Comparative analysis of gene regulatory networks: from network reconstruction to evolution.

Regulation of gene expression is central to many biological processes. Although reconstruction of regulatory circuits from genomic data alone is therefore desirable, this remains a major computational challenge. Comparative approaches that examine the conservation and divergence of circuits and their components across strains and species can help reconstruct circuits as well as provide insights into the evolution of gene regulatory processes and their adaptive contribution. In recent years, advances in genomic and computational tools have led to a wealth of methods for such analysis at the sequence, expression, pathway, module, and entire network level. Here, we review computational methods developed to study transcriptional regulatory networks using comparative genomics, from sequence to functional data. We highlight how these methods use evolutionary conservation and divergence to reliably detect regulatory components as well as estimate the extent and rate of divergence. Finally, we discuss the promise and open challenges in linking regulatory divergence to phenotypic divergence and adaptation.

The evolution, evolvability and engineering of gene regulatory DNA.

Mutations in non-coding regulatory DNA sequences can alter gene expression, organismal phenotype and fitness1-3. Constructing complete fitness landscapes, in which DNA sequences are mapped to fitness, is a long-standing goal in biology, but has remained elusive because it is challenging to generalize reliably to vast sequence spaces4-6. Here we build sequence-to-expression models that capture fitness landscapes and use them to decipher principles of regulatory evolution. Using millions of randomly sampled promoter DNA sequences and their measured expression levels in the yeast Saccharomyces cerevisiae, we learn deep neural network models that generalize with excellent prediction performance, and enable sequence design for expression engineering. Using our models, we study expression divergence under genetic drift and strong-selection weak-mutation regimes to find that regulatory evolution is rapid and subject to diminishing returns epistasis; that conflicting expression objectives in different environments constrain expression adaptation; and that stabilizing selection on gene expression leads to the moderation of regulatory complexity. We present an approach for using such models to detect signatures of selection on expression from natural variation in regulatory sequences and use it to discover an instance of convergent regulatory evolution. We assess mutational robustness, finding that regulatory mutation effect sizes follow a power law, characterize regulatory evolvability, visualize promoter fitness landscapes, discover evolvability archetypes and illustrate the mutational robustness of natural regulatory sequence populations. Our work provides a general framework for designing regulatory sequences and addressing fundamental questions in regulatory evolution.

Genome instability footprint under rapamycin and hydroxyurea treatments.

The mutational processes dictating the accumulation of mutations in genomes are shaped by genetic background, environment and their interactions. Accurate quantification of mutation rates and spectra under drugs has important implications in disease treatment. Here, we used whole-genome sequencing and time-resolved growth phenotyping of yeast mutation accumulation lines to give a detailed view of the mutagenic effects of rapamycin and hydroxyurea on the genome and cell growth. Mutation rates depended on the genetic backgrounds but were only marginally affected by rapamycin. As a remarkable exception, rapamycin treatment was associated with frequent chromosome XII amplifications, which compensated for rapamycin induced rDNA repeat contraction on this chromosome and served to maintain rDNA content homeostasis and fitness. In hydroxyurea, a wide range of mutation rates were elevated regardless of the genetic backgrounds, with a particularly high occurrence of aneuploidy that associated with dramatic fitness loss. Hydroxyurea also induced a high T-to-G and low C-to-A transversion rate that reversed the common G/C-to-A/T bias in yeast and gave rise to a broad range of structural variants, including mtDNA deletions. The hydroxyurea mutation footprint was consistent with the activation of error-prone DNA polymerase activities and non-homologues end joining repair pathways. Taken together, our study provides an in-depth view of mutation rates and signatures in rapamycin and hydroxyurea and their impact on cell fitness, which brings insights for assessing their chronic effects on genome integrity.

Intragenic repeat expansion in the cell wall protein gene HPF1 controls yeast chronological aging.

Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined Saccharomyces cerevisiae population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes FLO11 and HPF1 We found that massive expansion of intragenic tandem repeats within the N-terminal domain of HPF1 was sufficient to cause pronounced life span shortening. Life span impairment by HPF1 was buffered by rapamycin but not by calorie restriction. The HPF1 repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity.

Myeloid PTP1B deficiency protects against atherosclerosis by improving cholesterol homeostasis through an AMPK-dependent mechanism.

OBJECTIVE: Atherosclerosis is a chronic inflammatory process induced by the influx and entrapment of excess lipoproteins into the intima media of arteries. Previously, our lab demonstrated that systemic PTP1B inhibition protects against atherosclerosis in preclinical LDLR-/- models. Similarly, it was shown that myeloid-specific PTP1B ablation decreases plaque formation and ameliorates dyslipidaemia in the ApoE-/- model of atherosclerosis. We hypothesized that the relevant improvements in dyslipidaemia following modification of PTP1B activation may either result from changes in hepatic cholesterol biosynthesis and/or increased uptake and degradation by liver-resident macrophages. We examined this in animal models and patients with coronary artery disease. METHODS: In this study, we determined the cholesterol-lowering effect of myeloid-PTP1B deletion in mice fed a high-fat high-cholesterol diet and examined effects on total cholesterol levels and lipoprotein profiles. We also determined the effects of PTP1B inhibition to oxLDL-C challenge on foam cell formation and cholesterol efflux in human monocytes/macrophages. RESULTS: We present evidence that myeloid-PTP1B deficiency significantly increases the affinity of Kupffer cells for ApoB containing lipoproteins, in an IL10-dependent manner. We also demonstrate that PTP1B inhibitor, MSI-1436, treatment decreased foam cell formation in Thp1-derived macrophages and increased macrophage cholesterol efflux to HDL in an AMPK-dependent manner. We present evidence of three novel and distinct mechanisms regulated by PTP1B: an increase in cholesterol efflux from foam cells, decreased uptake of lipoproteins into intra-lesion macrophages in vitro and a decrease of circulating LDL-C and VLDL-C in vivo. CONCLUSIONS: Overall, these results suggest that myeloid-PTP1B inhibition has atheroprotective effects through improved cholesterol handling in atherosclerotic lesions, as well as increased reverse cholesterol transport. Trial registration Research registry, researchregistry 3235. Registered 07 November 2017, https://www.researchregistry.com/browse-the-registry#home/registrationdetails/5a01d0fce7e1904e93e0aac5/ .

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

GeNets: a unified web platform for network-based genomic analyses.

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing.

Yeasts are essential for many processes, including the production of some of our most beloved foods and beverages. Less is known to the public about the far-reaching impacts of yeasts on other products such as biofuels, pharmaceuticals, and industrial products. By leveraging and combining newly discovered yeast genetic diversity with now affordable and efficient genetic engineering and synthetic biology tools, academic and industrial yeast labs have designed yeast cell factories for a wide range of novel applications such as the production of medicines, components of human breast milk, heme for meat substitutes, bioplastics, and other biomaterials. This review covers the newest technologies developed for yeast research including synthetic biology and their use in the engineering of yeast cell factories for emerging applications.

Catabolic repression in early-diverging anaerobic fungi is partially mediated by natural antisense transcripts.

Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to optimize hydrolytic efficiency. Our analysis of anaerobic fungal transcriptomes reveals hundreds of cis-natural antisense transcripts (cis-NATs), which we hypothesize play an integral role in this regulation. Through integrated genomic and transcriptomic sequencing on a range of catabolic substrates, we validate these NATs in three species (Anaeromyces robustus, Neocallimasix californiae, and Piromyces finnis), and analyze their expression patterns and prevalence to gain insight into their function. NAT function was diverse and conserved across the three fungal genomes studied, with 10% of all metabolic process NATs associated with lignocellulose hydrolysis. Despite these similarities, however, only eleven gene targets were conserved orthologs. Several NATs were dynamically regulated by lignocellulosic substrates while their gene targets were unregulated. This observation is consistent with a hypothesized, but untested, regulatory mechanism where selected genes are exclusively regulated at the transcriptional/post-transcriptional level by NATs. However, only genes with high NAT relative expression levels displayed this phenomenon, suggesting a selection mechanism that favors larger dynamic ranges for more precise control of gene expression. In addition to this mode, we observed two other possible regulatory fates: canonical transcriptional regulation with no NAT response, and positive co-regulation of target mRNA and cognate NAT, which we hypothesize is a fine-tuning strategy to locally negate control outputs from global regulators. Our work reveals the complex contributions of antisense RNA to the catabolic response in anaerobic fungi, highlighting its importance in understanding lignocellulolytic activity for bioenergy applications. More importantly, the relative expression of NAT to target may form a critical determinant of transcriptional vs post-transcriptional (NAT) control of gene expression in primitive anaerobic fungi.

Natural gene expression variation studies in yeast.

The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants, mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap, such as mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, probably owing to genetic divergence. Future studies in this area will benefit from the access to a massive array of well-annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacological inhibition of protein tyrosine phosphatase 1B protects against atherosclerotic plaque formation in the LDLR-/- mouse model of atherosclerosis.

Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with type 1 or type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance, due to impaired insulin receptor (IR) signalling. Here, we demonstrate that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR prevents and reverses atherosclerotic plaque formation in an LDLR-/- mouse model of atherosclerosis. Acute (single dose) or chronic PTP1B inhibitor (trodusquemine) treatment of LDLR-/- mice decreased weight gain and adiposity, improved glucose homeostasis and attenuated atherosclerotic plaque formation. This was accompanied by a reduction in both, circulating total cholesterol and triglycerides, a decrease in aortic monocyte chemoattractant protein-1 (MCP-1) expression levels and hyperphosphorylation of aortic Akt/PKB and AMPKα. Our findings are the first to demonstrate that PTP1B inhibitors could be used in prevention and reversal of atherosclerosis development and reduction in CVD risk.

Individualised radial extracorporeal shock wave therapy (rESWT) for symptomatic calcific shoulder tendinopathy: a retrospective clinical study.

BACKGROUND: A retrospective single centre cohort analysis was performed to evaluate an individualised radial extracorporeal shock wave therapy (rESWT) protocol for treatment of symptomatic calcific shoulder tendinopathy. METHODS: 67 patients (79 Shoulders) were identified with 76 shoulders included for analysis. rESWT treatment protocol was adapted according to individual response to treatment. Variables included number of sessions, shockwave impulses, pressure and frequency. Success rate was estimated as the percentage of patients having ≥60% visual analogue score (VAS) pain decrease at follow-up. Recurrence at 1 year was recorded. RESULTS: Using this individualised symptom guided protocol, patients underwent a mean of 7 ± 1.5 rESWT sessions, with mean pressure of 1.7 ± 0.2 bar, mean frequency of 5 ± 0.3 Hz and 2175 ± 266 impulses. The mean pre-treatment VAS score of 6.7 ± 1.1 was significantly decreased to 3.2 ± 0.8 immediately post-treatment, 2.6 ± 0.9 at 1 month, 1.7 ± 1.0 at 3 months and 0.8 ± 1.0 at 1 year follow up (α = 0.05). One-year success rate was estimated at 92% and 1-year recurrence rate was 7%. CONCLUSIONS: We conclude that in this retrospective study an individualised rESWT protocol resulted in a high success rate with low number of recurrences. Randomised controlled trials to support these findings are recommended.

Arboretum: reconstruction and analysis of the evolutionary history of condition-specific transcriptional modules.

Comparative functional genomics studies the evolution of biological processes by analyzing functional data, such as gene expression profiles, across species. A major challenge is to compare profiles collected in a complex phylogeny. Here, we present Arboretum, a novel scalable computational algorithm that integrates expression data from multiple species with species and gene phylogenies to infer modules of coexpressed genes in extant species and their evolutionary histories. We also develop new, generally applicable measures of conservation and divergence in gene regulatory modules to assess the impact of changes in gene content and expression on module evolution. We used Arboretum to study the evolution of the transcriptional response to heat shock in eight species of Ascomycota fungi and to reconstruct modules of the ancestral environmental stress response (ESR). We found substantial conservation in the stress response across species and in the reconstructed components of the ancestral ESR modules. The greatest divergence was in the most induced stress, primarily through module expansion. The divergence of the heat stress response exceeds that observed in the response to glucose depletion in the same species. Arboretum and its associated analyses provide a comprehensive framework to systematically study regulatory evolution of condition-specific responses.

Comparative analysis of RNA sequencing methods for degraded or low-input samples.

RNA-seq is an effective method for studying the transcriptome, but it can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations or cadavers. Recent studies have proposed several methods for RNA-seq of low-quality and/or low-quantity samples, but the relative merits of these methods have not been systematically analyzed. Here we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and compared them against two control libraries. We found that the RNase H method performed best for chemically fragmented, low-quality RNA, and we confirmed this through analysis of actual degraded samples. RNase H can even effectively replace oligo(dT)-based methods for standard RNA-seq. SMART and NuGEN had distinct strengths for measuring low-quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.

Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses.

Formyl-peptide receptor type 2 (FPR2), also called ALX (the lipoxin A4 receptor), conveys the proresolving properties of lipoxin A4 and annexin A1 (AnxA1) and the proinflammatory signals elicited by serum amyloid protein A and cathelicidins, among others. We tested here the hypothesis that ALX might exist as homo- or heterodimer with FPR1 or FPR3 (the two other family members) and operate in a ligand-biased fashion. Coimmunoprecipitation and bioluminescence resonance energy transfer assays with transfected HEK293 cells revealed constitutive dimerization of the receptors; significantly, AnxA1, but not serum amyloid protein A, could activate ALX homodimers. A p38/MAPK-activated protein kinase/heat shock protein 27 signaling signature was unveiled after AnxA1 application, leading to generation of IL-10, as measured in vitro (in primary monocytes) and in vivo (after i.p. injection in the mouse). The latter response was absent in mice lacking the ALX ortholog. Using a similar approach, ALX/FPR1 heterodimerization evoked using the panagonist peptide Ac2-26, identified a JNK-mediated proapoptotic path that was confirmed in primary neutrophils. These findings provide a molecular mechanism that accounts for the dual nature of ALX and indicate that agonist binding and dimerization state contribute to the conformational landscape of FPRs.

Increased axonal bouton dynamics in the aging mouse cortex.

Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline.

Identification of a novel recycling sequence in the C-tail of FPR2/ALX receptor: association with cell protection from apoptosis.

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes ß-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation.

Evolution of regulatory networks in Candida glabrata: learning to live with the human host.

The opportunistic human fungal pathogen Candida glabrata is second only to C. albicans as the cause of Candida infections and yet is more closely related to Saccharomyces cerevisiae. Recent advances in functional genomics technologies and computational approaches to decipher regulatory networks, and the comparison of these networks among these and other Ascomycete species, have revealed both unique and shared strategies in adaptation to a human commensal/opportunistic pathogen lifestyle and antifungal drug resistance in C. glabrata. Recently, several C. glabrata sister species in the Nakeseomyces clade representing both human associated (commensal) and environmental isolates have had their genomes sequenced and analyzed. This has paved the way for comparative functional genomics studies to characterize the regulatory networks in these species to identify informative patterns of conservation and divergence linked to phenotypic evolution in the Nakaseomyces lineage.

Loss of GPR75 protects against non-alcoholic fatty liver disease and body fat accumulation.

Approximately 1 in 4 people worldwide have non-alcoholic fatty liver disease (NAFLD); however, there are currently no medications to treat this condition. This study investigated the role of adiposity-associated orphan G protein-coupled receptor 75 (GPR75) in liver lipid accumulation. We profiled Gpr75 expression and report that it is most abundant in the brain. Next, we generated the first single-cell-level analysis of Gpr75 and identified a subpopulation co-expressed with key appetite-regulating hypothalamic neurons. CRISPR-Cas9-deleted Gpr75 mice fed a palatable western diet high in fat adjusted caloric intake to remain in energy balance, thereby preventing NAFLD. Consistent with mouse results, analysis of whole-exome sequencing data from 428,719 individuals (UK Biobank) revealed that variants in GPR75 are associated with a reduced likelihood of hepatic steatosis. Here, we provide a significant advance in understanding of the expression and function of GPR75, demonstrating that it is a promising pharmaceutical target for NAFLD treatment.

Genomic Discovery and Structure-Activity Exploration of a Novel Family of Enzyme-Activated Covalent Cyclin-Dependent Kinase Inhibitors.

Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.