Purpose During sepsis, an excessive inflammatory immune reaction contributes to multi-organ dysfunction syndrome (MODS), a critical condition associated with high morbidity and mortality; however, the molecular mechanisms driving MODS remain elusive. Methods We used RNA sequencing to characterize transcriptional changes in the early phase of sepsis, at 6, 12, 24 hour time points in lung, kidney, liver, and heart tissues, in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis murine model. Results The CLP surgery induced significant changes (adj. p -value<0.05) in expression of hundreds of transcripts in the four organs tested, with the highest number exceeding 2,000 differentially expressed genes (DEGs) in all organs at 12 hours post-CLP. Over-representation analysis by functional annotations of DEGs to the Reactome database revealed the immune system, hemostasis, lipid metabolism, signal transduction, and extracellular matrix remodeling biological processes as significantly altered in at least two organs, while metabolism of proteins and RNA were revelaed as being liver tissue specific in the early phase of sepsis. Conclusion RNA sequencing across organs and time-points in the CLP murine model allowed us to study the trajectories of transcriptome changes demonstrating alterations common across multiple organs as well as biological pathways altered in an organ-specific manner. These findings could pave new directions in the research of sepsis-induced MODS and indicate new sepsis treatment strategies.

Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.

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Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation - related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPβ) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3'UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in MCPIP1 than inMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

The protein Swi6 in Saccharomyces cerevisiae is a cofactor in two complexes that regulate the transcription of the genes controlling the G1/S transition. It also ensures proper oxidative and cell wall stress responses. Previously, we found that Swi6 was crucial for the survival of genotoxic stress. Here, we show that a lack of Swi6 causes replication stress leading to double-strand break (DSB) formation, inefficient DNA repair and DNA content alterations, resulting in high cell mortality. Comparative genome hybridization experiments revealed that there was a random genome rearrangement in swi6Δ cells, whereas in diploid swi6Δ/swi6Δ cells, chromosome V is duplicated. SWI4 and PAB1, which are located on chromosome V and are known multicopy suppressors of swi6Δ phenotypes, partially reverse swi6Δ genome instability when overexpressed. Another gene on chromosome V, RAD51, also supports swi6Δ survival, but at a high cost; Rad51-dependent illegitimate recombination in swi6Δ cells appears to connect DSBs, leading to genome rearrangement and preventing cell death. This article has an associated First Person interview with the first author of the paper.