Professor Nicholas Hastie CBE FRS, Medical Research Council, UK
Professor Nicholas Hastie CBE FRS, Medical Research Council, UK
The evolution of global enhancers
Professor Denis Duboule, University of Geneva and EPFL Lausanne, Switzerland
In this paper, I will discuss potential mechanisms underlying the emergence of regulatory landscapes and their subsequent evolutionary recruitment in various developmental contexts. The long-range control of Hox genes during the development of the limbs and the external genitals will be used as an example, as well as the regulation of these genes during gut development.
The gastrointestinal tract is composed of a series of morphological sub-divisions, specialized in various functions associated with food breakdown and nutrients absorbtion. In mammals, the diet can considerably differ from one species to the other, depending on particular environmental adaptations. To digest the cellulose associated with vegetarian components, mammals belonging to the so-called hindgut fermenters, rely upon the caecum, a specialized gastrointestinal organ branching from the intestinal tract at the transition between the small and the large intestines. The molecular mechanisms underlying both the positioning, the budding and the extension of the caecum, which in some species can be longer than the gut itself, only starts to be understood, and was shown to require the concerted expression of several Hox genes, in particular members of the HoxA and HoxD clusters.
To try and understand how these genes are controlled during the morphogenesis of the caecum, we isolated and characterized the regulatory elements necessary for the transcription of Hoxd genes during the budding of this structure. We observe that an unusual number of enhancer sequences, with related specificities, are located within a gene desert flanking the HoxD cluster on its telomeric side. We also document the presence, within this large DNA interval, of a long non-coding RNA, speficically expressed in the growing caecum, which is involved in cis in the transcriptional regulation of Hoxd genes in this organ. We propose a model for the acquisition of multiple enhancer sequences with comparable activities, based on the existence of a regulatory structure that facilitates successive evolutionary recruitments of such sequences.
Saskia Delpretti, Thomas Montavon, Nicolas Lonfat, Ecole Polytechnique Fédérale, Switzerland
The pluripotent 3D genome
Professor Wouter de Laat, Hubrecht Institute, The Netherlands
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the unique capacity to differentiate into any somatic cell type. Microscopically, PSCs show minimal clustering of facultative heterochromatic regions such as centromeres and appear to have a more open chromatin structure, which is thought to reflect their ability to activate large portions of the genome upon differentiation. Yet, the three-dimensional organization of the pluripotent genome is not well understood. Here, we applied 4C technology to compare structural features of somatic and pluripotent genomes. In contrast to somatic cells, in PSCs, long-range contacts between inactive chromatin domains are nearly absent, however, active chromatin contacts do occur. Upon differentiation, cells lose the PSC-specific topology and adopt a somatic chromatin architecture. Conversely, the structural specifics of the pluripotent genome are regained during iPSC reprogramming. Finally, we describe a unique contact network of pluripotency genes centered around binding sites of the pluripotency factors Nanog, Oct 4 and Sox2. Collectively, our work reveals a PSC-specific DNA interaction network, which, we speculate, enhances the robustness of pluripotency.
Maps of open chromatin from association signals to function
Dr Panos Deloukas, Wellcome Trust Sanger Institute, UK
Genome-wide association (GWA) studies have been very successful in identifying genetic loci associated with complex traits, including disease. Many GWA signals map outside protein-coding regions suggesting that the underlying functional variants may influence phenotype through regulation of gene expression.
We used FAIRE (formaldehyde-assisted isolation of regulatory elements) coupled with sequencing to map nucleosome-depleted regions (NDRs), which mark active regulatory elements, in primary human megakaryocytes, erythroblasts and monocytes. Our data suggest that (i) cell type-specific NDRs can guide the identification of regulatory variants and (ii) sequence variants associated with the corresponding platelet and erythrocyte traits are enriched in NDRs in a cell type-dependent manner. As a proof-of-concept, we investigated the molecular mechanism of the 7q22.3 platelet volume locus.
We identified a megakaryocyte-specific NDR harbouring the index SNP which differentially binds the transcription factor EVI1 and affects PIK3CG gene expression in platelets. Gene expression profiling of Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet function.
Finally, we applied FAIRE maps in characterizing two low-frequency SNPs at the RBM8A locus, which is involved in thrombocytopenia with absent radii (TAR), a rare congenital malformation syndrome.
Deciphering the cis-regulatory code
Dr Eileen Furlong, EMBL, Germany
Embryonic development is controlled by precise patterns of temporal and spatial gene expression, which in turn are controlled by the activity of transcription factors converging on cis-regulatory modules (CRMs) or enhancer elements. The location and even combinatorial occupancy of CRMs can be experimentally measured using ChIP-seq at specific stages of development, at high-resolution. A current major challenge however, is to understand their function in terms of spatio-temporal enhancer activity. We have recently developed a new method to obtain cell-type specific signatures of chromatin state or transcription factor occupancy within the context of a developing multicellular embryo (Batch Tissue Specific ChIP: BiTS-ChIP). Using this method, we have measured chromatin modifications and RNA polymerase II occupancy in a specific tissue type at developmental stages before and after cell fate specification. The data revealed heterogeneous combinations of chromatin marks linked to active enhancers. Using a Bayesian network, we show that chromatin state is sufficient to predict, not just the location, but activity state of regulatory elements, accurately distinguishing between enhancers in an active versus inactive state. The model also revealed that Pol II occupancy and H3K79me3, a modification associated with Pol II elongation, are highly predictive for the precise timing of enhancer activity. Pol II occupancy is tightly correlated with both the timing and location of transcription factor occupancy suggesting that it may be recruited there by transcription factors. We are currently investigating how this relates to enhancer activation and three-dimensional looping interactions with promoter elements.