Caudal counter-represses Hunchback to regulateeven-skippedstripe 2 expression in Drosophila embryos

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped . While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by Caudal binding—this relationship is called counter-repression. We found evidence that this relationship is conserved by comparing predicted binding sites for Hunchback and Caudal across orthologous eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is depicted as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted by Caudal binding. We discuss the implications of this interaction for eve stripe 2 regulation and evolution

A mutation in theDrosophila melanogaster evestripe 2 minimal enhancer is buffered by flanking sequences

Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene’s endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogaster even-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site mutation in the minimal enhancer, the same gt-2 binding site mutation in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Mutating the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context

Caudal counter-represses Hunchback to regulate even-skipped stripe 2 expression in Drosophila embryos

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped . While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by Caudal binding—this relationship is called counter-repression. We found evidence that this relationship is conserved by comparing predicted binding sites for Hunchback and Caudal across orthologous eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is depicted as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted by Caudal binding. We discuss the implications of this interaction for eve stripe 2 regulation and evolution

A mutation in the Drosophila melanogaster eve stripe 2 minimal enhancer is buffered by flanking sequences

AbstractEnhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene’s endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogaster even-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site mutation in the minimal enhancer, the same gt-2 binding site mutation in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Mutating the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context

Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution

Yearly planning meetings: individualized development plans aren't just more paperwork.

The National Institutes of Health (NIH) encourages trainees to make Individualized Development Plans to help them prepare for academic and nonacademic careers. We describe our approach to building an Individualized Development Plan, the reasons we find them useful and empowering for both PIs and trainees, and resources to help other labs implement them constructively

Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution

Quantitative Data and Analyses for Vincent, et al. 2018

This fileset includes raw data and code for the analyses contained in Vincent, et al. 2018, "Hunchback is counter-repressed to regulate <i>even-skipped</i> stripe 2 expression in Drosophila embryos." In this study, we present evidence that the transcription factor Hunchback directly represses the <i>eve </i>stripe 3+7 enhancer and is counter-repressed in regulation of the <i>eve </i>stripe 2+7 enhancer. We collected quantitative, cellular resolution<i> </i>gene expression data in wild-type and perturbed Drosophila embryos containing reporter constructs for these enhancers. We also examined binding site enrichment in orthologous <i>eve </i>enhancers. Our results alter the canonical description of the <i>eve </i>stripe 2 enhancer where Hunchback is considered a direct activator; Hunchback instead represses this enhancer, and this repression is counteracted by additional sequence features.<div><br></div><div>The following files contain the data and the code used to generate the figures and tables contained in the study. We hope they will prove useful for the community.</div><div><br></div><div>Vincent2018_MATLAB: This is a compressed file containing raw data and code. This file contains subfiles organized this file by figure or table number, and each subfile contains commented MATLAB code, plots, and raw data in the form of pointclouds, or cellular resolution expression measurements from individual embryos.</div><div><br></div><div>PWMSforEnhancerMutations: This is a compressed file containing frequency position weight matrices for transcription factors expressed in the embryo. We used these matrices to predict binding sites for these factors with PATSER, a program available from the Stormo lab (http://stormo.wustl.edu/software.html). We generated these matrices from count matrices published in Noyes, et al. 2008 and Schroeder, et al. 2011 using a pseudocount of 1.</div><div><br></div><div>SitePredictionsforEnhancerMutations: This compressed file contains individual csv files with the binding site predictions used to design <i>eve </i>enhancers with binding site mutations. Each file all predicted binding sites within the enhancer, along with their coordinates and PATSER scores. We used a p-value cutoff of 0.003 to make these predictions.</div><div><br></div><div>PWMsforEnrichment: This is a compressed file containing frequency position weight matrices for Bicoid, Caudal, Hunchback and Krüppel. We used these matrices to predict binding sites for these factors with PATSER, a program available from the Stormo lab (http://stormo.wustl.edu/software.html). We generated these matrices from count matrices available through FlyFactorSurvey (Zhu, et al. 2011, http://mccb.umassmed.edu/ffs/) using a pseudocount of 0.1.</div><div><br></div><div>SitePredictionsforEnrichment: This compressed file contains individual csv files with the binding site predictions used to calculate binding site enrichment scores in orthologous <i>eve </i>enhancers (see Hare, et al. 2008 for enhancer sequences, and Wunderlich, et al. 2015 for the computational methods). Each file all predicted binding sites within the enhancer, along with their coordinates and PATSER scores. We used the default PATSER p-value cutoff to make these predictions. Scores for binding sites predicted in all DNAse accessible regions in stage 5 of embryogenesis (Thomas, et al. 2011) can be found in the corresponding folders in Vincent2018_MATLAB.<br></div