RESUMEN
Objective: Heterozygous mutations within the voltage-gated sodium channel α subunit (SCN1A) are responsible for the majority of cases of Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. Development of novel therapeutic approaches is mandatory in order to directly target the molecular consequences of the genetic defect. The aim of the present study was to investigate whether cis-acting long non-coding RNAs (lncRNAs) of SCN1A are expressed in brain specimens of children and adolescent with epilepsy as these molecules comprise possible targets for precision-based therapy approaches. Methods: We investigated SCN1A mRNA expression and expression of two SCN1A related antisense RNAs in brain tissues in different age groups of pediatric non-Dravet patients who underwent surgery for drug resistant epilepsy. The effect of different antisense oligonucleotides (ASOs) directed against SCN1A specific antisense RNAs on SCN1A expression was tested. Results: The SCN1A related antisense RNAs SCN1A-dsAS (downstream antisense, RefSeq identifier: NR_110598) and SCN1A-usAS (upstream AS, SCN1A-AS, RefSeq identifier: NR_110260) were widely expressed in the brain of pediatric patients. Expression patterns revealed a negative correlation of SCN1A-dsAS and a positive correlation of lncRNA SCN1A-usAS with SCN1A mRNA expression. Transfection of SK-N-AS cells with an ASO targeted against SCN1A-dsAS was associated with a significant enhancement of SCN1A mRNA expression and reduction in SCN1A-dsAS transcripts. Conclusion: These findings support the role of SCN1A-dsAS in the suppression of SCN1A mRNA generation. Considering the haploinsufficiency in genetic SCN1A related DS, SCN1A-dsAS is an interesting target candidate for the development of ASOs (AntagoNATs) based precision medicine therapeutic approaches aiming to enhance SCN1A expression in DS.
RESUMEN
Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. Here, we created an Sce knockout allele and show that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression.