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Assessing the effect of SUPT4H1 RNAi on the transcription of a repeat-containing reporter construct

ABSTRACT: SUPT4H1 is a transcription elongation factor comprising part of the RNA polymerase II complex. Recent studies propose a selective role for SUPT4H1 in transcription of repeat-containing transcripts, the translated products of which are a hallmark of neurodegeneration in disorders such as Huntington’s Disease and C9orf72-amyotrophic lateral sclerosis. To investigate the potential of SUPT4H1 as a therapeutic target in repeat-associated neurodegeneration, we engineered a reporter containing 71 hexanucleotide repeats (HNRs) encoding for pDi-amino acid repeats (DARPs) fused to EGFP. To approximate endogenous expression levels, the reporter also incorporated human C9orf72 intronic DNA immediately 5' of the hexanucleotide repeats. The reporter construct was integrated into the genome of HEK293t cells. We assessed the effect of depleting SUPT4H1 by RNAi interference on the transcriptome as a whole and on the DARP-EGFP reporter.

ORGANISM(S): Homo sapiens

PROVIDER: GSE121757 | GEO | 2019/01/01

REPOSITORIES: GEO

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C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD

Project description:Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100â??1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients. To compare the RNA Seq profiles from the cortex and spinal cord of transgenic mice expressing unexpanded human C9orf72 (F08, n=4), expanded human C9orf72 (F112, n=3/4), and nontransgenic controls (n=4).

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Project description:C9ORF72 Hexanucleotide Repeat Sequence Genotyping Project

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C9ORF72 Hexanucleotide Repeat Sequence Genotyping Project

Project description:C9ORF72 Hexanucleotide Repeat Sequence Genotyping Project

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