Tradeoffs and Optimality in the Evolution of Gene Regulation

Tradeoffs and Optimality in the Evolution of Gene Regulation: "Frank J. Poelwijk, Marjon G.J. de Vos, Sander J. Tans. Cellular regulation is believed to evolve in response to environmental variability. However, this has been difficult to test directly. Here, we show that a gene regulation system evolves to the op...."

Extensive characterization of NF-KappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits

Extensive characterization of NF-KappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits: "Background:
Genetic studies have provided ample evidence of the influence of non-coding DNA polymorphisms on trait variance, particularly those occurring within transcription factor binding sites. Protein binding microarrays and other platforms that can map these sites with great precision have enhanced our understanding of how a single nucleotide polymorphism can alter binding potential within an in vitro setting allowing for greater predictive capability of its effect on a transcription factor binding site.
Results:
We have used protein binding microarrays and Electrophoretic Mobility Shift Assay-Sequencing (EMSA-Seq), a deep sequencing based method we developed to analyze nine distinct human Nuclear Factor-kappaB (NF-kappaB) dimers. This family of transcription factors is one of the most extensively studied, but our understanding of its DNA binding preferences has been limited to the originally described consensus motif, GGRRNNYYCC. We highlight differences between NF-kappaB family members and also put under the spotlight non-canonical motifs that have so far received little attention. We utilize our data to interpret the binding of transcription factors between individuals across 1405 genomic regions laden with single nucleotide polymorphisms. We also associated binding correlations made using our data with risk alleles of disease and demonstrate its utility as a tool for functional studies of single nucleotide polymorphisms in regulatory regions.
Conclusions:
NF-kappaB dimers bind specifically to non-canonical motifs and these can be found within genomic regions in which a canonical motif is not evident. Binding affinity data generated with these different motifs can be used in conjunction with data from Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) to enable allele-specific analyses of expression and transcription factor-DNA interactions on a genome-wide scale."

Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways

Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways: "

by Xiaoyue Wang, Ralf J. Sommer

Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition of novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks."

Formation of the Long Range Dpp Morphogen Gradient

Formation of the Long Range Dpp Morphogen Gradient: "

by Gerald Schwank, Sascha Dalessi, Schu-Fee Yang, Ryohei Yagi, Aitana Morton de Lachapelle, Markus Affolter, Sven Bergmann, Konrad Basler

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution."

Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera) [RESEARCH ARTICLES]

Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera) [RESEARCH ARTICLES]: "Megan J. Wilson and Peter K. Dearden

Axis formation is a key step in development, but studies indicate that genes involved in insect axis formation are relatively fast evolving. Orthodenticle genes have conserved roles, often with hunchback, in maternal anterior patterning in several insect species. We show that two orthodenticle genes, otd1 and otd2, and hunchback act as maternal anterior patterning genes in the honeybee (Apis mellifera) but, unlike other insects, act to pattern the majority of the anteroposterior axis. These genes regulate the expression domains of anterior, central and posterior gap genes and may directly regulate the anterior gap gene giant. We show otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but that zen does not regulate the expression of honeybee gap genes. This suggests that interactions between anteroposterior and dorsal-ventral patterning are ancestral in holometabolous insects. Honeybee axis formation, and the function of the conserved anterior patterning gene orthodenticle, displays unique characters that indicate that, even when conserved genes pattern the axis, their regulatory interactions differ within orders of insects, consistent with relatively fast evolution in axis formation pathways.

"

Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos [RESEARCH ARTICLES]

Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos [RESEARCH ARTICLES]: "Bernhard Hampoelz, Yannick Azou-Gros, Roxane Fabre, Olga Markova, Pierre-Henri Puech, and Thomas Lecuit

Nuclear shape is different in stem cells and differentiated cells and reflects important changes in the mechanics of the nuclear envelope (NE). The current framework emphasizes the key role of the nuclear lamina in nuclear mechanics and its alterations in disease. Whether active stress controls nuclear deformations and how this stress interplays with properties of the NE to control NE dynamics is unclear. We address this in the early Drosophila embryo, in which profound changes in NE shape parallel the transcriptional activation of the zygotic genome. We show that microtubule (MT) polymerization events produce the elementary forces necessary for NE dynamics. Moreover, large-scale NE deformations associated with groove formation require concentration of MT polymerization in bundles organized by Dynein. However, MT bundles cannot produce grooves when the farnesylated inner nuclear membrane protein Kugelkern (Kuk) is absent. Although it increases stiffness of the NE, Kuk also stabilizes NE deformations emerging from the collective effect of MT polymerization forces concentrated in bundles. Finally, we report that MT-induced NE deformations control the dynamics of chromatin and its organization at steady state. Thus, the NE is a dynamic organelle, fluctuations of which increase chromatin dynamics. We propose that such mechanical regulation of chromatin dynamics by MTs might be important for gene regulation.

"

ADAPTIVE cis-REGULATORY CHANGES MAY INVOLVE FEW MUTATIONS

ADAPTIVE cis-REGULATORY CHANGES MAY INVOLVE FEW MUTATIONS: "

ABSTRACT

A long-standing debate in evo-devo research concerns the relative role of protein-coding and cis-regulatory regions in adaptation. Recent studies of genetic adaptation have revealed that the number of substitutions contributing to phenotypic variation is lower in cis-regulatory than in structural regions, which has led to the idea that cis-regulatory regions are less important in phenotypic adaptation. However, the number of substitutions is not the only important factor, the “size” of the adaptive contribution of these substitutions is important too. A geometrical reasoning predicts that, given their lesser pleiotropic effects, cis-regulatory substitutions should have a larger average adaptive contribution than protein-coding substitutions. Thus it is possible that even with a lower number of adaptive mutations, cis-regulatory regions may contribute at the same level or even more than protein-coding regions.

"

Rare and Common Regulatory Variation in Population-Scale Sequenced Human Genomes

Rare and Common Regulatory Variation in Population-Scale Sequenced Human Genomes: "

by Stephen B. Montgomery, Tuuli Lappalainen, Maria Gutierrez-Arcelus, Emmanouil T. Dermitzakis

Population-scale genome sequencing allows the characterization of functional effects of a broad spectrum of genetic variants underlying human phenotypic variation. Here, we investigate the influence of rare and common genetic variants on gene expression patterns, using variants identified from sequencing data from the 1000 genomes project in an African and European population sample and gene expression data from lymphoblastoid cell lines. We detect comparable numbers of expression quantitative trait loci (eQTLs) when compared to genotypes obtained from HapMap 3, but as many as 80% of the top expression quantitative trait variants (eQTVs) discovered from 1000 genomes data are novel. The properties of the newly discovered variants suggest that mapping common causal regulatory variants is challenging even with full resequencing data; however, we observe significant enrichment of regulatory effects in splice-site and nonsense variants. Using RNA sequencing data, we show that 46.2% of nonsynonymous variants are differentially expressed in at least one individual in our sample, creating widespread potential for interactions between functional protein-coding and regulatory variants. We also use allele-specific expression to identify putative rare causal regulatory variants. Furthermore, we demonstrate that outlier expression values can be due to rare variant effects, and we approximate the number of such effects harboured in an individual by effect size. Our results demonstrate that integration of genomic and RNA sequencing analyses allows for the joint assessment of genome sequence and genome function."

Protein Evolution in Cell and Tissue Development: Going Beyond Sequence and Transcriptional Analysis

Protein Evolution in Cell and Tissue Development: Going Beyond Sequence and Transcriptional Analysis: "Daniel J. Dickinson, William I. Weis, W. James Nelson. Studies of animal evolution often focus on sequence and transcriptional analysis, based on an assumption that the evolution of development is driven by changes in gene expression. We argue that bi...."

Enhancers: From Developmental Genetics to the Genetics of Common Human Disease

Enhancers: From Developmental Genetics to the Genetics of Common Human Disease: "Iain Williamson, Robert E. Hill, Wendy A. Bickmore. In mammals, long-range gene regulation became apparent through simple Mendelian disease genetics in human and developmental genetics in the mouse. Can the insights into gene control, provided by t...."

Shifting Patterns: Merging Molecules, Morphogens, Motility, and Methodology

Shifting Patterns: Merging Molecules, Morphogens, Motility, and Methodology: "Shawn C. Little, Eric F. Wieschaus. We highlight crucial technological progress of the past ten years that permits quantitative analysis of cellular behavior. Adapting these methods to the study of embryogenesis will be essential to...."

Coupled Evolution of Transcription and mRNA Degradation

Coupled Evolution of Transcription and mRNA Degradation: "

by Mally Dori-Bachash, Efrat Shema, Itay Tirosh

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast."

Quantitative dissection of the simple repression input-output function [Systems Biology]

Quantitative dissection of the simple repression input-output function [Systems Biology]: "We present a quantitative case study of transcriptional regulation in which we carry out a systematic dialogue between theory and measurement for an important and ubiquitous regulatory motif in bacteria, namely, that of simple repression. This architecture is realized by a single repressor binding site overlapping the promoter. From the theory point of view, this motif is described by a single gene regulation function based upon only a few parameters that are convenient theoretically and accessible experimentally. The usual approach is turned on its side by using the mathematical description of these regulatory motifs as a predictive tool to determine the number of repressors in a collection of strains with a large variation in repressor copy number. The predictions and corresponding measurements are carried out over a large dynamic range in both expression fold change (spanning nearly four orders of magnitude) and repressor copy number (spanning about two orders of magnitude). The predictions are tested by measuring the resulting level of gene expression and are then validated by using quantitative immunoblots. The key outcomes of this study include a systematic quantitative analysis of the limits and validity of the input–output relation for simple repression, a precise determination of the in vivo binding energies for DNA–repressor interactions for several distinct repressor binding sites, and a repressor census for Lac repressor in Escherichia coli."

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression [Genetics]

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression [Genetics]: "Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary—whereas the entire N terminus of the protein is dispensable—for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors."

Determinants and dynamics of genome accessibility

Determinants and dynamics of genome accessibility: "

Determinants and dynamics of genome accessibility

Nature Reviews Genetics 12, 554 (2011).
doi:10.1038/nrg3017

Authors: Oliver Bell, Vijay K. Tiwari, Nicolas H. Thomä & Dirk Schübeler

In eukaryotes, all DNA-templated reactions occur in the context of chromatin. Nucleosome packaging inherently restricts DNA accessibility for regulatory proteins but also provides an opportunity to regulate DNA-based processes through modulating nucleosome positions and local chromatin structure. Recent advances in genome-scale methods are yielding increasingly

"

Comparative Genomics of the Odorant-Binding and Chemosensory Protein Gene Families across the Arthropoda: Origin and Evolutionary History of the Chemosensory System

Comparative Genomics of the Odorant-Binding and Chemosensory Protein Gene Families across the Arthropoda: Origin and Evolutionary History of the Chemosensory System: "

Chemoreception is a biological process essential for the survival of animals, as it allows the recognition of important volatile cues for the detection of food, egg-laying substrates, mates, or predators, among other purposes. Furthermore, its role in pheromone detection may contribute to evolutionary processes, such as reproductive isolation and speciation. This key role in several vital biological processes makes chemoreception a particularly interesting system for studying the role of natural selection in molecular adaptation. Two major gene families are involved in the perireceptor events of the chemosensory system: the odorant-binding protein (OBP) and chemosensory protein (CSP) families. Here, we have conducted an exhaustive comparative genomic analysis of these gene families in 20 Arthropoda species. We show that the evolution of the OBP and CSP gene families is highly dynamic, with a high number of gains and losses of genes, pseudogenes, and independent origins of subfamilies. Taken together, our data clearly support the birth-and-death model for the evolution of these gene families with an overall high gene turnover rate. Moreover, we show that the genome organization of the two families is significantly more clustered than expected by chance and, more important, that this pattern appears to be actively maintained across the Drosophila phylogeny. Finally, we suggest the homologous nature of the OBP and CSP gene families, dating back their most recent common ancestor after the terrestrialization of Arthropoda (380--450 Ma) and we propose a scenario for the origin and diversification of these families.

"

Review. Adaptation and habitat selection

Review. Adaptation and habitat selection: "

The struggle for existence occurs through the vital rates of population growth. This basic fact demonstrates the tight connection between ecology and evolution that defines the emerging field of eco-evolutionary dynamics. An effective synthesis of the interdependencies between ecology and evolution is grounded in six principles. The mechanics of evolution specifies the origin and rules governing traits and evolutionary strategies. Traits and evolutionary strategies achieve their selective value through their functional relationships with fitness. Function depends on the underlying structure of variation and the temporal, spatial and organizational scales of evolution. An understanding of how changes in traits and strategies occur requires conjoining ecological and evolutionary dynamics. Adaptation merges these five pillars to achieve a comprehensive understanding of ecological and evolutionary change. I demonstrate the value of this world-view with reference to the theory and practice of habitat selection. The theory allows us to assess evolutionarily stable strategies and states of habitat selection, and to draw the adaptive landscapes for habitat-selecting species. The landscapes can then be used to forecast future evolution under a variety of climate change and other scenarios.

"

Response to “The Reality of Pervasive Transcription”

Response to “The Reality of Pervasive Transcription”: "

by Harm van Bakel, Corey Nislow, Benjamin J. Blencowe, Timothy R. Hughes

"

The Reality of Pervasive Transcription

The Reality of Pervasive Transcription: "

by Michael B. Clark, Paulo P. Amaral, Felix J. Schlesinger, Marcel E. Dinger, Ryan J. Taft, John L. Rinn, Chris P. Ponting, Peter F. Stadler, Kevin V. Morris, Antonin Morillon, Joel S. Rozowsky, Mark B. Gerstein, Claes Wahlestedt, Yoshihide Hayashizaki, Piero Carninci, Thomas R. Gingeras, John S. Mattick

"

Enhancer-PRE communication contributes to the expansion of gene expression domains in proliferating primordia [RESEARCH ARTICLES]

Enhancer-PRE communication contributes to the expansion of gene expression domains in proliferating primordia [RESEARCH ARTICLES]: "Lidia Perez, Lara Barrio, David Cano, Ulla-Maj Fiuza, Mariana Muzzopappa, and Marco Milan

Trithorax-group and Polycomb-group proteins interact with chromosomal elements, termed PRE/TREs, to ensure stable heritable maintenance of the transcriptional state of nearby genes. Regulatory elements that bind both groups of proteins are termed maintenance elements (MEs). Some of these MEs maintain the initial activated transcriptional state of a nearby reporter gene through several rounds of mitosis during development. Here, we show that expression of hedgehog in the posterior compartment of the Drosophila wing results from the communication between a previously defined ME and a nearby cis-regulatory element termed the C enhancer. The C enhancer integrates the activities of the Notch and Hedgehog signalling pathways and, from the early wing primordium stage, drives expression to a thin stripe in the posterior compartment that corresponds to the dorsal-ventral compartment boundary. The ME maintains the initial activated transcriptional state conferred by the C enhancer and contributes to the expansion, by growth, of its expression domain throughout the posterior compartment. Communication between the ME and the C enhancer also contributes to repression of gene expression in anterior cells. Most interestingly, we present evidence that enhancers and MEs of different genes are interchangeable modules whose communication is involved in restricting and expanding the domains of gene expression. Our results emphasize the modular role of MEs in regulation of gene expression within growing tissues.

"

The Same Regulatory Point Mutation Changed Seed-Dispersal Structures in Evolution and Domestication

The Same Regulatory Point Mutation Changed Seed-Dispersal Structures in Evolution and Domestication: "Nicolas Arnaud, Tom Lawrenson, Lars Østergaard, Robert Sablowski. It is unclear whether gene regulatory changes that drive evolution at the population and species levels [1–3] can be extrapolated to higher taxonomic levels [4, 5]. Here, we investigated the role ...."

Rapid Evolutionary Rewiring of a Structurally Constrained Eye Enhancer

Rapid Evolutionary Rewiring of a Structurally Constrained Eye Enhancer: "Christina I. Swanson, David B. Schwimmer, Scott Barolo. BackgroundEnhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcriptio...."

Interactions between Glucocorticoid Treatment and Cis-Regulatory Polymorphisms Contribute to Cellular Response Phenotypes

Interactions between Glucocorticoid Treatment and Cis-Regulatory Polymorphisms Contribute to Cellular Response Phenotypes: "

by Joseph C. Maranville, Francesca Luca, Allison L. Richards, Xiaoquan Wen, David B. Witonsky, Shaneen Baxter, Matthew Stephens, Anna Di Rienzo

Glucocorticoids (GCs) mediate physiological responses to environmental stress and are commonly used as pharmaceuticals. GCs act primarily through the GC receptor (GR, a transcription factor). Despite their clear biomedical importance, little is known about the genetic architecture of variation in GC response. Here we provide an initial assessment of variability in the cellular response to GC treatment by profiling gene expression and protein secretion in 114 EBV-transformed B lymphocytes of African and European ancestry. We found that genetic variation affects the response of nearby genes and exhibits distinctive patterns of genotype-treatment interactions, with genotypic effects evident in either only GC-treated or only control-treated conditions. Using a novel statistical framework, we identified interactions that influence the expression of 26 genes known to play central roles in GC-related pathways (e.g. NQO1, AIRE, and SGK1) and that influence the secretion of IL6."

Building Enhancers from the Ground Up: A Synthetic Biology Approach

Building Enhancers from the Ground Up: A Synthetic Biology Approach: "Roee Amit, Hernan G. Garcia, Rob Phillips, Scott E. Fraser. A challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhanc...."

Slow protein evolutionary rates are dictated by surface-core association [Evolution]

Slow protein evolutionary rates are dictated by surface-core association [Evolution]: "Why do certain proteins evolve much slower than others? We compared not only rates per protein, but also rates per position within individual proteins. For ∼90% of proteins, the distribution of positional rates exhibits three peaks: a peak of slow evolving residues, with average log2[normalized rate], log2μ, of ca. −2, corresponding primarily to core residues; a peak of fast evolving residues (log2μ ∼ 0.5) largely corresponding to surface residues; and a very fast peak (log2μ ∼ 2) associated with disordered segments. However, a unique fraction of proteins that evolve very slowly exhibit not only a negligible fast peak, but also a peak with a log2μ ∼ −4, rather than the standard core peak of −2. Thus, a “freeze” of a protein's surface seems to stop core evolution as well. We also observed a much higher fraction of substitutions in potentially interacting residues than expected by chance, including substitutions in pairs of contacting surface-core residues. Overall, the data suggest that accumulation of surface substitutions enables the acceptance of substitutions in core positions. The underlying reason for slow evolution might therefore be a highly constrained surface due to protein–protein interactions or the need to prevent misfolding or aggregation. If the surface is inaccessible to substitutions, so becomes the core, thus resulting in very slow overall rates."

Sexually dimorphic regulation of the Wingless morphogen controls sex-specific segment number in Drosophila [Developmental Biology]

Sexually dimorphic regulation of the Wingless morphogen controls sex-specific segment number in Drosophila [Developmental Biology]: "Sexual dimorphism is widespread throughout the metazoa and plays important roles in mate recognition and preference, sex-based niche partitioning, and sex-specific coadaptation. One notable example of sex-specific differences in insect body morphology is presented by the higher diptera, such as Drosophila, in which males develop fewer abdominal segments than females. Because diversity in segment number is a distinguishing feature of major arthropod clades, it is of fundamental interest to understand how different numbers of segments can be generated within the same species. Here we show that sex-specific and segment-specific regulation of the Wingless (Wg) morphogen underlies the development of sexually dimorphic adult segment number in Drosophila. Wg expression is repressed in the developing terminal male abdominal segment by the combination of the Hox protein Abdominal-B (Abd-B) and the sex-determination regulator Doublesex (Dsx). The subsequent loss of the terminal male abdominal segment during pupation occurs through a combination of developmental processes including segment compartmental transformation, apoptosis, and suppression of cell proliferation. Furthermore, we show that ectopic expression of Wg is sufficient to rescue this loss. We propose that dimorphic Wg regulation, in concert with monomorphic segment-specific programmed cell death, are the principal mechanisms of sculpting the sexually dimorphic abdomen of Drosophila."

Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq [METHOD]

Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq [METHOD]: "

Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples. Single-cell RNA-seq expression profiles were generated, and clustered to form a two-dimensional cell map onto which expression data were projected. The resulting cell map integrates three levels of organization: the whole population of cells, the functionally distinct subpopulations it contains, and the single cells themselves—all without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the transcriptomes of 85 single cells of two distinct types. We believe this strategy will enable the unbiased discovery and analysis of naturally occurring cell types during development, adult physiology, and disease.

"

Genome-wide identification of conserved regulatory function in diverged sequences [METHOD]

Genome-wide identification of conserved regulatory function in diverged sequences [METHOD]: "

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term 'covert.'

"

Combinatorial binding of transcription factors in the pluripotency control regions of the genome [RESEARCH]

Combinatorial binding of transcription factors in the pluripotency control regions of the genome [RESEARCH]: "

The pluripotency control regions (PluCRs) are defined as genomic regions that are bound by POU5F1, SOX2, and NANOG in vivo. We utilized a high-throughput binding assay to record more than 270,000 different DNA/protein binding measurements along incrementally tiled windows of DNA within these PluCRs. This high-resolution binding map is then used to systematically define the context of POU factor binding, and reveals patterns of cooperativity and competition in the pluripotency network. The most prominent pattern is a pervasive binding competition between POU5F1 and the forkhead transcription factors. Like many transcription factors, POU5F1 is co-expressed with a paralog, POU2F1, that shares an apparently identical binding specificity. By analyzing thousands of binding measurements, we discover context effects that discriminate POU2F1 from POU5F1 binding. Proximal NANOG binding promotes POU5F1 binding, whereas nearby SOX2 binding favors POU2F1. We demonstrate by cross-species comparison and by chromatin immunoprecipitation (ChIP) that the contextual sequence determinants learned in vitro are sufficient to predict POU2F1 binding in vivo.

"

Genome-wide identification of conserved regulatory function in diverged sequences [METHOD]

Genome-wide identification of conserved regulatory function in diverged sequences [METHOD]: "

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term 'covert.'

"

Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line

Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line: "

by Velia Siciliano, Filippo Menolascina, Lucia Marucci, Chiara Fracassi, Immacolata Garzilli, Maria Nicoletta Moretti, Diego di Bernardo

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the “switch off” times, as comparared to the nonautoregulatated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour."