Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds

Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds:

Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds

Nature 477, 7366 (2011). doi:10.1038/nature10391

Authors: Zhe Wang, Rebecca L. Young, Huiling Xue & Günter P. Wagner

Morphological characters are the result of developmental gene expression. The identity of a character is ultimately grounded in the gene regulatory network directing development and thus whole-genome gene expression data can provide evidence about character identity. This approach has been successfully used to assess cell-type identity. Here we use transcriptomic data to address a long-standing uncertainty in evolutionary biology, the identity of avian wing digits. Embryological evidence clearly identifies the three wing digits as developing from digit positions 2, 3 and 4 (ref. 6), whereas palaeontological data suggest that they are digits I, II and III. We compare the transcriptomes of the wing and foot digits and find a strong signal that unites the first wing digit with the first foot digit, even though the first wing digit develops from embryological position 2. Interestingly, our transcriptomic data of the posterior digits show a higher degree of differentiation among forelimb digits compared with hindlimb digits. These data show that in the stem lineage of birds the first digit underwent a translocation from digit position 1 to position 2, and further indicate that the posterior wing digits have unique identities contrary to any model of avian digit identity proposed so far.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design:

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Nature 477, 7365 (2011). doi:10.1038/nature10403

Authors: Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Héloïse Muller, Narayana Annaluru, William J. Blake, Joy W. Schwerzmann, Junbiao Dai, Derek L. Lindstrom, Annabel C. Boeke, Daniel E. Gottschling, Srinivasan Chandrasegaran, Joel S. Bader & Jef D. Boeke

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Premetazoan Ancestry of the Myc-Max Network

Premetazoan Ancestry of the Myc-Max Network:

The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.

MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda [Evolution]

MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda [Evolution]: Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g., spiders, insects, and their allies) into a monophyletic group of invertebrates with walking appendages known as the Panarthropoda. However, molecular data generally do not support the inclusion of tardigrades within the Panarthropoda, but instead place them closer to Nematoda (roundworms). Here we present results from the analyses of two independent genomic datasets, expressed sequence tags (ESTs) and microRNAs (miRNAs), which congruently resolve the phylogenetic relationships of Tardigrada. Our EST analyses, based on 49,023 amino acid sites from 255 proteins, significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations—comparisons of model fit, signal dissection, and taxonomic pruning—we show that support for a Tardigrada + Nematoda group derives from the phylogenetic artifact of long-branch attraction. Our small RNA libraries fully support our EST results; no miRNAs were found to link Tardigrada and Nematoda, whereas all panarthropods were found to share one unique miRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second miRNA (miR-305). Our study confirms the monophyly of the legged ecdysozoans, shows that past support for a Tardigrada + Nematoda group was due to long-branch attraction, and suggests that the velvet worms are the sister group to the arthropods.

lincRNAs act in the circuitry controlling pluripotency and differentiation

lincRNAs act in the circuitry controlling pluripotency and differentiation:

lincRNAs act in the circuitry controlling pluripotency and differentiation

Nature 477, 7364 (2011). doi:10.1038/nature10398

Authors: Mitchell Guttman, Julie Donaghey, Bryce W. Carey, Manuel Garber, Jennifer K. Grenier, Glen Munson, Geneva Young, Anne Bergstrom Lucas, Robert Ach, Laurakay Bruhn, Xiaoping Yang, Ido Amit, Alexander Meissner, Aviv Regev, John L. Rinn, David E. Root & Eric S. Lander

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the

The Snail Repressor Inhibits Release, Not Elongation, of Paused Pol II in the Drosophila Embryo

The Snail Repressor Inhibits Release, Not Elongation, of Paused Pol II in the Drosophila Embryo: Jacques P. Bothma, Joe Magliocco, Michael Levine. The development of the precellular Drosophila embryo is characterized by exceptionally rapid transitions in gene activity, with broadly distributed maternal regulatory gradients giving way ....

Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification

Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification: Minjia Tan, Hao Luo, Sangkyu Lee, Fulai Jin, Jeong Soo Yang, Emilie Montellier, Thierry Buchou, Zhongyi Cheng, Sophie Rousseaux, Nisha Rajagopal, Zhike Lu, Zhen Ye, Qin Zhu, Joanna Wysocka, Yang Ye, Saadi Khochbin, Bing Ren, Yingming Zhao. We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine....

Two types of cis-trans compensation in the evolution of transcriptional regulation [Evolution]

Two types of cis-trans compensation in the evolution of transcriptional regulation [Evolution]: Because distant species often share similar macromolecules, regulatory mutations are often considered responsible for much of their biological differences. Recently, a large portion of regulatory changes has been attributed to cis-regulatory mutations. Here, we examined an alternative possibility that the putative contribution of cis-regulatory changes was, in fact, caused by compensatory action of cis- and trans-regulatory elements. First, we show by stochastic simulations that compensatory cis-trans evolution maintains the binding affinity of a transcription factor at a constant level, thereby spuriously exaggerating the contribution of cis-regulatory mutations to gene expression divergence. This exaggeration was not observed when changes in the binding affinity were compensated by variable transcription factor concentration. Second, using reciprocal introgressions of Drosophila, we show that relative expression of heterozygous alleles from two distinct species often varied significantly between different species backgrounds, indicating the possible action of cis-trans compensation. Taken together, we propose that cis-trans hybrid incompatibilities are accumulating much faster than generally considered.

Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera) [Evolution]

Alternative splicing of a single transcription factor drives selfish reproductive behavior in honeybee workers (Apis mellifera) [Evolution]: In eusocial insects the production of daughters is generally restricted to mated queens, and unmated workers are functionally sterile. The evolution of this worker sterility has been plausibly explained by kin selection theory [Hamilton W (1964) J Theor Biol 7:1–52], and many traits have evolved to prevent conflict over reproduction among the females in an insect colony. In honeybees (Apis mellifera), worker reproduction is regulated by the queen, brood pheromones, and worker policing. However, workers of the Cape honeybee, Apis mellifera capensis, can evade this control and establish themselves as social parasites by activating their ovaries, parthenogenetically producing diploid female offspring (thelytoky) and producing queen-like amounts of queen pheromones. All these traits have been shown to be strongly influenced by a single locus on chromosome 13 [Lattorff HMG, et al. (2007) Biol Lett 3:292–295]. We screened this region for candidate genes and found that alternative splicing of a gene homologous to the gemini transcription factor of Drosophila controls worker sterility. Knocking out the critical exon in a series of RNAi experiments resulted in rapid worker ovary activation—one of the traits characteristic of the social parasites. This genetic switch may be controlled by a short intronic splice enhancer motif of nine nucleotides attached to the alternative splice site. The lack of this motif in parasitic Cape honeybee clones suggests that the removal of nine nucleotides from the altruistic worker genome may be sufficient to turn a honeybee from an altruistic worker into a parasite.

Transcription factor RBPJ/CSL: A genome-wide look at transcriptional regulation [Commentary]

Transcription factor RBPJ/CSL: A genome-wide look at transcriptional regulation [Commentary]: The transcriptional regulator RBPJ, otherwise known as CSL (“CBF-1, Suppressor of Hairless, Lag-2,” after its mammalian, Drosophila, and Caenorhabditis elegans orthologues) is a highly conserved DNA-binding protein that plays a central role in cell fate decisions in metazoa. RBPJ mediates canonical Notch signaling (1). In the absence of active Notch, RBPJ is thought to be primarily a transcriptional repressor that exists in complexes with corepressors (2). When bound to the active intracellular domain of Notch (NICD), RBPJ recruits a coactivator complex, including a Mastermind homologue (MAML1-3 in mammals), and drives a complex transcriptional program with pervasive phenotypic effects. This program...

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer [RESEARCH ARTICLES]

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer [RESEARCH ARTICLES]: Paolo Struffi, Maria Corado, Leah Kaplan, Danyang Yu, Christine Rushlow, and Stephen Small

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo.

Use of a Drosophila genome-wide conserved sequence database to identify functionally related cis-regulatory enhancers

Use of a Drosophila genome-wide conserved sequence database to identify functionally related cis-regulatory enhancers:

Abstract

Phylogenetic footprinting has revealed that cis-regulatory modules consist of clusters of conserved DNA sequences. We have generated a Drosophila melanogaster genomic database of conserved sequence clusters (CSCs) to facilitate enhancer discovery and analysis of their sub-structure. The database consists of >100,000 CSCs gleaned from EvoPrints spanning over 90% of the genome. To identify related enhancers based on shared conserved sequence elements, we have developed database search and alignment algorithms, collectively known as cis-Decoder. These web-accessible tools initially identify conserved repeat elements within an EvoPrinted input enhancer and then search the database for CSCs that score highly against the input enhancer. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. The genome-wide CSC database and cis-Decoder algorithms can be used to discover and analyze cis-regulatory DNA involved in any developmental process. Developmental Dynamics, 2011. © 2011 Wiley-Liss, Inc.

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes [RESEARCH]

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes [RESEARCH]:

Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

Positive and Negative Selection in Murine Ultraconserved Noncoding Elements

Positive and Negative Selection in Murine Ultraconserved Noncoding Elements:

There are many more selectively constrained noncoding than coding nucleotides in the mammalian genome, but most mammalian noncoding DNA is subject to weak selection, on average. One of the most striking discoveries to have emerged from comparisons among mammalian genomes is the hundreds of noncoding elements of more than 200 bp in length that show absolute conservation among mammalian orders. These elements represent the tip of the iceberg of a much larger class of conserved noncoding elements (CNEs). Much evidence suggests that CNEs are selectively constrained and not mutational cold-spots, and there is evidence that some CNEs play a role in the regulation of development. Here, we quantify negative and positive selection acting in murine CNEs by analyzing within-species nucleotide variation and between-species divergence of CNEs that we identified using a phylogenetically independent comparison. The distribution of fitness effects of new mutations in CNEs, inferred from within-species polymorphism, suggests that CNEs receive a higher number of strongly selected deleterious mutations and many fewer nearly neutral mutations than amino acid sites of protein-coding genes or regulatory elements close to genes. However, we also show that CNEs experience a far higher proportion of adaptive substitutions than any known category of genomic sites in murids. The absolute rate of adaptation of CNEs is similar to that of amino acid sites of proteins. This result suggests that there is widespread adaptation in mammalian conserved noncoding DNA elements, some of which have been implicated in the regulation of crucially important processes, including development.

Evolution of Sex-Specific Traits through Changes in HOX-Dependent doublesex Expression

Evolution of Sex-Specific Traits through Changes in HOX-Dependent doublesex Expression:
by Kohtaro Tanaka, Olga Barmina, Laura E. Sanders, Michelle N. Arbeitman, Artyom Kopp


Almost every animal lineage is characterized by unique sex-specific traits, implying that such traits are gained and lost frequently in evolution. However, the genetic mechanisms responsible for these changes are not understood. In Drosophila, the activity of the sex determination pathway is restricted to sexually dimorphic tissues, suggesting that spatial regulation of this pathway may contribute to the evolution of sex-specific traits. We examine the regulation and function of doublesex (dsx), the main transcriptional effector of the sex determination pathway, in the development and evolution of Drosophila sex combs. Sex combs are a recent evolutionary innovation and show dramatic diversity in the relatively few Drosophila species that have them. We show that dsx expression in the presumptive sex comb region is activated by the HOX gene Sex combs reduced (Scr), and that the male isoform of dsx up-regulates Scr so that both genes become expressed at high levels in this region in males but not in females. Precise spatial regulation of dsx is essential for defining sex comb position and morphology. Comparative analysis of Scr and dsx expression reveals a tight correlation between sex comb morphology and the expression patterns of both genes. In species that primitively lack sex combs, no dsx expression is observed in the homologous region, suggesting that the origin and diversification of this structure were linked to the gain of a new dsx expression domain. Two other, distantly related fly lineages that independently evolved novel male-specific structures show evolutionary gains of dsx expression in the corresponding tissues, where dsx may also be controlled by Scr. These findings suggest that changes in the spatial regulation of sex-determining genes are a key mechanism that enables the evolution of new sex-specific traits, contributing to some of the most dramatic examples of phenotypic diversification in nature.

Evidence for Hitchhiking of Deleterious Mutations within the Human Genome

Evidence for Hitchhiking of Deleterious Mutations within the Human Genome:

by Sung Chun, Justin C. Fay

Deleterious mutations present a significant obstacle to adaptive evolution. Deleterious mutations can inhibit the spread of linked adaptive mutations through a population; conversely, adaptive substitutions can increase the frequency of linked deleterious mutations and even result in their fixation. To assess the impact of adaptive mutations on linked deleterious mutations, we examined the distribution of deleterious and neutral amino acid polymorphism in the human genome. Within genomic regions that show evidence of recent hitchhiking, we find fewer neutral but a similar number of deleterious SNPs compared to other genomic regions. The higher ratio of deleterious to neutral SNPs is consistent with simulated hitchhiking events and implies that positive selection eliminates some deleterious alleles and increases the frequency of others. The distribution of disease-associated alleles is also altered in hitchhiking regions. Disease alleles within hitchhiking regions have been associated with auto-immune disorders, metabolic diseases, cancers, and mental disorders. Our results suggest that positive selection has had a significant impact on deleterious polymorphism and may be partly responsible for the high frequency of certain human disease alleles.

Pioneer factors: directing transcriptional regulators within the chromatin environment

Pioneer factors: directing transcriptional regulators within the chromatin environment: Luca Magnani, Jérôme Eeckhoute, Mathieu Lupien. Chromatin is a well-known obstacle to transcription as it controls DNA accessibility, which directly impacts the recruitment of the transcriptional machinery. The recent burst of functional genomi....

[Report] optix Drives the Repeated Convergent Evolution of Butterfly Wing Pattern Mimicry

[Report] optix Drives the Repeated Convergent Evolution of Butterfly Wing Pattern Mimicry: Heliconius butterfly wing pattern mimicry is driven by cis-regulatory variation of the optix gene.

Authors: Robert D. Reed, Riccardo Papa, Arnaud Martin, Heather M. Hines, Brian A. Counterman, Carolina Pardo-Diaz, Chris D. Jiggins, Nicola L. Chamberlain, Marcus R. Kronforst, Rui Chen, Georg Halder, H. Frederik Nijhout, W. Owen McMillan

Bistability versus Bimodal Distributions in Gene Regulatory Processes from Population Balance

Bistability versus Bimodal Distributions in Gene Regulatory Processes from Population Balance:

by Che-Chi Shu, Anushree Chatterjee, Gary Dunny, Wei-Shou Hu, Doraiswami Ramkrishna

In recent times, stochastic treatments of gene regulatory processes have appeared in the literature in which a cell exposed to a signaling molecule in its environment triggers the synthesis of a specific protein through a network of intracellular reactions. The stochastic nature of this process leads to a distribution of protein levels in a population of cells as determined by a Fokker-Planck equation. Often instability occurs as a consequence of two (stable) steady state protein levels, one at the low end representing the “off” state, and the other at the high end representing the “on” state for a given concentration of the signaling molecule within a suitable range. A consequence of such bistability has been the appearance of bimodal distributions indicating two different populations, one in the “off” state and the other in the “on” state. The bimodal distribution can come about from stochastic analysis of a single cell. However, the concerted action of the population altering the extracellular concentration in the environment of individual cells and hence their behavior can only be accomplished by an appropriate population balance model which accounts for the reciprocal effects of interaction between the population and its environment. In this study, we show how to formulate a population balance model in which stochastic gene expression in individual cells is incorporated. Interestingly, the simulation of the model shows that bistability is neither sufficient nor necessary for bimodal distributions in a population. The original notion of linking bistability with bimodal distribution from single cell stochastic model is therefore only a special consequence of a population balance model.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes:

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

Nature Methods 8, 737 (2011).
doi:10.1038/nmeth.1662

Authors: Koen J T Venken, Karen L Schulze, Nele A Haelterman, Hongling Pan, Yuchun He, Martha Evans-Holm, Joseph W Carlson, Robert W Levis, Allan C Spradling, Roger A Hoskins & Hugo J Bellen

MicroRNAs can generate thresholds in target gene expression

MicroRNAs can generate thresholds in target gene expression:

MicroRNAs can generate thresholds in target gene expression

Nature Genetics 43, 854 (2011).
doi:10.1038/ng.905

Authors: Shankar Mukherji, Margaret S Ebert, Grace X Y Zheng, John S Tsang, Phillip A Sharp & Alexander van Oudenaarden

Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features [RESEARCH]

Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features [RESEARCH]:

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates.

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes [RESEARCH]

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes [RESEARCH]:

Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

Insulators form gene loops by interacting with promoters in Drosophila [RESEARCH ARTICLES]

Insulators form gene loops by interacting with promoters in Drosophila [RESEARCH ARTICLES]: Maksim Erokhin, Anna Davydova, Olga Kyrchanova, Alexander Parshikov, Pavel Georgiev, and Darya Chetverina

Chromatin insulators are regulatory elements involved in the modulation of enhancer-promoter communication. The 1A2 and Wari insulators are located immediately downstream of the Drosophila yellow and white genes, respectively. Using an assay based on the yeast GAL4 activator, we have found that both insulators are able to interact with their target promoters in transgenic lines, forming gene loops. The existence of an insulator-promoter loop is confirmed by the fact that insulator proteins could be detected on the promoter only in the presence of an insulator in the transgene. The upstream promoter regions, which are required for long-distance stimulation by enhancers, are not essential for promoter-insulator interactions. Both insulators support basal activity of the yellow and white promoters in eyes. Thus, the ability of insulators to interact with promoters might play an important role in the regulation of basal gene transcription.

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression [RESEARCH ARTICLES]

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression [RESEARCH ARTICLES]: Leslie Dunipace, Anil Ozdemir, and Angelike Stathopoulos

It has been shown in several organisms that multiple cis-regulatory modules (CRMs) of a gene locus can be active concurrently to support similar spatiotemporal expression. To understand the functional importance of such seemingly redundant CRMs, we examined two CRMs from the Drosophila snail gene locus, which are both active in the ventral region of pre-gastrulation embryos. By performing a deletion series in a ~25 kb DNA rescue construct using BAC recombineering and site-directed transgenesis, we demonstrate that the two CRMs are not redundant. The distal CRM is absolutely required for viability, whereas the proximal CRM is required only under extreme conditions such as high temperature. Consistent with their distinct requirements, the CRMs support distinct expression patterns: the proximal CRM exhibits an expanded expression domain relative to endogenous snail, whereas the distal CRM exhibits almost complete overlap with snail except at the anterior-most pole. We further show that the distal CRM normally limits the increased expression domain of the proximal CRM and that the proximal CRM serves as a `damper' for the expression levels driven by the distal CRM. Thus, the two CRMs interact in cis in a non-additive fashion and these interactions may be important for fine-tuning the domains and levels of gene expression.

Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling [RESEARCH ARTICLES]

Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling [RESEARCH ARTICLES]: Cyril Charles, Maria Hovorakova, Youngwook Ahn, David B. Lyons, Pauline Marangoni, Svatava Churava, Brian Biehs, Andrew Jheon, Herve Lesot, Guive Balooch, Robb Krumlauf, Laurent Viriot, Renata Peterkova, and Ophir D. Klein

Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.