Project description:The function and retention/reprogramming of epigenetic marks during the germline-to-embryo transition is a key issue in developmental and cellular biology, with relevance to stem cell programming and trans-generational inheritance. In zebrafish, DNAme patterns are programmed in transcriptionally-quiescent early cleavage embryos; paternally-inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal pattern. Here we show that a ‘placeholder’ nucleosome, containing the histone H2A variant H2A.Z(FV) and H3K4me1, occupies virtually all regions lacking DNAme in both sperm and cleavage embryos – residing at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with the Placeholder become either active H3K4me3-marked or silent H3K4me3/K27me3-marked (bivalent). Importantly, functional perturbation causing Placeholder loss confers DNAme acquisition, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent stages (gamete-zygote-cleavage), an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNAme, poising parental genes for either gene-specific activation or facultative repression.
Project description:The function and retention/reprogramming of epigenetic marks during the germline-to-embryo transition is a key issue in developmental and cellular biology, with relevance to stem cell programming and trans-generational inheritance. In zebrafish, DNAme patterns are programmed in transcriptionally-quiescent early cleavage embryos; paternally-inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal pattern. Here we show that a ‘placeholder’ nucleosome, containing the histone H2A variant H2A.Z(FV) and H3K4me1, occupies virtually all regions lacking DNAme in both sperm and cleavage embryos – residing at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with the Placeholder become either active H3K4me3-marked or silent H3K4me3/K27me3-marked (bivalent). Importantly, functional perturbation causing Placeholder loss confers DNAme acquisition, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent stages (gamete-zygote-cleavage), an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNAme, poising parental genes for either gene-specific activation or facultative repression.
Project description:The function and retention/reprogramming of epigenetic marks during the germline-to-embryo transition is a key issue in developmental and cellular biology, with relevance to stem cell programming and trans-generational inheritance. In zebrafish, DNAme patterns are programmed in transcriptionally-quiescent early cleavage embryos; paternally-inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal pattern. Here we show that a ‘placeholder’ nucleosome, containing the histone H2A variant H2A.Z(FV) and H3K4me1, occupies virtually all regions lacking DNAme in both sperm and cleavage embryos – residing at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with the Placeholder become either active H3K4me3-marked or silent H3K4me3/K27me3-marked (bivalent). Importantly, functional perturbation causing Placeholder loss confers DNAme acquisition, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent stages (gamete-zygote-cleavage), an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNAme, poising parental genes for either gene-specific activation or facultative repression.
Project description:Background & aims: MicroRNAs (miRNAs) encapsulated in EVs are potential diagnostic and prognostic biomarkers. However, discrepancies on miRNA patterns and their validation are still frequent due to differences in sample origin, EVs isolation, miRNA extraction and sequencing methods. Selecting appropriate EVs isolation methods is therefore a critical step for miRNA-based biomarker discovery. The aim of the present study is to find the most suitable EVs isolation method for miRNAs sequencing adequate for clinical application. Material & Methods EVs were isolated by Size Exclusion Chromatography (SEC), iodixanol gradients (GRAD) and the combination of both (SEC+GRAD), using the same plasma sample, in triplicate isolation assays. Isolated EVs were characterized and RNA was extracted. Three different protocols for miRNA library preparation were compared (NEBNext, NEXTFlex and SMARTer smRNA-seq) and miRNAs encapsulated on EVs were sequenced using NextSeq 500 system (Illumina). Finally, the yield, abundance and diversity of miRNAs using the three different EVs isolation protocols were analyzed and compared between them. Results The majority of lipoproteins, total cholesterol and plasma proteins were removed from the EVs-containing fractions by using SEC, GRAD, and SEC+GRAD. SEC method recovered a larger amount of EVs followed by GRAD and SEC+GRAD, while GRAD and SEC+GRAD yielded the purest vesicles. NEBNext was the library preparation kit that showed the highest reproducibility among replicas, higher number of reads corresponding to miRNAs and more different miRNAs, followed by NEXTFlex and SMARTer smRNA-seq. GRAD method showed the highest reproducibility among replicas, a higher number of reads corresponding to miRNAs and more different miRNAs, followed by SEC and SEC+GRAD methods. Conclusions These results render the GRAD method to isolate EVs as one of the most appropriate to detect miRNAs from Evs.