Targeting the fusion protein RIOX1-dSpCas9 to the promoter region of B4GALT1 for removal of the histone mark H3K4me3

Abstract

Šest epigenetičkih mehanizama simultanim djelovanjem regulira gensku ekspresiju, a njihove su interakcije, zastupljenosti i efekti na transkripcijski status određenog gena specifični u odnosu na kromatinski „okoliš“. Cilj istraživanja bio je ukloniti histonsku oznaku H3K4me3 u promotorskoj regiji gena B4GALT1 pomoću fuzijskog proteina RIOX1-dSpCas9 navođenog na specifičnu regiju sa šest različitih molekula sgRNA (od engl. single guide RNA). Također, cilj je bio dizajnirati testove za analizu stupnja metilacije pirosekvenciranjem ove regije. Metodom kromatinske precipitacije CUT&RUN, nakon koje slijedi qPCR, utvrđeno je da nije došlo do statistički značajnog smanjenja u količini histonske oznake H3K4me3 kao ni do povećanja stupnja metilacije DNA u analiziranoj regiji. Metodom kvantitativnog PCR je utvrđeno da nije došlo do promjene u genskoj ekspresiji. Gen B4GALT1 ima složenu genetičku i epigenetičku regulaciju, a efekt fuzijskog proteina RIOX1-dSpCas9 mogao je ostati nezapažen zbog uskog područja koje je analizirano, ali i zbog već prisutnih epigenetičkih modifikacija i strukture kromatina koji su mogli onemogućiti njegovo vezanje ili katalitičku aktivnost.Gene expression is regulated by six epigenetic mechanisms that work simultaneously. Their interactions as well as their effects on the transcriptional status of a certain gene are specific and depend on chromatin context. The goal of this research was to remove the histone mark H3K4me3 from the promoter region of the B4GALT1 gene using the fusion protein RIOX1-dSpCas9, which was guided to a specific region by six different sgRNA molecules. Also, the goal was to design assays for pyrosequencing in order to analyze DNA methylation level in this region. The CUT&RUN chromatin precipitation method followed with qPCR revealed no statistically significant change in the amount of histone mark H3K4me3 nor in DNA methylation level of the analyzed region. Quantitative PCR showed no change in the the B4GALT1 gene transcriptional activity. The B4GALT1 gene has complex genetic and epigenetic regulation, and the effect of the RIOX1-dSpCas9 fusion protein could have gone unnoticed due to the narrow region in the gene promoter that was analyzed. Also, the already present epigenetic modifications and chromatin structure in the targeted region could have prevent the binding or catalytic activity of RIOX1-dSpCas9