Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis1–9. Dynamic histone modifications may have important roles in MZT10–13, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps14 or require a highly specialized microfluidics device that is not readily available15. We developed a micro-scale chromatin immunoprecipitation and sequencing (μChlP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%-60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive role of histone marks for ZGA. In zebrafish, pre-ZGA development for ten cell cycles provides an opportunity to examine whether genomic enrichment in modified histones is present before initiation of transcription. By profiling histone H3 trimethylation on all zebrafish promoters before and after ZGA, we demonstrate here an epigenetic prepatterning of developmental gene expression. This involves pre-ZGA marking of transcriptionally inactive genes involved in homeostatic and developmental regulation by permissive H3K4me3 with or without repressive H3K9me3 or H3K27me3. Our data suggest that histone modifications are instructive for the developmental gene expression program.

Excessive alcohol intake can alter the gut microbiota, which may underlie the pathophysiology of alcohol-related diseases. We examined gut microbiota composition and functions in patients with alcohol overconsumption for >10 years, compared to a control group of patients with a history of no or low alcohol intake. Faecal microbiota composition was assessed by 16S rRNA sequencing. Gut microbiota functions were evaluated by quantification of short-chain fatty acids (SCFAs) and predictive metagenome profiling (PICRUSt). Twenty-four patients, mean age 64.8 years (19 males), with alcohol overconsumption, and 18 control patients, mean age 58.2 years (14 males) were included. The two groups were comparable regarding basic clinical variables. Nutritional assessment revealed lower total score on the screening tool Mini Nutritional Assessment, lower muscle mass as assessed by handgrip strength, and lower plasma vitamin C levels in the alcohol overconsumption group. Bacteria from phylum Proteobacteria were found in higher relative abundance, while bacteria from genus Faecalibacterium were found in lower relative abundance in the group of alcohol overconsumers. The group also had higher levels of the genera Sutterella, Holdemania and Clostridium, and lower concentration and percentage of butyric acid. When applying PICRUSt to predict the metagenomic composition, we found that genes related to invasion of epithelial cells were more common in the group of alcohol overconsumers. We conclude that gut microbiota composition and functions in patients with alcohol overconsumption differ from patients with low consumption of alcohol, and seem to be skewed into a putative pro-inflammatory direction.

Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs.

Embryo development proceeds from a cascade of gene activation and repression events controlled by epigenetic modifications of DNA and histones. Little is known about epigenetic states in the developing zebrafish, despite its importance as a model organism. We report here DNA methylation and histone modification profiles of promoters of developmentallyregulated genes (pou5f1, sox2, sox3, klf4, nnr, otx1b, nes, vasa), as well as tert and bactin2, in zebrafish embryos at the mid-late blastula transition, shortly after embryonic genome activation. We identify four classes of promoters based on the following profiles: (i) those enriched in marks of active genes (H3K9ac, H4ac, H3K4me3) without transcriptionally repressing H3K9me3 or H3K27me3; (ii) those enriched in H3K9ac, H4ac and H3K27me3, without H3K9me3; one such gene was klf4, shown by in situ hybridization to be mosaically expressed, likely accounting for the detection of both activating and repressive marks on its promoter; (iii) those enriched in H3K4me3 and H3K27me3 without acetylation; and (iv) those enriched in all histone modifications examined. Culture of embryo-derived cells under differentiation conditions leads to H3K9 and H4 deacetylation and H3K9 and H3K27 trimethylation on genes that are inactivated, yielding an epigenetic profile similar to those of fibroblasts or muscle. All promoters however retain H3K4me3, indicating an uncoupling of H3K4me3 occupancy and gene expression. All non-CpG island developmentallyregulated promoters are DNA unmethylated in embryos, but hypermethylated in fibroblasts. Our results suggest that differentially expressed embryonic genes are regulated by various patterns of histone modifications on unmethylated DNA, which create a developmentally permissive chromatin state.

Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions.I n yeast and in more complex eukaryotes, cell growth is restricted by the rate of mRNA translation and ribosome biogenesis, which depend on the transcription of ribosomal protein genes (RPGs), tRNAs and rRNAs. Synthesis of rRNA, tRNAs, and 5S rRNA represents 75% of total cellular transcription, whereas transcription of RPGs corresponds to 50% of RNA polymerase II (RNAPII) initiation events (1). These processes consume a significant portion of the cell's resources, making nutrient availability a limiting factor to cell growth and proliferation (2). The conserved rapamycin-sensitive target of rapamycin complex 1 (TORC1) is a master regulator of the cellular nutrient response (2, 3). Under nitrogen-rich conditions, TORC1 promotes growth-related processes, like protein synthesis, ribosome biogenesis, and tRNA synthesis, while inhibiting catabolic processes, like autophagy (2). Conversely, inhibition of TORC1 activity by nitrogen depletion (or addition of the TORC1 inhibitor rapamycin) results in a metabolic switch from anabolism to catabolism, which involves many cellular processes, including down-regulation of transcription of RPGs, rRNA and tRNA genes (2, 3).A key downstream target of TORC1 in regulation of tRNA transcription is the conserved RNAPIII inhibitor Maf1, which is phosphorylated and maintained in the cytoplasm under nitrogenrich conditions (2, 3). Maf1 becomes hypophosphorylated under conditions that inhibit TORC1, allowing it to enter the nucleus where it associates with TFIIIB. The interaction between Maf1 and TFIIIB prevents the recruitment of RNAPIII and precludes transcription reinitiation at 5S rRNA and tRNA genes (4, 5). However, expression of an unphosphorylatable maf1 mutant does not completely repress tRNA expression in nutrient-replete cells (6), suggesting that dephosphorylation of Maf1 alone is not sufficient to fully inhibit RNAPIII. Indeed, inhibition of TORC1 also results in phospho...

Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA+ RNA content fluctuates significantly during zebrafish early developmental stages. As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA+ RNA.