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RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells

ABSTRACT: To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on two indepedent Htt-null stable cell lines and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion. Conclusion: Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178467 | GEO | 2021/06/19

REPOSITORIES: GEO

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Project description:The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington’s disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD. To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results. The unbiased approach used in our study provides novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD. Keywords: cell type comparison we generate four Hdh-HET MEF cell lines and four Hdh-KO MEF cell lines, and performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray.

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RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells

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