The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factorinduced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:b-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor-and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Summary SIRT3 is a member of the Sirtuin family of NAD+-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations, however the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis.

Long range regulatory interactions among distal enhancers and target genes are important for tissue-specific gene expression. Genome-scale identification of these interactions in a cell line-specific manner, especially using the fewest possible datasets, is a significant challenge. We develop a novel computational approach, Regulatory Interaction Prediction for Promoters and Long-range Enhancers (RIPPLE), that integrates published Chromosome Conformation Capture (3C) data sets with a minimal set of regulatory genomic data sets to predict enhancer-promoter interactions in a cell line-specific manner. Our results suggest that CTCF, RAD21, a general transcription factor (TBP) and activating chromatin marks are important determinants of enhancer-promoter interactions. To predict interactions in a new cell line and to generate genome-wide interaction maps, we develop an ensemble version of RIPPLE and apply it to generate interactions in five human cell lines. Computational validation of these predictions using existing ChIA-PET and Hi-C data sets showed that RIPPLE accurately predicts interactions among enhancers and promoters. Enhancer-promoter interactions tend to be organized into subnetworks representing coordinately regulated sets of genes that are enriched for specific biological processes and cis-regulatory elements. Overall, our work provides a systematic approach to predict and interpret enhancer-promoter interactions in a genome-wide cell-type specific manner using a few experimentally tractable measurements.

Legumes are essential components of agricultural systems because they enrich the soil in nitrogen and require little environmentally deleterious fertilizers. A complex symbiotic association between legumes and nitrogen-fixing soil bacteria called rhizobia culminates in the development of root nodules, where rhizobia fix atmospheric nitrogen and transfer it to their plant host. Here we describe a quantitative proteomic atlas of the model legume Medicago truncatula and its rhizobial symbiont Sinorhizobium meliloti, which includes more than 23,000 proteins, 20,000 phosphorylation sites, and 700 lysine acetylation sites. Our analysis provides insight into mechanisms regulating symbiosis. We identify a calmodulin-binding protein as a key regulator in the host and assign putative roles and targets to host factors (bioactive peptides) that control gene expression in the symbiont. Further mining of this proteomic resource may enable engineering of crops and their microbial partners to increase agricultural productivity and sustainability.

SUMMARY Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.