Muhire, B. | bioRxiv Science

bioRxiv2022

Stimulation-responsive enhancers regulate inflammatory gene activation through retention and modification of H2A.Z-variant accessible nucleosomes

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies1,2. The mechanism of gene regulation in response to dmPGE2 is poorly understood. Here, we show that dmPGE2 regulates inflammatory gene induction by modulating the chromatin architecture and activity of enhancer elements in human HSPCs. We identified the specific genomic reorganization at stimuli-responsive enhancers that permits rapid transcriptional activation. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes that are permissive to binding of the transcription factor CREB. CREB binding to enhancer nucleosomes is concomitant with deposition of the histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone variant nucleosomes retained within inducible enhancers facilitate transcription factor (TF) binding. Acetylation of histone variant nucleosomes by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism by which inflammatory mediators such as dmPGE2 lead to acute transcriptional changes and alter HSPC behavior.

developmental biology↗

bioRxiv2022

Combined single-sample metabolomics and RNAseq reveals a hepatic pyrimidine metabolic response to acute viral infection

ObjectiveMetabolomics and RNA sequencing (RNAseq) each provide powerful readouts of phenotype, and integration of these data can provide information greater than the sum of their parts. The ability to conduct such analysis on a single sample has many practical advantages, especially when dealing with rare or difficult-to-obtain samples. While methods exist to isolate multiple biomolecular subclasses from the same sample, in-depth analysis of the suitability of these approaches for multi- omics readouts is lacking. MethodsMice were injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh) control and liver tissue was harvested 2.5-days later. RNA was isolated from aliquots of pulverized liver tissue either following metabolite extraction using 80% methanol (MetRNA) or directly from frozen tissue (RNA). RNA sequencing data was evaluated by differential expression analysis via edgeR and dispersion using Ginis mean differences. Differential metabolite abundance was assessed using LIMMA. Pathway enrichment analysis was conducted on metabolomics and RNAseq data using MetaboAnalysts joint-integration tools. ResultsPrior metabolite extraction had no deleterious effects on quality or quantity of isolated RNA. RNA and MetRNA generated from the same sample clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Of the 2,169 genes that were differentially expressed between LCMV and Veh, the vast majority (n=1,848) were shared between extraction method, with the remainder evenly divided between RNA (n=165) and MetRNA (n=156). These differentially expressed genes unique to extraction method were attributed to randomness around the false discovery rate (FDR) = 0.05 cutoff and stochastic changes in variance estimation. Gini analysis further revealed that extraction method had no effect on the dispersion of detected transcripts across the entire dataset. To demonstrate the power of multi-omics integration on interrogated metabolic phenotypes, we next performed integrated pathway enrichment analysis on RNAseq data and metabolomics data. Our analysis revealed pyrimidine metabolism as the most impacted pathway by LCMV infection. Plotting up- and down-regulated genes and metabolites on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pyrimidine pathway exposed a pattern enzymatic degradation of pyrimidine nucleotides to generate the nucleobase uracil. Further, uracil was among the most differentially abundant metabolite in serum of LCMV infected mice, suggesting a novel mechanism of hepatic uracil export in acute infection response. ConclusionsWe demonstrate that prior metabolite extraction does not have a deleterious effect on RNAseq quality, which enables investigators to confidently perform metabolomics and RNAseq on the same sample. Implementation of this approach revealed a novel involvement of the hepatic pyrimidine metabolism during acute viral infection.

molecular biology↗