Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors

Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors–Prospero (Pros), D-Pax2, and Delilah (Dei)–that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.

Biological Prescience: The Role of Anticipation in Organismal Processes

Anticipation is the act of using information about the past and present to make predictions about future scenarios. As a concept, it is predominantly associated with the psychology of the human mind; however, there is accumulating evidence that diverse taxa without complex neural systems, and even biochemical networks themselves, can respond to perceived future conditions. Although anticipatory processes, such as circadian rhythms, stress priming, and cephalic responses, have been extensively studied over the last three centuries, newer research on anticipatory genetic networks in microbial species shows that anticipatory processes are widespread, evolutionarily old, and not simply reserved for neurological complex organisms. Overall, data suggest that anticipatory responses represent a unique type of biological processes that can be distinguished based on their organizational properties and mechanisms. Unfortunately, an empirically based biologically explicit framework for describing anticipatory processes does not currently exist. This review attempts to fill this void by discussing the existing examples of anticipatory processes in non-cognitive organisms, providing potential criteria for defining anticipatory processes, as well as their putative mechanisms, and drawing attention to the often-overlooked role of anticipation in the evolution of physiological systems. Ultimately, a case is made for incorporating an anticipatory framework into the existing physiological paradigm to advance our understanding of complex biological processes.

Allelic correlation is a marker of tradeoffs between barriers to transmission of expression variability and signal responsiveness in genetic networks

Accurately functioning genetic networks should be responsive to signals but prevent transmission of stochastic bursts of expression. Existing data in mammalian cells suggests that such transcriptional "noise" is transmitted by some genes and not others, suggesting that noise transmission is tunable, perhaps at the expense of other signal processing capabilities. However, systematic claims about noise transmission in genetic networks have been limited by the inability to directly measure noise transmission. Here we build a mathematical framework capable of modeling allelic correlation and noise transmission. We find that allelic correlation and noise transmission correspond across a broad range of model parameters and network architectures. We further find that limiting noise transmission comes with the trade-off of being unresponsive to signals, and that within the parameter regimes that are responsive to signals, there is a further trade-off between response time and basal noise transmission. Using a published allele specific single cell RNA-sequencing dataset, we found that genes with high allelic odds ratios are enriched for cell-type specific functions, and that within multiple signaling pathways, factors which are upstream in the pathway have higher allelic odds ratios than downstream factors. Overall, our findings suggest that some degree of noise transmission is required to be responsive to signals, but that minimization of noise transmission can be accomplished by trading-off for a slower response time.

Genome-Wide Screening and Genetic Networks in Pleiotropic Drug Resistance, Oxidative Stress, and Drug Targets in S. Cerevisiae

<p>One of the major problems in biology is to identify genes that are involved in specific processes. Classical genetics and biochemistry, although powerful and informative, can be very labour intensive and do not necessarily characterise networked genes in processes that may overarch numerous biochemical pathways. Here we utilised genomic tools that are capable of defining networks to identify genes involved the complex target mode-of-action of a novel antifungal compound, neothyonidioside and in regulating specific stress processes and the PDR phenotype. The first part of this study investigated the mode-of-action of the antifungal compound, neothyonidioside (neo). We developed a neo resistant mutant strain then utilising a modification of SGAM, a genetic mapping tool, and application of genome-wide chemical-genetic profiling, we identified the neo resistant locus NCP1. This gene acts at a late step in ergosterol biosynthesis but is not the target of neo. The finding that many of the component genes in the ESCRT complex were necessary for neo resistance allowed us to predict and verify by high-content fluorescence microcopy that interruptions in the endosome-multivesicular body pathway were involved. From the known function of the ESCRT proteins and that neo binds ergosterol only above threshold concentrations of ergosterol (explaining the mutant phenotype) we concluded that neo disruption of membrane curvature and fusion capability in the endosome-vacuole pathway is its target. In the second part of this study we identified genes in a genome-wide fashion that modulate the pleiotropic drug resistance (PDR) phenotype and oxidative stress response. Many PDR targets are well studied ABC transporters (e.g. PDR5 , YOR1), but the modulating events between xenobiotic sensing and transcription factor activation, and possible crosstalk between PDR and other stress responses such as oxidative stress are not well characterised. To identify specific genes involved in the PDR and oxidative stress processes, we developed a fluorescent reporter screen for effects on the PDR-target ABC-transporters, Pdr5p and Yor1p tagged with GFP. For the oxidative stress response, the oxidative stress (OS) transcription factor Yap1p tagged with GFP was used. Each reporter was placed in the yeast non-essential gene deletion background of ~4800 strains which were then subjected to either xenobiotic treatments (PDR –GFP reporters) or oxidant treatments (Yap1p-GFP). We then screened for gene deletions which prevented the normal upregulation of PDR reporters in the presence of xenobiotics. Controls were included in the screens that assured we were assessing genes that must contribute to or act before the transcription of the ABC-transporters. A similar screening strategy was pursued for identifying gene deletions that prevent the normal nuclear re-localisation of Yap1p in the presence of oxidants. A major finding in this study was identification of genes contributing to the PDR phenotype that involved signalling (Rho-GTPase, MAPK), that were involved in RNA polymerase II mediator complexes and chromatin modification (subunits of ADA and SAGA histone acetyltransferase complexes), and that were involved in sphingo/phosphorlipids biosynthesis. Secondary screens comprising spot dilution growth assays and Western blots of Pdr5p abundance confirmed key genes of the primary screen and showed that these were specific and not global transcriptional effects.For some of the gene-dependencies, our results can only be construed to indicate the existence of alternative pathways underpinning the PDR phenotype in a Pdr1p/Pdr3p independent manner. We then supposed that if in fact PDR phenotypes are the result of genetic networks, then genes known to interact with the most highly connected hubs from our PDR screen results should also to some extent contribute to the PDR phenotype (spot dilution growth assays, Western blot abundance). A selection of 18 such genes that also appeared in our primary screen but were deemed to be below the cut-off point were phenotype tested and in 60% of the cases showed similar phenotypes to the genes already identified. This result not only proved the validity of the screening methods but validated the original supposition, i.e. that PDR phenotypes can be affected, through gene networks.</p>

Genome-Wide Screening and Genetic Networks in Pleiotropic Drug Resistance, Oxidative Stress, and Drug Targets in S. Cerevisiae

<p>One of the major problems in biology is to identify genes that are involved in specific processes. Classical genetics and biochemistry, although powerful and informative, can be very labour intensive and do not necessarily characterise networked genes in processes that may overarch numerous biochemical pathways. Here we utilised genomic tools that are capable of defining networks to identify genes involved the complex target mode-of-action of a novel antifungal compound, neothyonidioside and in regulating specific stress processes and the PDR phenotype. The first part of this study investigated the mode-of-action of the antifungal compound, neothyonidioside (neo). We developed a neo resistant mutant strain then utilising a modification of SGAM, a genetic mapping tool, and application of genome-wide chemical-genetic profiling, we identified the neo resistant locus NCP1. This gene acts at a late step in ergosterol biosynthesis but is not the target of neo. The finding that many of the component genes in the ESCRT complex were necessary for neo resistance allowed us to predict and verify by high-content fluorescence microcopy that interruptions in the endosome-multivesicular body pathway were involved. From the known function of the ESCRT proteins and that neo binds ergosterol only above threshold concentrations of ergosterol (explaining the mutant phenotype) we concluded that neo disruption of membrane curvature and fusion capability in the endosome-vacuole pathway is its target. In the second part of this study we identified genes in a genome-wide fashion that modulate the pleiotropic drug resistance (PDR) phenotype and oxidative stress response. Many PDR targets are well studied ABC transporters (e.g. PDR5 , YOR1), but the modulating events between xenobiotic sensing and transcription factor activation, and possible crosstalk between PDR and other stress responses such as oxidative stress are not well characterised. To identify specific genes involved in the PDR and oxidative stress processes, we developed a fluorescent reporter screen for effects on the PDR-target ABC-transporters, Pdr5p and Yor1p tagged with GFP. For the oxidative stress response, the oxidative stress (OS) transcription factor Yap1p tagged with GFP was used. Each reporter was placed in the yeast non-essential gene deletion background of ~4800 strains which were then subjected to either xenobiotic treatments (PDR –GFP reporters) or oxidant treatments (Yap1p-GFP). We then screened for gene deletions which prevented the normal upregulation of PDR reporters in the presence of xenobiotics. Controls were included in the screens that assured we were assessing genes that must contribute to or act before the transcription of the ABC-transporters. A similar screening strategy was pursued for identifying gene deletions that prevent the normal nuclear re-localisation of Yap1p in the presence of oxidants. A major finding in this study was identification of genes contributing to the PDR phenotype that involved signalling (Rho-GTPase, MAPK), that were involved in RNA polymerase II mediator complexes and chromatin modification (subunits of ADA and SAGA histone acetyltransferase complexes), and that were involved in sphingo/phosphorlipids biosynthesis. Secondary screens comprising spot dilution growth assays and Western blots of Pdr5p abundance confirmed key genes of the primary screen and showed that these were specific and not global transcriptional effects.For some of the gene-dependencies, our results can only be construed to indicate the existence of alternative pathways underpinning the PDR phenotype in a Pdr1p/Pdr3p independent manner. We then supposed that if in fact PDR phenotypes are the result of genetic networks, then genes known to interact with the most highly connected hubs from our PDR screen results should also to some extent contribute to the PDR phenotype (spot dilution growth assays, Western blot abundance). A selection of 18 such genes that also appeared in our primary screen but were deemed to be below the cut-off point were phenotype tested and in 60% of the cases showed similar phenotypes to the genes already identified. This result not only proved the validity of the screening methods but validated the original supposition, i.e. that PDR phenotypes can be affected, through gene networks.</p>

Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Taiji-reprogram: a framework to uncover cell-type specific regulators and predict cellular reprogramming cocktails

Cellular reprogramming is a promising technology to develop disease models and cell-based therapies. Identification of the key regulators defining the cell type specificity is pivotal to devising reprogramming cocktails for successful cell conversion but remains a great challenge. Here, we present a systems biology approach called Taiji-reprogram to efficiently uncover transcription factor (TF) combinations for conversion between 154 diverse cell types or tissues. This method integrates the transcriptomic and epigenomic data to construct cell-type specific genetic networks and assess the global importance of TFs in the network. Comparative analysis across cell types revealed TFs that are specifically important in a particular cell type and often tightly associated with cell-type specific functions. A systematic search of TFs with differential importance in the source and target cell types uncovered TF combinations for desired cell conversion. We have shown that Taiji-reprogram outperformed the existing methods to better recover the TFs in the experimentally validated reprogramming cocktails. This work not only provides a comprehensive catalog of TFs defining cell specialization but also suggests TF combinations for direct cell conversion.

Genetic Networks That Govern Sexual Reproduction in the Pezizomycotina

Sexual development in filamentous fungi is a complex process that relies on the precise control of and interaction between a variety of genetic networks and pathways. The mating-type ( MAT ) genes are the master regulators of this process and typically act as transcription factors, which control the expression of genes involved all stages of the sexual cycle.