA but not translated into protein. The discovery that these two distinct mutations cause largely similar clinical syndromes put emphasis on the molecular properties they have in common, namely, RNA transcripts containing expanded, non-translated repeats. The mutant RNA transcripts of DM1 and DM2 aberrantly affect the splicing of the same target RNAs, such as chloride channel 1 (ClC-1) and insulin receptor (INSR), resulting in their shared myotonia and insulin resistance. Whether the entire disease pathology of DM1 and DM2 is caused by interference in RNA processing remains to be seen. This review focuses on the molecular significance of the similarities and differences between DM1 and DM2 in understanding the disease pathology of myotonic dystrophy.
Asunto(s)
Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Animales , Expansión de las Repeticiones de ADN , Humanos , Distrofia Miotónica/clasificación , Distrofia Miotónica/etiologíaRESUMEN
Prior studies of the DM1 locus have shown that the CTG repeats are a component of a CTCF-dependent insulator element and that repeat expansion results in conversion of the region to heterochromatin. We now show that the DM1 insulator is maintained in a local heterochromatin context: an antisense transcript emanating from the adjacent SIX5 regulatory region extends into the insulator element and is converted into 21 nucleotide (nt) fragments with associated regional histone H3 lysine 9 (H3-K9) methylation and HP1gamma recruitment that is embedded within a region of euchromatin-associated H3 lysine 4 (H3-K4) methylation. CTCF restricts the extent of the antisense RNA at the wild-type (wt) DM1 locus and constrains the H3-K9 methylation to the nucleosome associated with the CTG repeat, whereas the expanded allele in congenital DM1 is associated with loss of CTCF binding, spread of heterochromatin, and regional CpG methylation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Sitios de Carácter Cuantitativo/genética , ARN sin Sentido/biosíntesis , Proteínas Represoras/metabolismo , Transcripción Genética/genética , Repeticiones de Trinucleótidos/genética , Factor de Unión a CCCTC , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Fibroblastos/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Elementos Aisladores/genética , Metilación , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN sin Sentido/genética , Proteínas Represoras/genéticaRESUMEN
Myotonic dystrophy 1 (DM1) is the most common inherited neuromuscular disease in adults. The disorder, characterized by myotonia, muscle wasting and weakness, cataract, insulin resistance, and mental impairment, is caused by the expansion of an unstable CTG repeat located in the 3' untranslated region of DMPK. The repeat expansion suppresses the expression of the homeobox gene SIX5. We describe here an experimental system to identify downstream transcriptional targets of mouse Six5 in order to elucidate the role of SIX5 in the pathogenesis of DM1 and development. By overexpressing a constitutively active Six5 (VP16-Six5wt) using adenovirus-mediated gene transfer in P19 cells and subsequent expression profiling using cDNA arrays, 21 genes, whose expression level increased by the treatment, were identified as potential target genes. Genes expressed in the somites, skeletal muscles, brain and meninges comprised the majority, suggesting the role of Six5 in the development and function of mesodermal tissues and brain. We provide evidence that Igfbp5 encoding a component of IGF signaling is a direct Six5-target. Moreover, the overall expression level of Igfbp5 was decreased in Six5-deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD-induced muscle conversion was altered in cells of DM1 patients. Our results not only identify Six5 as an activator that directs Igfbp5 expression but also suggest that reduced SIX5 expression in DM1 might contribute to specific aspects of the DM1 phenotype.
