RESUMEN
Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.
Asunto(s)
Azauridina , Proteína Huntingtina , Enfermedad de Huntington , Proteínas Mutantes , Mutación , Proteínas Nucleares , Fenotipo , Proteínas Represoras , Factores de Elongación Transcripcional , Alelos , Animales , Azauridina/farmacología , Células Cultivadas , Expansión de las Repeticiones de ADN , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Proteína Huntingtina/biosíntesis , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Mediciones Luminiscentes , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Proteínas Represoras/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
SUPT4H1 is a transcription elongation factor that makes up part of the RNA polymerase II complex. Recent studies propose a selective role for SUPT4H1 in the transcription of repeat-containing DNA, the translated products of which contribute to neurodegenerative disorders such as C9orf72-amyotrophic lateral sclerosis. To investigate the potential of SUPT4H1 as a therapeutic target in repeat-associated neurodegeneration, we depleted SUPT4H1 by RNA interference to inhibit the function of the SUPT4H1/SUPT5H transcription elongation complex. Depletion of SUPT4H1 leads to a global reduction in all cellular RNA, highlighting the significant challenges that are associated with targeting this molecule for the treatment of human disease. Any requirement of SUPT4H1 for transcription of specific transcripts should be interpreted in the context of global modulatory effects on the transcriptome.