Gene regulatory evolution in cold-adapted fly populations neutralizes plasticity and may undermine genetic canalization

The relationships between adaptive evolution, phenotypic plasticity, and canalization remain incompletely understood. Theoretical and empirical studies have made conflicting arguments on whether adaptive evolution may enhance or oppose the plastic response. Gene regulatory traits offer excellent potential to study the relationship between plasticity and adaptation, and they can now be studied at the transcriptomic level. Here we take advantage of three closely-related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. We measure the transcriptome-wide plasticity in gene expression levels and alternative splicing (intron usage) between warm and cold laboratory environments. We find that suspected adaptive changes in both gene expression and alternative splicing tend to neutralize the ancestral plastic response. Further, we investigate the hypothesis that adaptive evolution can lead to decanalization of selected gene regulatory traits. We find strong evidence that suspected adaptive gene expression (but not splicing) changes in cold-adapted populations are more vulnerable to the genetic perturbation of inbreeding than putatively neutral changes. We find some evidence that these patterns may reflect a loss of genetic canalization accompanying adaptation, although other processes including hitchhiking recessive deleterious variants may contribute as well. Our findings augment our understanding of genetic and environmental effects on gene regulation in the context of adaptive evolution.

Exercise reprograms the inflammatory landscape of multiple stem cell compartments during mammalian aging

Exercise has the ability to rejuvenate stem cells and improve tissue homeostasis and regeneration in aging animals. However, the cellular and molecular changes elicited by exercise have not been systematically studied across a broad range of cell types in stem cell compartments. To gain better insight into the mechanisms by which exercise affects niche and stem cell function, we subjected young and old mice to aerobic exercise and generated a single cell transcriptomic atlas of muscle, neural and hematopoietic stem cells with their niche cells and progeny. Complementarily, we also performed whole transcriptome analysis of single myofibers from these animals. We identified common and unique pathways that are compromised across these tissues and cell types in aged animals. We found that exercise has a rejuvenating effect on subsets of stem cells, and a profound impact in the composition and transcriptomic landscape of both circulating and tissue resident immune cells. Exercise ameliorated the upregulation of a number of inflammatory pathways as well as restored aspects of cell-cell communication within these stem cell compartments. Our study provides a comprehensive view of the coordinated responses of multiple aged stem cells and niche cells to exercise at the transcriptomic level.

RNA sequencing of chronic GVHD skin lesions defines shared and unique inflammatory pathways characterizing lichen planus and morphea

Cutaneous involvement of chronic graft-versus-host disease (cGVHD) has a wide range of manifestations including a lichenoid form with a currently assumed mixed Th1/Th17 signature and a sclerotic form with Th1 signature. Despite substantial heterogeneity of innate and adaptive immune cells recruited to the skin and of the different clinical manifestations, treatment depends mainly on the severity of the skin involvement, and relies on systemic, high-dose glucocorticoids alone or in combination with a calcineurin inhibitor. We performed the first study using RNAseq to profile and compare the transcriptome of lichen planus cGVHD (n=8), morphea cGVHD (n=5), and healthy controls (n=6). Our findings revealed shared and unique inflammatory pathways to each cGVHD subtype that are both pathogenic and targetable. In particular, the deregulation of IFN signaling pathway was strongly associated with cutaneous cGVHD, whereas the triggering receptor expressed on myeloid cells-1 (TREM-1) pathway was found to be specific of lichen planus and likely contributes to its pathogenesis. The results were confirmed at a protein level by performing immunohistochemistry staining and at a transcriptomic level using Real-Time quantitative PCR.

Characterization of metabolic compartmentalization in the liver using spatially resolved metabolomics

Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI and achieved broad coverage of central carbon, nucleotide, and lipid metabolism pathways. We used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic compartmentalization in livers, which becomes disrupted under nutrient stress conditions. Fasting caused changes in glucose metabolism and increased the levels of fatty acids in the circulation. In contrast, a prolonged high-fat diet (HFD) caused lipid accumulation within liver tissues with clear zonal patterns. Fatty livers had higher levels of purine and pentose phosphate related metabolites, which generates reducing equivalents to counteract oxidative stress. This MALDI MSI approach allowed the visualization of liver metabolic compartmentalization at high resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo .

Derivedness Index for Estimating Degree of Phenotypic Evolution of Embryos: A Study of Comparative Transcriptomic Analyses of Chordates and Echinoderms

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named “derivedness index” to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.

Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum

Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. Results We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. Conclusions The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions

Intratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

Multi-Omic Analysis of Chronic Myelomonocytic Leukaemia Monocytes Reveals Immune Dysregulation Mediated By TGF-β Activation

Introduction: Chronic myelomonocytic leukaemia (CMML) is a clonal haematological neoplasm characterised by persistent monocytosis and myeloid dysplasia. Treatment options are few and hampered by incomplete understanding of its core biology. CMML is genetically homogeneous compared to most cancers, with >90% of patients displaying recurrent mutations in a small group of epigenetic regulator genes. Despite this, CMML exhibits substantial clinical heterogeneity, suggesting an important role for epigenetic dysregulation in CMML biology. However, the CMML epigenome remains little studied. Methods: We performed multi-omic analyses on primary CD14 + monocytes from up to 13 CMML patients and 3 age-matched healthy controls, to identify regions of epigenetic dysregulation unique to CMML. Monocytes represent the defining downstream malignant cell population in CMML, contributing important disease features and supportive crosstalk with disease-initiating CMML stem cells; their targeting thus has therapeutic potential. We integrated RNA-seq, ATAC-seq and ChIP-seq for four histone marks, encompassing both activating and repressive marks (Fig 1A), to evaluate CMML monocytes at both the chromatin and transcriptome levels. Results: All datasets clearly separated CMML from control monocytes by principal component analysis, whilst revealing substantial epigenetic heterogeneity between patients (Fig 1B). Most of the differentially accessible regions were distal to genes, suggestive of widespread enhancer dysregulation in CMML. ROSE analysis identified novel superenhancers specific to CMML monocytes, including several mapping to genes previously implicated in CMML biology (e.g. CXCL8). Further analysis of differentially bound or accessible regions suggested consistent dysregulation of various pathways, including JAK/STAT, AKT and TREM1 signalling and the DNA damage response. Notably, there was strong epigenetic activation of the TGF-β pathway, with motifs for SMAD2, SMAD3, SMAD4 and FOXH1 consistently and strongly enriched across multiple datasets (Fig 1C, left). A signature of TGF-β target genes in monocytes, including many pro-survival genes, was also enriched in the matched RNA-seq data, suggesting a role for TGF-β activation in CMML monocytosis (Fig 1D, top). TGF-β signalling has been implicated in the monocyte-to-macrophage transition, but not previously as a driver of CMML biology. Concurrently, there was strong epigenetic downregulation of the NF-κB pathway, evidenced by loss of chromatin accessibility at NF-κB binding elements in CMML monocytes (Fig 1C, right). This suggests a block in the inflammatory response of monocytes, expected to result in a tolerant phenotype. The block in NF-κB signalling was not directly evident in the RNA-seq data, likely reflecting absence of inflammatory stimuli at sampling. Extensive crosstalk between TGF-β and NF-κB signalling is recognised, implicating TGF-β activation in the observed repression of the NF-κB pathway in our data. A tolerant phenotype in monocytes has been previously linked to increased mitochondrial biogenesis. Indeed, RNA-seq highlighted higher expression of genes encoding components of the oxidative phosphorylation machinery in CMML monocytes (Fig 1D, bottom), including ATP5J, COX7A2 and NDUFB1. Discussion: Combined transcriptomic and epigenomic analysis revealed profound dysregulation of the epigenetic landscape and of multiple signalling pathways in primary CMML monocytes. Whereas the ATAC-seq and ChIP-seq datasets aligned closely, significant discordance from the RNA-seq demonstrates the value of integrating multiple approaches for a complete picture of epigenetic dysregulation. Discordant changes identified at the chromatin but not transcriptomic level likely reflect poised potential. We describe TGF-β pathway activation in CMML for the first time, highlighting a potentially tractable therapeutic strategy. We propose a model whereby TGF-β activation directly represses NF-κB signalling potential in these cells, promoting a tolerant phenotype whilst conferring resistance to apoptosis (Fig 1E). This may be germane to the immune dysfunction (and propensity to autoimmunity) characteristic of CMML. Validation of lead candidate targets will be presented, highlighting novel therapeutic approaches for this disease of unmet clinical need. Figure 1 Figure 1. Disclosures Somervaille: Novartis: Consultancy, Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Astex: Research Funding; StemLine: Consultancy; Novartis: Consultancy; Takeda: Consultancy.

Potentiating antibiotic efficacy via perturbation of non-essential gene expression

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.