Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.

Foxp3+ TREG cells have been at the focus of intense investigation for their recognized roles in preventing autoimmunity, facilitating tissue recuperation following injury, and orchestrating a tolerance to innocuous non-self-antigens. To perform these critical tasks, TREG cells undergo deep epigenetic, transcriptional, and post-transcriptional changes that allow them to adapt to conditions found in tissues both at steady-state and during inflammation. The path leading TREG cells to express these tissue-specialized phenotypes begins during thymic development, and is further driven by epigenetic and transcriptional modifications following TCR engagement and polarizing signals in the periphery. However, this process is highly regulated and requires TREG cells to adopt strategies to avoid losing their regulatory program altogether. Here, we review the origins of tissue-resident TREG cells, from their thymic and peripheral development to the transcriptional regulators involved in their tissue residency program. In addition, we discuss the distinct signalling pathways that engage the inflammatory adaptation of tissue-resident TREG cells, and how they relate to their ability to recognize tissue and pathogen-derived danger signals.

During infections, the timings of effector differentiation of pulmonary immune responses are of paramount importance, as pathogen persistence and unsuppressed inflammation can rapidly lead to a loss of function, increased frailty, and death. Thus, both an efficient clearance of the danger and a rapid resolution of inflammation are critical to host survival. We now know that tissue-localized FoxP3+ regulatory T cells, a subset of CD4+ T cells, are highly attuned to the type of immune response, acquiring unique phenotypic characteristics that allow them to adapt their suppressive functions with the nature of inflammatory cells. To achieve this, activated effector TREG cells acquire specialized TH 1, TH 2, and TH 17-like characteristics that allow them to migrate, survive, and time their function(s) through refined mechanisms. Herein, we describe how this process requires a unique developmental path that includes the acquisition of master transcription factors and the expression of receptors adapted to sense local danger signals that are found during pulmonary inflammation. In turn, we offer an overview of how these characteristics promote the capacity of local effector TREG cells to proliferate, survive, and display suppressive strategies to resolve lung injury.

Genome editing technologies including the CRISPR/Cas9 system have greatly improved our knowledge of gene function and biological processes, however, these approaches have also brought new challenges to determining genotype-phenotype correlations. In this chapter, we briefly review gene-editing technologies used in zebrafish and discuss the differences in phenotypes that can arise when gene expression is inhibited by anti-sense or by gene editing techniques. We outline possible explanations for why knockout phenotypes are milder, tissue-restricted, or even absent, compared with severe knockdown phenotypes. One proposed explanation is transcriptional adaptation, a form of genetic robustness that is induced by deleterious mutations but not gene knockdowns. Although much is unknown about what triggers this process, its relevance in shaping genome expression has been shown in multiple animal models. We recently explored if transcriptional adaptation could explain genotype-phenotype discrepancies seen between two zebrafish models of the centrosomal protein Cep290 deficiency. We compared cilia-related phenotypes in knockdown (anti-sense) and knockout (mutation) Cep290 models and showed that only cep290 gene mutation induces the upregulation of genes encoding the cilia-associated small GTPases Arl3, Arl13b, and Unc119b. Importantly, the ectopic expression of Arl3, Arl13b, and Unc119b in cep290 morphant zebrafish embryos rescued cilia defects. Here we provide protocols and experimental approaches that can be used to explore if transcriptional adaptation may be modulating gene expression in a zebrafish ciliary mutant model.

Bacteria adapt to their host by mutating specific genes and by reprogramming their gene expression. Different strains of a bacterial species often mutate the same genes during infection, demonstrating convergent genetic adaptation. However, there is limited evidence for convergent adaptation at the transcriptional level. To this end, we utilize genomic data of 114 Pseudomonas aeruginosa strains, derived from patients with chronic pulmonary infection, and the P. aeruginosa transcriptional regulatory network. Relying on loss-of-function mutations in genes encoding transcriptional regulators and predicting their effects through the network, we demonstrate predicted expression changes of the same genes in different strains through different paths in the network, implying convergent transcriptional adaptation. Furthermore, through the transcription lens we associate yet-unknown processes, such as ethanol oxidation and glycine betaine catabolism, with P. aeruginosa host adaptation. We also find that known adaptive phenotypes, including antibiotic resistance, which were identified before as achieved by specific mutations, are achieved also through transcriptional changes. Our study has revealed novel interplay between the genetic and transcriptional levels in host adaptation, demonstrating the versatility of the adaptive arsenal of bacterial pathogens and their ability to adapt to the host conditions in a myriad of ways. IMPORTANCE Pseudomonas aeruginosa causes significant morbidity and mortality. The pathogen\'s remarkable ability to establish chronic infections greatly depends on its adaptation to the host environment. Here, we use the transcriptional regulatory network to predict expression changes during adaptation. We expand the processes and functions known to be involved in host adaptation. We show that the pathogen modulates the activity of genes during adaptation, including genes implicated in antibiotic resistance, both directly via genomic mutations and indirectly via mutations in transcriptional regulators. Furthermore, we detect a subgroup of genes whose predicted changes in expression are associated with mucoid strains, a major adaptive phenotype in chronic infections. We propose that these genes constitute the transcriptional arm of the mucoid adaptive strategy. Identification of different adaptive strategies utilized by pathogens during chronic infection has major promise in the treatment of persistent infections and opens the door to personalized tailored antibiotic treatment in the future.

Understanding the way genes work amongst individuals and across generations to shape form and function is a common theme for many genetic studies. The recent advances in genetics, genome engineering and DNA sequencing reinforced the notion that genes are not the only players that determine a phenotype. Due to physiological or pathological fluctuations in gene expression, even genetically identical cells can behave and manifest different phenotypes under the same conditions. Here, we discuss mechanisms that can influence or even disrupt the axis between genotype and phenotype; the role of modifier genes, the general concept of genetic redundancy, genetic compensation, the recently described transcriptional adaptation, environmental stressors, and phenotypic plasticity. We furthermore highlight the usage of induced pluripotent stem cells (iPSCs), the generation of isogenic lines through genome engineering, and sequencing technologies can help extract new genetic and epigenetic mechanisms from what is hitherto considered \'noise\'.

Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissue-specific clinical cancers. These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution. In this study, we mimicked histone H3 at the 56th lysine (H3K56) mutant incorporation in mouse embryonic stem cells (mESCs) by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation. The data show that two types of H3K56 mutants, namely H3 lysine 56-to-methionine (H3K56M) and H3 lysine 56-to-alanine (H3K56A), promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants. Under this condition, the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases, especially in the Mycl1 region, a known molecular marker frequently occurring in multiple malignant cancers. Additionally, we found the disruption of H3K56 acetylation distribution in the copy-gain regions, which indicates a probable epigenetic mechanism of H3K56M and H3K56A. We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription; genes involved in the mitogen-activated protein kinase pathway are partially upregulated, whereas genes associated with intrinsic apoptotic function show obvious downregulation. The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas. This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.

Genetic manipulations with a robust and predictable outcome are critical to investigate gene function, as well as for therapeutic genome engineering. For many years, knockdown approaches and reagents including RNA interference and antisense oligonucleotides dominated functional studies; however, with the advent of precise genome editing technologies, CRISPR-based knockout systems have become the state-of-the-art tools for such studies. These technologies have helped decipher the role of thousands of genes in development and disease. Their use has also revealed how limited our understanding of genotype-phenotype relationships is. The recent discovery that certain mutations can trigger the transcriptional modulation of other genes, a phenomenon called transcriptional adaptation, has provided an additional explanation for the contradicting phenotypes observed in knockdown versus knockout models and increased awareness about the use of each of these approaches. In this review, we first cover the strengths and limitations of different gene perturbation strategies. Then we highlight the diverse ways in which the genotype-phenotype relationship can be discordant between these different strategies. Finally, we review the genetic robustness mechanisms that can lead to such discrepancies, paying special attention to the recently discovered phenomenon of transcriptional adaptation.

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1 h u2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the \"leading edge\" differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher\'s assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1 h u2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

Purple acid phosphatases (PAP)-encoding genes form a complex network that play a critical role in plant phosphate (Pi) homeostasis. Mostly, the functions of PAPs were investigated individually. However, the interactions of most of these genes in response to various concentrations of available Pi remain unknown. In this study, the roles of AtPAP17 and AtPAP26 genes, and their relationship within Pi homeostasis context were investigated. Surprisingly, atpap17 and atpap26 mutants not only showed no obvious developmental defects, but also produced higher biomass in compare to wild type (WT) plants under normal growth conditions. Comparing gene expression patterns of these mutants with WT plant, we identified a set of genes up-regulated in mutant plants but not in WT. Based on these unexpected results and up-regulation of AtPAP17 and AtPAP26 genes by the loss of function of each other, the hypothesis of compensation relationship between these genes in Pi homeostasis was assessed by generating atpap17/atpap26 double mutants. Observation of developmental defects in atpap17/atpap26 mutant but not in single mutants indicated a compensation relationship between AtPAP17 and AtPAP26 genes in Pi homeostasis network. Taken together, these results demonstrate the activation of AtPAP17 and AtPAP26 genes to buffer against the loss of function of each other, and this compensation relationship is vital for Arabidopsis growth and development.