Enhancer Responses to Similarly Distributed Antagonistic Gradients in Development

Formation of spatial gene expression patterns in development depends on transcriptional responses mediated by gene control regions, enhancers. Here, we explore possible responses of enhancers to overlapping gradients of antagonistic transcriptional regulators in the Drosophila embryo. Using quantitative models based on enhancer structure, we demonstrate how a pair of antagonistic transcription factor gradients with similar or even identical spatial distributions can lead to the formation of distinct gene expression domains along the embryo axes. The described mechanisms are sufficient to explain the formation of the anterior and the posterior knirps expression, the posterior hunchback expression domain, and the lateral stripes of rhomboid expression and of other ventral neurogenic ectodermal genes. The considered principles of interaction between antagonistic gradients at the enhancer level can also be applied to diverse developmental processes, such as domain specification in imaginal discs, or even eyespot pattern formation in the butterfly wing

Divergence in cis-regulatory networks: taking the 'species' out of cross-species analysis

Significant differences between species in genomic occupancy of conserved transcription factors are mostly due to species-specificity of cis-regulatory sequences

Studying the functional conservation of cis-regulatory modules and their transcriptional output

<p>Abstract</p> <p>Background</p> <p><it>Cis</it>-regulatory modules (CRMs) are distinct, genomic regions surrounding the target gene that can independently activate the promoter to drive transcription. The activation of a CRM is controlled by the binding of a certain combination of transcription factors (TFs). It would be of great benefit if the transcriptional output mediated by a specific CRM could be predicted. Of equal benefit would be identifying <it>in silico </it>a specific CRM as the driver of the expression in a specific tissue or situation. We extend a recently developed biochemical modeling approach to manage both prediction tasks. Given a set of TFs, their protein concentrations, and the positions and binding strengths of each of the TFs in a putative CRM, the model predicts the transcriptional output of the gene. Our approach predicts the location of the regulating CRM by using predicted TF binding sites in regions near the gene as input to the model and searching for the region that yields a predicted transcription rate most closely matching the known rate.</p> <p>Results</p> <p>Here we show the ability of the model on the example of one of the CRMs regulating the <it>eve </it>gene, MSE2. A model trained on the MSE2 in <it>D. melanogaster </it>was applied to the surrounding sequence of the <it>eve </it>gene in seven other <it>Drosophila </it>species. The model successfully predicts the correct MSE2 location and output in six out of eight <it>Drosophila </it>species we examine.</p> <p>Conclusion</p> <p>The model is able to generalize from <it>D. melanogaster </it>to other <it>Drosophila </it>species and accurately predicts the location and transcriptional output of MSE2 in those species. However, we also show that the current model is not specific enough to function as a genome-wide CRM scanner, because it incorrectly predicts other genomic regions to be MSE2s.</p

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

Template Requirements for Telomerase Translocation in Kluyveromyces lactis

Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component. In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence. To determine the role of the 5-nt repeats defining the ends of the K. lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats. These template mutations typically result in telomeres that are shorter than those of wild-type cells. The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed. Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp. Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K. lactis. In addition, the region immediately 3′ of the template may play an important role in translocation of the enzyme

Evolution of 3D chromatin organization in metazoan species

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en Sevilla (España) del 18 al 21 de septiembre de 2023.Metazoan genomes are organized into topologically associating domains (TADs), which ensure an appropriate interaction between regulatory elements and their target genes. Disruptions in TADs can drive human disease, including developmental malformations or cancer, but also serve as a substrate for phenotypical adaptation. Despite their functional significance, studies conducted across diverse animal models have revealed remarkable differences in how these domains are formed. Invertebrates, TADs mainly result from a mechanism of loop extrusion, derived from the interplay between the cohesin complex and the insulator protein CCCTC-binding factor (CTCF). However, in certain invertebrates such as flies, these domains seem to primarily depend on chromatin and transcriptional state, with a less prominent role for CTCF. This discrepancy raises fundamental questions on how the mechanisms of 3D chromatin organization may have evolved. We have recently investigated the divergence of 3D chromatin organization throughout metazoan evolution. For that, we have performed CTCF orthologous replacements from species of different taxa, in both mouse embryonic stem cells and flies. This setup has enabled us to evaluate the impact of these substitutions on 3D chromatin organization, transcriptional activity, and resulting phenotypes. This talk will discuss our recent results, which allowed us to uncouple two distinct functions of CTCF as a transcriptional regulator and as a mediator of 3D chromatin organization. Our findings also suggest a model where CTCF has played a major role in the evolution of 3D chromatin by shifting its chromatin binding preferences and partners.Peer reviewe

Evolution of the Ventral Midline in Insect Embryos

SummaryThe ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D.mel.) and the mosquito Anopheles gambiae (A.gam.), the midline is narrow and spans just 1–2 cells. However, in the honeybee, Apis mellifera (A.mel.), the ventral midline is broad and encompasses 5–6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D.mel., but it is activated solely by Twi in A.mel. We suggest that the Twi-only mode of regulation—and the broad ventral midline—represents the ancestral form of CNS patterning in Holometabolous insects