Discovery of New Secondary Metabolites by Epigenetic Regulation and NMR Comparison from the Plant Endophytic Fungus Monosporascus eutypoides

Overexpression of the histone acetyltransferase and the 1H NMR spectroscopic experiments of the endophytic fungus Monosporascus eutypoides resulted in the isolation of two new compounds, monosporasols A (1) and B (2), and two known compounds, pestaloficin C (3) and arthrinone (4). Their planar structures and absolute configurations were determined by spectroscopic analysis including high resolution electrospray ionization mass spectroscopy (HRESIMS), one-dimensional (1D) and two-dimensional (2D) NMR, and calculated electronic circular dichroism data. Compounds 1–2 were screened in cytotoxic bioassays against HeLa, HCT-8, A549 and MCF-7 cells. Our work highlights the enormous potential of epigenetic manipulation along with the NMR comparison as an effective strategy for unlocking the chemical diversity encoded by fungal genomes.

Importance of parental genome balance in the generation of novel yet heritable epigenetic and transcriptional states during doubled haploid breeding

BackgroundDoubling the genome contribution of haploid plants has accelerated breeding in most cultivated crop species. Although plant doubled haploids are isogenic in nature, they frequently display unpredictable phenotypes, thus limiting the potential of this technology. Therefore, being able to predict the factors implicated in this phenotypic variability could accelerate the generation of desirable genomic combinations and ultimately plant breeding.ResultsWe use computational analysis to assess the transcriptional and epigenetic dynamics taking place during doubled haploids generation in the genome of Brassica oleracea. We observe that doubled haploid lines display unexpected levels of transcriptional and epigenetic variation, and that this variation is largely due to imbalanced contribution of parental genomes. We reveal that epigenetic modification of transposon-related sequences during DH breeding contributes to the generation of unpredictable yet heritable transcriptional states. Targeted epigenetic manipulation of these elements using dCas9-hsTET3 confirms their role in transcriptional regulation. We have uncovered a hitherto unknown role for parental genome balance in the transcriptional and epigenetic stability of doubled haploids.ConclusionsThis is the first study that demonstrates the importance of parental genome balance in the transcriptional and epigenetic stability of doubled haploids, thus enabling predictive models to improve doubled haploid-assisted plant breeding.

Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system

Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.