Project description:Endomitosis is a non-canonical cell cycle in which cells undergo all four phases of a canonical cell cycle (G1, S, G2 and M), but do not initiate or complete cytokinesis during M phase. To identify the transcriptional differences between endomitosis and canonical cell cycles in C. elegans, we isolated cells from L1 larvae of a strain expressing intestinal-specific mCherry (elt-2p::mCherry-PH-P2A-H2B-mCherry) grown for 6 hours at 20ºC. We sorted intestinal and non-intestinal cells both with a 4N DNA content by FACS based on the mCherry expression and the Hoechst-34580 profile. We performed RNA-seq in intestinal (3 replicates) and non-intestinal cells (3 replicates) and differential gene expression analysis to identify the genes that are upregulated or downregulated in intestinal endomitosis compared to canonical cell cycles.
Project description:Endomitosis is a non-canonical cell cycle in which cells undergo all four phases of a canonical cell cycle (G1, S, G2 and M), but do not initiate or complete cytokinesis during M phase. We found that cytokinesis genes, as well as many cell-cycle genes, are transcriptionally downregulated during endomitosis of the C. elegans intestine. To understand the mechanisms underlying cell-cycle gene repression, we isolated cells from L1 larvae of a strain expressing intestinal-specific mCherry (elt-2p::mCherry-PH-P2A-H2B-mCherry) grown for one hour at 20ºC. We sorted intestinal and non-intestinal cells by FACS based on the mCherry expression. We performed ChIC-seq (or CUT&RUN) to map the H3K4me3, H3K9me3, H3K27me3 and H3K36me3 histone modifications and analyze whether promoter regions of cell-cycle genes are more enriched with active or repressive marks in intestinal cells undergoing endomitosis.
Project description:Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. Knockouts of the O-GlcNAc cycling enzymes in C. elegans are viable and fertile, allowing a global analysis of the impact of GlcNAcylation. Whole genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show phenotypically altered lifespan and susceptibility to UV stress. L1 larvae were synchronized by starvation for two days after hatching in sterile buffer. Wild type and mutant animals were collected and total RNA isolated for gene expression analysis
Project description:Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signalling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset – lips-15 – and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signalling pheromones. Together, our data implicate a soluble signalling molecule in density sensing by L1 stage C. elegans, and provide guidance for design of experiments focused on early developmental gene regulation.
Project description:Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. Knockouts of the O-GlcNAc cycling enzymes in C. elegans are viable and fertile, allowing a global analysis of the impact of GlcNAcylation. Here we compare gene expression in wild type and O-GlcNAc mutants (ogt-1 and oga-1) in synchronized, fed L1 animals. Whole genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show phenotypically altered lifespan and susceptibility to UV stress.