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Analysis of total RNA and ribosome-associated RNA in HTT KO MEF versus control.

ABSTRACT: Purpose: We previously highlighted that the wild-type protein Huntingtin (HTT) interacts with the ribosome in mouse embryonic fibroblasts (MEF). We previously showed that HTT does not control control new protein synthesis at a global level. Hence, we sought to determine whether HTT regulates translation of specific mRNA subsets. To this end, we performed a translatome analysis in HTT-deleted versus control samples. Methods: From HTT-deleted and control MEF, we isolated total cellular RNA via Trizol/chloroform extraction. From matching samples, we performed ribosome purification and isolated ribosome-associated RNA via Trizol/chloroform extraction of ribosome pellets. From n=3 independent pairs of replicates for total RNA and n=3 independent pairs of replicates for ribosome-associated RNA, libraries were prepared and sequenced (Genewiz) (total 12 samples). Read alignment and counting were performed using STAR and HTSeqCount softwares. We performed differential expression analysis comparing HTT-deleted samples to control samples for total RNA on one hand, and HTT-deleted samples to control samples for ribosome-associated RNA on the other hand. We did so using EdgeR with RUVseq normalization based on residuals (k=2). Results: For each RNA hit, we analysed differential association to the ribosome normalized to differential expression, by comparing HTT-deleted and control samples. From this analysis, we identified 115 genes displaying differential ribosome association upon HTT deletion, but for which total RNA expression level was unchanged (cut-offs: false discovery rate (FDR) p- value < 0.05, |log2 fold-change| > 1). Among the pool of translationally-only regulated hits, we found genes with decreased association with the ribosome in absence of HTT (Rxfp1, Kng1, Cxcl9, Trpm6, Wnt2b); and with increased association with the ribosome in absence of HTT (Trpm2, Tox2, Ip6k3, Csf3r, Tlr1). Conclusions: Our results highlight differential ribosome association at a single transcript level upon HTT deletion. Altogether, these data support the hypothesis that HTT regulates the translation of a specific subset of mRNA.

ORGANISM(S): Mus musculus

PROVIDER: GSE209946 | GEO | 2023/08/09

REPOSITORIES: GEO

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Project description:Despite extensive progress in Huntingtonâ??s Disease (HD) research, very little is known about the association of epigenetic variation and HD pathogenesis in human brain tissues. Moreover, its contribution to the tissue-specific transcriptional regulation of the huntingtin gene (HTT), in which HTT expression levels are highest in brain and testes, is currently unknown. To investigate the role of DNA methylation in HD pathogenesis and tissue-specific expression of HTT, we utilized the Illumina HumanMethylation450K BeadChip array to measure DNA methylation in a cohort of age-matched HD and control human cortex and liver tissues. In cortex samples, we found minimal evidence of HD-associated DNA methylation at probed sites after correction for cell heterogeneity but did observe an association to age of disease onset. By contrast, comparison of matched cortex and liver samples revealed tissue-specific DNA methylation of the HTT gene region at 38 sites (FDR < 0.05). Importantly, we identified a novel differentially methylated binding site in the HTT proximal promoter for the transcription factor CTCF. This CTCF site displayed increased occupancy in cortex, where HTT expression is higher, compared to liver. Additionally, CTCF silencing reduced the activity of a HTT promoter-reporter construct, suggesting that CTCF plays a role in regulating HTT promoter function. Overall, although we were unable to detect HD-associated DNA methylation alterations at queried sites, we found that DNA methylation may be correlated to age of disease onset in cortex tissues. Moreover, our data suggest that DNA methylation may, in part, contribute to tissue-specific HTT transcription through differential CTCF occupancy. The Illumina Infinium HumanMethylation450 Beadchip was used to obtain genome-wide DNA methylation measures in human cortex tissue (n = 13), with a subset of matched liver tissues (n = 5) , from a cohort of HD (n = 7) and control (n = 6) individuals in order to identify potential HD-related DNA methylation aberration in the brain as well as tissue-specific DNA methylation variation at the HTT gene locus.

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Analysis of total RNA and ribosome-associated RNA in HTT KO MEF versus control.

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