Low-Affinity Binding Sites and the Transcription Factor Specificity Paradox in Eukaryotes.

Eukaryotic transcription factors (TFs) from the same structural family tend to bind similar DNA sequences, despite the ability of these TFs to execute distinct functions in vivo. The cell partly resolves this specificity paradox through combinatorial strategies and the use of low-affinity binding sites, which are better able to distinguish between similar TFs. However, because these sites have low affinity, it is challenging to understand how TFs recognize them in vivo. Here, we summarize recent findings and technological advancements that allow for the quantification and mechanistic interpretation of TF recognition across a wide range of affinities. We propose a model that integrates insights from the fields of genetics and cell biology to provide further conceptual understanding of TF binding specificity. We argue that in eukaryotes, target specificity is driven by an inhomogeneous 3D nuclear distribution of TFs and by variation in DNA binding affinity such that locally elevated TF concentration allows low-affinity binding sites to be functional.

The Importance of Timing.

Building a nervous system requires a precise sequence of genetic transitions, mediated in part by the temporal and spatial regulation of transcription factors. Quan et al. add to our understanding of this regulation by describing an evolutionarily conserved post-translational mechanism that rapidly extinguishes proneural protein activity in neural precursors.

Deconvolving the recognition of DNA shape from sequence.

Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the 3D shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins.

Low affinity binding site clusters confer hox specificity and regulatory robustness.

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins.

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.

Dense and pleiotropic regulatory information in a developmental enhancer.

Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Origins of specificity in protein-DNA recognition.

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 when the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those when 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 the 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 a region of the minor groove is narrow). Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Bank, 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, whereas shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.

SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues.

Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein-DNA binding in vivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, we developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, we identified the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx-DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, we suggest that SpyTag-SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional in vivo applications that were previously impractical.

Homothorax controls a binary Rhodopsin switch in Drosophila ocelli.

Visual perception of the environment is mediated by specialized photoreceptor (PR) neurons of the eye. Each PR expresses photosensitive opsins, which are activated by a particular wavelength of light. In most insects, the visual system comprises a pair of compound eyes that are mainly associated with motion, color or polarized light detection, and a triplet of ocelli that are thought to be critical during flight to detect horizon and movements. It is widely believed that the evolutionary diversification of compound eye and ocelli in insects occurred from an ancestral visual organ around 500 million years ago. Concurrently, opsin genes were also duplicated to provide distinct spectral sensitivities to different PRs of compound eye and ocelli. In the fruit fly Drosophila melanogaster, Rhodopsin1 (Rh1) and Rh2 are closely related opsins that originated from the duplication of a single ancestral gene. However, in the visual organs, Rh2 is uniquely expressed in ocelli whereas Rh1 is uniquely expressed in outer PRs of the compound eye. It is currently unknown how this differential expression of Rh1 and Rh2 in the two visual organs is controlled to provide unique spectral sensitivities to ocelli and compound eyes. Here, we show that Homothorax (Hth) is expressed in ocelli and confers proper rhodopsin expression. We find that Hth controls a binary Rhodopsin switch in ocelli to promote Rh2 expression and repress Rh1 expression. Genetic and molecular analysis of rh1 and rh2 supports that Hth acts through their promoters to regulate Rhodopsin expression in the ocelli. Finally, we also show that when ectopically expressed in the retina, hth is sufficient to induce Rh2 expression only at the outer PRs in a cell autonomous manner. We therefore propose that the diversification of rhodpsins in the ocelli and retinal outer PRs occurred by duplication of an ancestral gene, which is under the control of Homothorax.

Control of tissue morphogenesis by the HOX gene Ultrabithorax.

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.

A lexicon for homeodomain-DNA recognition.

Decoding the cis-regulatory logic of eukaryotic genomes requires knowledge of the DNA-binding specificities of all transcription factors. New work (Berger et al., 2008; Noyes et al., 2008) provides individual specificities for nearly all Drosophila and mouse homeodomains, key DNA-binding domains in many transcription factors. The data underscore the complexity of determining target specificities in vivo.

Low affinity binding sites in an activating CRM mediate negative autoregulation of the Drosophila Hox gene Ultrabithorax.

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Disentangling the many layers of eukaryotic transcriptional regulation.

Regulation of gene expression in eukaryotes is an extremely complex process. In this review, we break down several critical steps, emphasizing new data and techniques that have expanded current gene regulatory models. We begin at the level of DNA sequence where cis-regulatory modules (CRMs) provide important regulatory information in the form of transcription factor (TF) binding sites. In this respect, CRMs function as instructional platforms for the assembly of gene regulatory complexes. We discuss multiple mechanisms controlling complex assembly, including cooperative DNA binding, combinatorial codes, and CRM architecture. The second section of this review places CRM assembly in the context of nucleosomes and condensed chromatin. We discuss how DNA accessibility and histone modifications contribute to TF function. Lastly, new advances in chromosomal mapping techniques have provided increased understanding of intra- and interchromosomal interactions. We discuss how these topological maps influence gene regulatory models.

To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins.

Hox proteins are key regulatory transcription factors that act in different tissues of the embryo to provide specific spatial and temporal coordinates to each cell. These patterning functions often depend on the presence of the TALE-homeodomain class cofactors, which form cooperative DNA-binding complexes with all Hox proteins. How this family of cofactors contributes to the highly diverse and specific functions of Hox proteins in vivo remains an important unsolved question. We review here the most recent advances in understanding the molecular mechanisms underlying Hox-TALE function. In particular, we discuss the role of DNA shape, DNA-binding affinity, and protein-protein interaction flexibility in dictating Hox-TALE specificity. We propose several models to explain how these mechanisms are integrated with each other in the context of the many distinct functions that Hox and TALE factors carry out in vivo.

Genome-wide prediction of minor-groove electrostatic potential enables biophysical modeling of protein-DNA binding.

Protein-DNA binding is a fundamental component of gene regulatory processes, but it is still not completely understood how proteins recognize their target sites in the genome. Besides hydrogen bonding in the major groove (base readout), proteins recognize minor-groove geometry using positively charged amino acids (shape readout). The underlying mechanism of DNA shape readout involves the correlation between minor-groove width and electrostatic potential (EP). To probe this biophysical effect directly, rather than using minor-groove width as an indirect measure for shape readout, we developed a methodology, DNAphi, for predicting EP in the minor groove and confirmed the direct role of EP in protein-DNA binding using massive sequencing data. The DNAphi method uses a sliding-window approach to mine results from non-linear Poisson-Boltzmann (NLPB) calculations on DNA structures derived from all-atom Monte Carlo simulations. We validated this approach, which only requires nucleotide sequence as input, based on direct comparison with NLPB calculations for available crystal structures. Using statistical machine-learning approaches, we showed that adding EP as a biophysical feature can improve the predictive power of quantitative binding specificity models across 27 transcription factor families. High-throughput prediction of EP offers a novel way to integrate biophysical and genomic studies of protein-DNA binding.

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression.

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, we show that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, we map a motif in the posterior Hox protein Abdominal-A (AbdA) that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism.