Reverse-Engineering a Transcriptional Enhancer: A Case Study inDrosophila

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.

Rapid Evolutionary Rewiring of a Structurally Constrained Eye Enhancer

“…spa is directly bound and regulated by the Notch pathway effector Suppressor of Hairless [Su(H)], the Runx-family protein Lozenge (Lz),and two Ets-family EGFR/MAPK pathway effectors, PointedP2 (PntP2) and Yan/Aop, via motifs resembling Su(H)/Runx/Ets consensus binding sites (Figure 1a) [37]. Regions 1, 4, 5, and 6a of spa , which contain no binding sites for the above TFs, also harbor multiple essential regulatory sequences [8, 41]. The linear organization and spacing (“grammar”) of these regulatory sites is critically important for both robust transcriptional activation and correct cell-type specific expression [8].…”

Deciphering the genome's regulatory code: The many languages of DNA

“…[1] This means that we have to expand our knowledge about CRM architecture by thoroughly dissecting a larger number of individual CRMs. Taken together, the increasing knowledge obtained from the detailed dissection of individual CRMs, in combination with genome-wide ChIP-on-chip datasets, will allow us to make further progress toward understanding the cis -regulatory code.…”

“…Yet, TF binding data obtained from in vitro experiments and mutational CRM structure-function analysis have neither been sufficient to predict CRMs, nor have they been able to predict the activity of a given CRM. [1] One of the central aims of current genome biology is to unravel the general principles of TF targeting and information integration on CRMs that determine developmental patterns in vivo . As CRMs do not act in isolation, we also need to understand the complex gene regulatory networks in which key TFs dynamically bind to hundreds of CRMs and cause feedback on other CRMs depending on the developmental context.…”

A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design

“…As our understanding of gene regulation grows so to does the predictive capability of transcriptional models. The methods we describe here now dramatically expand the use of synthetic biology to create artificial enhancers [7, 8]. …”

“…The multimerization of known transcription factor binding sites (TFBSs) [8], for example failed to produce synthetic enhancers that mimic the expression of four Drosophila regulatory elements. While small (100–200 bp) synthetic cis -regulatory elements have been successfully used in the analysis of short-range repression in the Drosophila embryo [11, 12], such elements lack the complexity of wild type enhancers such as the even-skipped ( eve ) minimum stripe 2 element (MSE2), which contain, to date, thirteen characterized regulatory sites bound by five different transcription factors [13, 14].…”