Mechanisms of aberrant HTT splicing and its implications on RNA homeostasis in Huntington’s disease

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Date

2025-01-10

Authors

Hoschek, Franziska

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Dissertation

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Abstract

Huntington disease (HD) is an autosomal dominant monogenic neurological disorder. The elongation of the CAG repeat in exon 1 of huntingtin (HTT) leads to the production of a short novel HTT isoform called HTT1a. HTT1a is a functional polyadenylated mRNA that contains the sequences of HTT exon 1 and a part of intron 1. Both the HTT1a mRNA as well as the encoded HTT exon 1 protein are known to be toxic. The generation of HTT1a is CAG repeat length dependent but the exact mechanism leading to HTT1a production remains unknown. In addition, it is unclear whether there is a tissue specific expression profile of HTT1a outside of the brain. Thus, a first approach in this study was to detect HTT1a in several different tissues and cell types. In addition to the involvement of the splicing machinery also regulators of transcription like R-loops and epigenetic modifications could contribute to the generation of HTT1a. Therefore, the involvement of DNA methylation, histone H3K27 trimethylations and R-loops at the HTT1a genomic region was analysed. The role of R-loops and epigenetics is not only interesting with regards to the production of HTT1a but also general transcriptional dysregulation in HD. Hence, the genome wide distribution of differentially methylated regions (DMRs), H3K27me3 marks and R-loops was investigated. For the detection of the HTT1a transcript in human peripheral tissues and brain a highly sensitive digital PCR (dPCR) assay was established. The analysis of transcriptional changes, epigenetics and R-loops was performed in primary human fibroblasts derived from healthy controls, adult onset and juvenile onset HD patients. Amplification-free, long read Nanopore sequencing was used for the analysis of the transcriptome and methylome. R-loops and H3K27me3 were detected by the novel method Cleavage under targets and tagmentation (CUT&Tag). Using dPCR, the HTT1a transcript could be detected not only in human post-mortem tissue in several brain regions but also in human muscle tissue, peripheral blood mononuclear cells (PBMCs) and fibroblasts. A significant, positive linear correlation between the expression levels of HTT1a and the CAG repeat length was found in the motor cortex and in PBMCs. Consequently, the measurement of HTT1a levels in easily accessible PBMCs could be a feasible biomarker in studies aiming at HTT lowering. The analysis of DNA methylation, H3K27me3 and R-loops in HTT exon 1 and intron 1 resulted in no significant differences neither between control and adult onset nor control and juvenile onset HD human fibroblasts. Based on the data obtained, the effect of the studied epigenetic factors on the generation of HTT1a in fibroblasts could not be clarified. However, fibroblasts are much less affected by transcriptional and possibly also epigenetic changes in HD than neurons and therefore these factors may well play a role in the generation of HTT1a in neuronal cells. On the genome wide level, the biggest differences in the transcriptome, epigenome and R-loops were found between control and juvenile onset HD fibroblasts. In contrast, HD mutation state was associated with fewer significant differences in the transcriptome, epigenome and R-loops. This was most likely due to the distinct findings in adult and juvenile onset HD fibroblasts. Overall, the number of significant differentially expressed genes or enriched epigenetic/R-loop regions was low. Only a few genes were identified that had a significant differentially enriched region of more than one type. Moreover, the overlap of genes with significant differentially enriched epigenetic or R-loop regions and differentially expressed genes was low. Nevertheless, TBX5 which was found to be significantly upregulated on the transcript level in juvenile onset HD fibroblasts also showed reduced H3K27me3 and DNA methylation, an epigenetic state associated with active transcription, at its transcription start site. TBX5, encoding T-box binding transcription factor 5, is interacting with transcription factors involved in HD pathogenesis. Consequently, TBX5 might be an interesting candidate to study in the context of transcriptional dysregulation in HD. The gene ontology and pathway enrichment analysis of the differentially expressed genes and genes containing at least one DMR or differential H3K27me3 region or R-loop resulted in multiple inconclusive pathways and processes. However, SUZ12 and EZH2 were identified as potential regulators in all juvenile HD onset gene sets. This is especially interesting as they are both part of the polycomb repressive complex 2, which is discussed to play a role in transcriptional dysregulation in HD. To sum up, in this work the CAG length dependent expression of HTT1a in PBMCs was shown which implies the assessment of HTT1a as potential future biomarker in clinical HD studies. Additionally, this study could further broaden the knowledge about transcriptional dysregulation in HD by analysing genome wide changes in epigenetic marks and R-loops.

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Faculties

Medizinische Fakultät

Institutions

UKU. Klinik für Neurologie
UKU. Abteilung für Gentherapie
UKU. Klinik für Herz-, Thorax- und Gefäßchirurgie

Citation

DFG Project uulm

EU Project THU

Other projects THU

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CC BY 4.0 International

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DOI external

DOI external

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DFG Project THU

item.page.thu.projectEU

item.page.thu.projectOther

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Keywords

Epigenetics, Transcription regulation, Huntington-Chorea, Epigenetik, Transkriptionsfaktor, Huntington disease, Transcription factors, Epigenomics, DDC 610 / Medicine & health