Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale.DOI: http://dx.doi.org/10.7554/eLife.08445.001

has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the and genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins.

The neural crest is a transient population of cells that forms within the developing central nervous system and migrates away to generate a wide range of derivatives throughout the body during vertebrate embryogenesis. These cells are of evolutionary and clinical interest, constituting a key defining trait in the evolution of vertebrates and alterations in their development are implicated in a high proportion of birth defects and craniofacial abnormalities. In the hindbrain and the adjacent cranial neural crest cells (cNCCs), nested domains of Hox gene expression provide a combinatorial'Hox-code' for specifying regional properties in the developing head. Hox genes have been shown to play important roles at multiple stages in cNCC development, including specification, migration, and differentiation. However, relatively little is known about the underlying gene-regulatory mechanisms involved, both upstream and downstream of Hox genes. Furthermore, it is still an open question as to how the genes of the neural crest GRN are linked to Hox-dependent pathways. In this review, we describe Hox gene expression, function and regulation in cNCCs with a view to integrating these genes within the emerging gene regulatory network for cNCC development. We highlight early roles for Hox1 genes in cNCC specification, proposing that this may be achieved, in part, by regulation of the balance between pluripotency and differentiation in precursor cells within the neuro-epithelium. We then describe what is known about the regulation of Hox gene expression in cNCCs and discuss this from the perspective of early vertebrate evolution.

Hoxa1 has diverse functional roles in differentiation and development. We have identified and characterized properties of regions bound by Hoxa1 on a genome-wide basis in differentiating mouse ES cells. Hoxa1 bound regions are enriched for clusters of consensus binding motifs for Hox, Pbx and Meis and many display co-occupancy of Pbx and Meis. Pbx and Meis are members of the TALE family and genome-wide analysis of multiple TALE members (Pbx, Meis, TGIF, Prep1 and Prep2) show that nearly all Hoxa1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins defines distinct classes of Hoxa1 targets and indicates a role as cofactors in modulating the specificity of Hox proteins. We also discovered extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes. This study provides new insight into a regulatory network involving combinatorial interactions between Hoxa1 and TALE proteins.