Tissue injury triggers activation of mesenchymal lineage cells into wound-repairing myofibroblasts, whose unrestrained activity leads to fibrosis. Although this process is largely controlled at the transcriptional level, whether the main transcription factors involved have all been identified has remained elusive. Here, we report multi-omics analyses unraveling Basonuclin 2 (BNC2) as a myofibroblast identity transcription factor. Using liver fibrosis as a model for in-depth investigations, we first show that BNC2 expression is induced in both mouse and human fibrotic livers from different etiologies and decreases upon human liver fibrosis regression. Importantly, we found that BNC2 transcriptional induction is a specific feature of myofibroblastic activation in fibrotic tissues. Mechanistically, BNC2 expression and activities allow to integrate pro-fibrotic stimuli, including TGFβ and Hippo/YAP1 signaling, towards induction of matrisome genes such as those encoding type I collagen. As a consequence, Bnc2 deficiency blunts collagen deposition in livers of mice fed a fibrogenic diet. Additionally, our work establishes BNC2 as potentially druggable since we identified the thalidomide derivative CC-885 as a BNC2 inhibitor. Altogether, we propose that BNC2 is a transcription factor involved in canonical pathways driving myofibroblastic activation in fibrosis.

The unique functional versatility of the liver is paramount for organismal homeostasis. Both liver development and adult functions are controlled by tightly regulated transcription factor networks, within which nuclear receptors (NRs) regulate essential functions of parenchymal and non-parenchymal cells. Acting as transcription factors sensitive to extracellular cues such as steroidal hormones, lipid metabolites and xenobiotics and modulated by intracellular signaling pathways, NRs orchestrate many aspects of hepatic physiology. While liver functional zonation and adaptability to fluctuating conditions are known to rely on a sophisticated cellular architecture, a comprehensive knowledge of NRs functions in the different liver cell types is still lacking. As a first step toward the accurate mapping of NR functions in mouse liver, we characterized their levels of expression in whole liver as a function of time and diet, and explored NRs isoform expression in hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells and liver sinusoidal cells. In addition, we leveraged liver single cell RNAseq studies to provide here an up-to-date compendium of NR expression in mouse liver in space and time.

The functional versatility of the liver is paramount for organismal homeostasis. Adult liver functions are controlled by a tightly regulated transcription factor network including nuclear receptors (NRs), which orchestrate many aspects of hepatic physiology. NRs are transcription factors sensitive to extracellular cues such as hormones, lipids, xenobiotics etc. and are modulated by intracellular signaling pathways. While liver functional zonation and adaptability to fluctuating conditions rely on a sophisticated cellular architecture, a comprehensive knowledge of NR functions within liver cell populations is still lacking. As a step toward the accurate mapping of NR functions in liver, we characterized their levels of expression in whole liver from C57Bl6/J male mice as a function of time and diet. Nr1d1 (Rev-erbα), Nr1d2 (Rev-erbβ), Nr1c2 (Pparβ/δ) and Nr1f3 (Rorγ) exhibited a robust cyclical expression in ad libitum-fed mice which was, like most cyclically expressed NRs, reinforced upon time-restricted feeding. In a few instances, cyclical expression was lost or gained as a function of the feeding regimen. NR isoform expression was explored in purified hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells and liver sinusoidal cells. The expression of some NR isoforms, such as Nr1h4 (Fxrα) and Nr1b1 (Rarα) isoforms, was markedly restricted to a few cell types. Leveraging liver single cell RNAseq studies yielded a zonation pattern of NRs in hepatocytes, liver sinusoidal cells and stellate cells, establishing a link between NR subtissular localization and liver functional specialization. In summary, we provide here an up-to-date compendium of NR expression in mouse liver in space and time.

IntroductionDespite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful.MethodsTo this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established.Results and discussionMutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.