One prediction from this model is that such enhancers should tolerate some evolutionary changes in TFBS content, while having little or no effect on TF occupancy across species, as recently observed for the distal-less enhancer occupied by a Hox TF collective ( Uhl et al., 2016).
NUCLEOTIDE SEQUENCE FULL
Unlike billboard enhancers, a TF collective does not require the presence of motifs for every TF to be occupied by the full collective, as observed in cardioblast ( Junion et al., 2012), leg precursor ( Uhl et al., 2016) and dopaminergic neuron ( Doitsidou et al., 2013) enhancers. We recently proposed the TF collective model, where TFs bind to enhancers cooperatively through a combination of protein::DNA and protein::protein interactions allowing for a flexible combination of TFBSs. At the other extreme the ‘billboard’ model ( Arnosti and Kulkarni, 2005 Kulkarni and Arnosti, 2003) proposes that TFs bind additively or in subgroups to an enhancer with relatively independent effects on gene expression, and therefore have flexibility in the relative positions of their TFBSs ( Khoueiry et al., 2010). The best studied, and perhaps only true enhancesome, is the interferon beta enhancer, a 57 bp core element with a very strict arrangement of TFBS ( Panne et al., 2007 Thanos and Maniatis, 1995). Mutations or sequence variants that affect either the motif quality or the motif arrangement results in loss of enhancer activity. The enhanceosome describes elements that are bound by many TFs through cooperative interactions facilitated by the very precise relative orientation, spacing and helical phasing of the TF’s binding sites (TFBSs) within the enhancer. Several models have been proposed to explain enhancer function. This often-surprising gap between changes in genotype and phenotype in developmental contexts highlights the current challenge in understanding both enhancer function and evolutionary constraints. The loss or gain of TF binding sites in the otx enhancer in ascidians ( Oda-Ishii et al., 2005), the Endo 16 enhancer in sea urchins ( Balhoff and Wray, 2005), and the eve enhancer in Drosophila ( Ludwig et al., 1998), for example, have only marginal effects on enhancer activity. In other cases, however, changes in enhancer sequence appear to have limited impact on phenotype. In sticklebacks, for example, the recurrent deletion of an entire enhancer driving pelvic pitx expression leads to the lack of spines in several freshwater populations ( Chan et al., 2010 Shapiro et al., 2004). Changes in TF binding sites within an enhancer can lead to phenotypic differences between individuals ( Prud'homme et al., 2007 Rogers et al., 2013), and between species ( Levine and Davidson, 2005 Wray, 2007). Transcription factors (TFs) act largely through enhancer elements - modular sequences that instruct genes when and where to be expressed ( Levine and Davidson, 2005 Slattery et al., 2014 Spitz and Furlong, 2012) and provide robustness and precision to developmental programs ( Cannavò et al., 2016 Frankel et al., 2010 Perry et al., 2010). Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood.
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