A feed-forward relay integrates the regulatory activities of Bicoid and Orthodenticle via sequential binding to suboptimal sites.

The K50 (lysine at amino acid position 50) homeodomain (HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otd's ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes, including otd Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning.

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a context-specific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity.

A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity.

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network.

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous cooccupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide insights into the transcriptional codes regulating muscle gene expression and offer a generalizable approach for similar studies in other systems.

Dual transcriptional activator and repressor roles of TBX20 regulate adult cardiac structure and function.

The ongoing requirement in adult heart for transcription factors with key roles in cardiac development is not well understood. We recently demonstrated that TBX20, a transcriptional regulator required for cardiac development, has key roles in the maintenance of functional and structural phenotypes in adult mouse heart. Conditional ablation of Tbx20 in adult cardiomyocytes leads to a rapid onset and progression of heart failure, with prominent conduction and contractility phenotypes that lead to death. Here we describe a more comprehensive molecular characterization of the functions of TBX20 in adult mouse heart. Coupling genome-wide chromatin immunoprecipitation and transcriptome analyses (RNA-Seq), we identified a subset of genes that change expression in Tbx20 adult cardiomyocyte-specific knockout hearts which are direct downstream targets of TBX20. This analysis revealed a dual role for TBX20 as both a transcriptional activator and a repressor, and that each of these functions regulates genes with very specialized and distinct molecular roles. We also show how TBX20 binds to its targets genome-wide in a context-dependent manner, using various cohorts of co-factors to either promote or repress distinct genetic programs within adult heart. Our integrative approach has uncovered several novel aspects of TBX20 and T-box protein function within adult heart. Sequencing data accession number (http://www.ncbi.nlm.nih.gov/geo): GSE30943.

Why STAR Protocols authors make excellent protocol reviewers.

When researchers submit a protocol for peer review and publication, they receive feedback from reviewers to help improve the usability of the protocol. These authors can be the perfect peer reviewers helping propel research forward. They can use their technical expertise and sharpened writing skills to help improve the main aspects of published protocols, namely their clarity and reproducibility. This backstory chronicles the journey of Dr. Guillaume Blot, from a junior researcher and author to a protocol reviewer. For complete details, please refer to Blot et al. (2021).

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA.

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

A Comprehensive Drosophila melanogaster Transcription Factor Interactome.

Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.

DNA overstretching in the presence of glyoxal: structural evidence of force-induced DNA melting.

When a long DNA molecule is stretched beyond its B-form contour length, a transition occurs in which its length increases by a factor of 1.7, with very little force increase. A quantitative model was proposed to describe this transition as force-induced melting, where double-stranded DNA is converted into single-stranded DNA. The force-induced melting model accurately describes the thermodynamics of DNA overstretching as a function of solution conditions and in the presence of DNA binding ligands. An alternative explanation suggests a transformation into S-DNA, a double-stranded form which preserves the interstrand base pairing. To determine the extent to which DNA base pairs are exposed to solution during the transition, we held DNA overstretched to different lengths within the transition in the presence of glyoxal. If overstretching involved strand separation, then force-melted basepairs would be glyoxal-modified, thus essentially permanently single-stranded. Subsequent stretches confirm that a significant fraction of the DNA melted by force is permanently melted. This result demonstrates that DNA overstretching is accompanied by a disruption of the DNA helical structure, including a loss of hydrogen bonding.

Survey of variation in human transcription factors reveals prevalent DNA binding changes.

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation.

Shaping the Future of Research: a perspective from junior scientists.

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 (nd) and 3 (rd), 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers' synthesis of the outcomes.

Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.

Notch and MAML-1 complexation do not detectably alter the DNA binding specificity of the transcription factor CSL.

BACKGROUND: Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined. PRINCIPAL FINDINGS: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins. CONCLUSIONS/SIGNIFICANCE: These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit.

The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY.

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.

Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA.

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.