Recurrent disruptive mutations in chromodomain helicase DNA-binding protein 2 and 8 (CHD2 and CHD8) are emerging as prominent risk factors for Epilepsy and ASD, respectively. While both CHD2 and CHD8 play important roles in chromatin regulation and transcription, not fully dissected are the molecular consequences of the inactivating mutations described in patients. Here, we first investigated how chromatin reacts to CHD8 suppression by analysing a panel of histone modifications in human induced pluripotent stem cell-derived neural progenitors (hiNPC). CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affected highly expressed, CHD8-bound genes. Strikingly, the levels of transcription in cells presenting reduced H3K36me3 in the gene body appeared unchanged, however the process of alternative splicing was instead significantly affected. In particular, we have found aberrant alternative splicing patterns – mainly affecting alternative first and exon skipping events - of ~ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”. Mechanistically, mass-spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator Heterogeneous Nuclear Ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain CHD8-suppression splicing phenotype, partially implicating SETD2, H3K36me3 methyltransferase. Most of the mutations in CHD2 and CHD8 genes are disruptive, leading to protein haploinsufficiency. Thus, any molecular manipulation eliciting an increase in CHD2/CHD8 proteins, could prove beneficial for therapeutic development. Here, we intended to provide a Proof-of-Principle of how SINEUP - recently described class of non-coding RNAs able to augment, in a specific and controlled way, the expression of target proteins - can increase the translation of CHD2/CHD8 proteins and possibly rescue haploinsufficiency-associated phenotypes. With this purpose, we designed and cloned SINEUP targeting human CHD2/CHD8 isoforms and tested their efficacy in human induced pluripotent stem (iPS), induced neural progenitor cells (hiNPC) expressing normal and reduced levels of the target proteins and in patients’ fibroblasts bearing CHD8 heterozygous loss-of-function mutations. While stimulation by different CHD2/CHD8-SINEUP molecules didn’t elicit any effect in wild-type cells with physiologic levels of CHD2/CHD8, CHD2/CHD8-SINEUP were fully effective in haploinsufficient conditions, when reduced levels of the target proteins were expressed. Functionally, CHD8-SINEUP were able to revert molecular phenotypes associated to CHD8-suppression, i.e. the transcriptional dysregulation of the ASD-related genes, MBD3 and SHANK3, and restore the genome-wide reduction of H3K36me3 enrichment. Strikingly, chd8-SINEUP injection in vivo, in chd8-morpholino treated, developing zebrafish embryos, confirmed that stimulation of internal methionine could rescue chd8-suppression induced macrocephaly phenotype. In conclusion, CHD2/CHD8-SINEUP molecules represent a Proof-of-Concept towards the development of an RNA-based therapy for neurodevelopmental syndromes, with implications for and beyond ASD and epilepsy, surely relevant to a large repertory of presently incurable brain genetic diseases.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/338434 |
| Date | 28 April 2022 |
| Creators | Arnoldi, Michele |
| Contributors | Arnoldi, Michele, Biagioli, Marta, Denti, Michela Alessandra |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/embargoedAccess |
| Relation | firstpage:1, lastpage:228, numberofpages:228 |
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