Rapid Evolutionary Rewiring of a Structurally Constrained Eye Enhancer

SummaryBackgroundEnhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as—in some cases—the arrangement and spacing of binding sites for these factors. Here, we examine evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye.ResultsDespite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. Our data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that we observe. Furthermore, stereotypical spatial relationships between certain binding sites (“grammar elements”) can be identified in all sparkling orthologs—although the sites themselves are often recently derived. We also find that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in noncone cells.ConclusionsRapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. Our findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale

A model of spatially restricted transcription in opposing gradients of activators and repressors

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102118/1/msb201248-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102118/2/msb201248.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102118/3/msb201248.reviewer_comments.pd

Developing Future Biologists: Developmental Biology for Undergraduates from Underserved Communities

Developing Future Biologists (DFB) is an inclusive, trainee-run organization that strives to excite and engage the next generation of biologists, regardless of race, gender or socioeconomic status, in the field of developmental biology. DFB offers a week-long course consisting of active lectures, hands-on laboratory sessions, and professional development opportunities through interactions with scientists from a variety of backgrounds and careers. A major goal of DFB is to propel undergraduate students from underserved communities to pursue biomedical research opportunities and advanced degrees in science. To achieve this goal, we provide DFB participants with continuing access to a diverse network of scientists that students can utilize to secure opportunities and foster success throughout multiple stages of their research careers. Here, we describe the flourishing DFB program at the University of Michigan to encourage other institutions to create their own DFB programs

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

AbstractThe highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1

Reverse-Engineering a Transcriptional Enhancer: A Case Study in Drosophila

Abstract Enhancers, or cis-regulatory elements, are the principal determinants of spatiotemporal patterning of gene expression. For reasons of clinical and research utility, it is desirable to build customized enhancers that drive novel gene expression patterns, but currently, we largely rely on “found” genomic elements. Synthetic enhancers, assembled from transcription factor binding sites taken from natural signal-regulated enhancers, generally fail to behave like their wild-type counterparts when placed in transgenic animals, suggesting that important aspects of enhancer function are still unexplored. As a step toward the creation of a truly synthetic regulatory element, we have undertaken an extensive structure–function study of an enhancer of the Drosophila decapentaplegic (dpp) gene that drives expression in the developing visceral mesoderm (VM). Although considerable past efforts have been made to dissect the dppVM enhancer, transgenic experiments presented here indicate that its activity cannot be explained by the known regulators alone. dppVM contains multiple, previously uncharacterized, regulatory sites, some of which exhibit functional redundancy. The results presented here suggest that even the best-studied enhancers must be further dissected before they can be fully understood, and before faithful synthetic elements based on them can be created. Implications for developmental genetics, mathematical modeling, and therapeutic applications are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63213/1/ten.tea.2008.0074.pd

Using the Game of Mastermind to Teach, Practice, and Discuss Scientific Reasoning Skills

The code-breaking game Mastermind, which can be played in minutes at no cost, creates opportunities for students to discuss scientific reasoning, hypothesis-testing, effective experimental design, and sound interpretation of results

An ancient yet flexible cis-regulatory architecture allows localized Hedgehog tuning by patched/Ptch1

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure—one of the oldest cis-regulatory features discovered in animal genomes—explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways. DOI: http://dx.doi.org/10.7554/eLife.13550.00

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

<p>Abstract</p> <p>Background</p> <p>In male <it>Drosophila melanogaster</it>, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.</p> <p>Results</p> <p>MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in <it>Drosophila</it>. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity <it>in vitro </it>and UAS-DsRed activation in <it>Drosophila</it>. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-<it>lacZ </it>and UAS-<it>arm-lacZ </it>reporter genes. The latter utilizes the constitutive promoter from the <it>arm </it>gene to drive <it>lacZ </it>expression. In contrast to the strong induction of UAS-DsRed expression, UAS-<it>arm-lacZ </it>expression increased by about 2-fold in both sexes.</p> <p>Conclusions</p> <p>Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-<it>DsRed </it>but not UAS-<it>arm-lacZ </it>genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.</p

The Gene Regulatory Cascade Linking Proneural Specification with Differentiation in Drosophila Sensory Neurons

Temporal expression profiling of sensory precursor cells reveals how the atonal proneural transcription factor regulates a specialized neuronal differentiation pathway