Professor Douglas Epstein, University of Pennsylvania, USA
Remote control of Shh gene expression in the limb bud
Professor Robert Hill, MRC IGMM, University of Edinburgh, Scotland
Multi-species conserved non-coding elements occur in the vertebrate genome and are clustered in the vicinity of developmentally regulated genes. Many act as cis-regulators of transcription and may reside at long distances from the genes they regulate. The relationship of conserved sequence to encoded regulatory information and indeed, the mechanism by which these contribute to long-range transcriptional regulation is not well understood. The ZRS, a highly conserved cis-regulator, is a paradigm for long-range gene regulation acting over ~1Mb to control spatiotemporal expression of Shh in the limb bud. In addition mutations in this regulator account for a number of limb abnormalities which include polydactyly, tibial hypoplasia and syndactyly. We describe the modular nature of this developmental regulator and show that a number of activities are encoded by this enhancer. Restriction of the expression pattern in the limb can, at least in part, be attributed to distinct binding sites in highly conserved domains that lie in the ZRS. Members of two groups of ETS transcription factors mediate a differential effect on Shh expression, defining the parameters of the expression pattern. Occupancy at multiple GABP/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. In addition analyses over longer sequence stretches dissect the ZRS into two distinct activities; one that regulates spatiotemporal activity and one that controls the long-range activity. Spatiotemporal activity is encoded within an element which functions efficiently only from a close range; whereas, long range activity is encoded by a second element which transmits the spatiotemporal activity over a large genomic distance. These two encoded regulatory activities integrate to control the number of digits and morphologically, ensure a stable limb phenotype based on a pattern of five digits.
Charting the genome regulatory architecture with transposons
Dr Francois Spitz, European Molecular Biology Laboratory, Germany
Vertebrate genomes are characterized by the presence of cis-regulatory elements located at great distances from the genes they control. Genomic rearrangements found in humans suggest that the specific organization of large loci is not random, but contributes importantly to implement the specific activities of these remote enhancers. To determine the organization of the mammalian genome and identify elements and genomic parameters that define enhancer regulatory activities, we have developed an in vivo approach, building on the controlled mobilization of a Sleeping Beauty transposon to distribute a regulatory sensor throughout the mouse genome. Analysis of a large genome-wide collection of insertions revealed principles of the genome regulatory architecture. Furthermore, the properties of Sleeping Beauty, in combination with in vivo chromosomal engineering, allows investigation of the fine-scale structure of loci of interests, shedding light on how remote enhancers may control target gene expression.
High Throughput Enhancer Assessment In Vivo
De Len Pennacchio, Lawrence Berkeley National Laboratory, USA
The paucity of a defined collection of mammalian transcriptional enhancers has largely precluded both our ability to develop computational methods for predicting additional tissue-specific enhancers in the human genome and to assess such sequences for their role in human disease. In ongoing studies, we are leveraging extreme evolutionary sequence conservation as well as next generation ChIP-Sequencing to identify putative gene regulatory elements and are characterizing their in vivo enhancer activity in a transgenic mouse assay. To date we have tested over 2000 such sequences in animals, and observed that >1000 function reproducibly as tissue-specific enhancers of gene expression. As a community resource, we have established a database to visualize and query the activity of these enhancer sequences (http://enhancer.lbl.gov/) and continue to generate additional in vivo enhancer data for >300 sequences per year. In recent studies directly from human tissues, we show that the conservation of enhancers across mammals varies widely depending on the specific tissue of examination. In particular, enhancers of the nervous system have high levels of evolutionary constraint while enhancers of the heart are largely not conserved. These findings highlight the importance of enhancer identification directly from human tissues for certain organs and hence disease states. In addition, this growing set of enhancers with in vivo-defined activities provides a molecular toolbox that can be used to experimentally target gene expression to organs and tissues in animals and constitutes a starting point for studying the role of regulatory elements in human disease.
Transcriptional enhancers in development and variation of the human face
Professor Joanna Wysocka, Stanford School of Medicine, USA
The face is at the center of our identity: it is the feature that best distinguishes an individual, while also connecting each of us to our broader ethnic ancestry and to the genetic inheritance from our parents, often evident in familial resemblances. And yet we know very little about the genetic basis of human facial variation. Nonetheless, a growing number of reports documents a link between enhancer mutations and complex human diseases. Moreover, evidence from model organisms begins to emerge that genetic variation in cis-regulatory elements underlies much of morphological evolution and diversity. Although human craniofacial development is extremely complex, the central bauplan of facial morphology is established early in embryogenesis by the neural crest cells and their derivatives. We recently developed an in vitro model of human neural crest formation and used it to epigenomically annotate enhancer repertoire if this unique cell type. I will discuss our hypothesis that allelic sequence variants of neural crest enhancers regulate normal-range variation of craniofacial features and confer susceptibility for malformations.