Genome binding/occupancy profiling by high throughput sequencing
Summary
In order to get insight in the role of metabolic perturbations during the first steps of cell differentiation, the phenotype, gene expression profile and chromatin accessibility of CD34+ cells were examined using bulk- and single-cell techniques during the initial 96 hours of in vitro differentiation. Specific enzyme inhibitors targeting key steps of the energy metabolism were employed to induce perturbations and assess their effect. The results support a model whereby energy requirements play the role of initiator of differentiation. According to it, metabolic perturbations induce rapid and non-specific, enabling a state of multi-primed gene expression. A selective re-compaction of the chromatin constrained by the availability of key metabolites essential for repressive epigenetic modifications and by the action of transcription regulator proteins consolidates the phenotypic response of the cells.
Overall design
Human hematopoietic CD34+ cells were grown in Xvivo medium supplemented with cytokines with or without energy metabolism inhibitor (DON, 2-DG or AOA) during 03h, 12h or 24h. For each time point and condition, at least three biological replicates were examined (three different donors). For the control condition without inhibitor (CTRL), five replicates were used. 00h Xvivo time point corresponds to CD34+ cells grown without cytokines in the medium during a short period (less than 03h). Files containing the read count per detected peak per sample (one sample = merged biological replicates for the same time point) after the union of all peaks list (Reduce function) and DEseq normalization : DEseq_readcount_union_allsamples.rds (grange object to be used in R) DEseq_readcount_union_allsamples.csv (table to be read in excel)
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