The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanisms: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analyzing the three dimensional structures of protein-DNA complexes, we show that the binding of arginines to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a striking example of this effect. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings suggest that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA binding specificity.

Specific interactions between proteins and DNA are fundamental to many biological processes. In this review, we provide a revised view of protein-DNA interactions that emphasizes the importance of the three-dimensional structures of both macromolecules. We divide protein-DNA interactions into two categories: those where the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those where the protein recognizes a sequence-dependent DNA shape (shape readout). We further divide base readout into those interactions that occur in the major groove from those that occur in the minor groove. Analogously, the readout of DNA shape is subdivided into global shape recognition, for example when the DNA helix exhibits an overall bend, and local shape recognition, for example when a base pair step is kinked or when a region of the minor groove is narrow. Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Base, we argue that individual DNA binding proteins combine multiple readout mechanisms to achieve DNA binding specificity. Specificity that distinguishes between families frequently involves base readout in the major groove while shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.

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Summary Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. Functional analysis in transgenic mice and Drosophila embryos demonstrated that the conserved elements are involved in a positive autoregulatory loop dependent on labial (lab) family members. Binding of Hoxb-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for pbx1, a homolog of the Drosophila extradenticle (exd) homoedomain protein. In vitro exd/pbx serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of Hoxb-1 with exd/pbx proteins as cofactors.

We show that homothorax (hth) is required for the Hox genes to pattern the body of the fruit fly, Drosophila melanogaster. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (EXD), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. MEIS1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of EXD in cell culture and Drosophila embryos. Thus, Meis1 is a murine homolog of hth. MEIS1/HTH also specifically binds to EXD with high affinity in vitro. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein-protein interaction between EXD and HTH results in EXD's nuclear translocation.