Stephen S. Gisselbrecht

Ras Pathway Specificity Is Determined by the Integration of Multiple Signal-Activated and Tissue-Restricted Transcription Factors

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.

Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal sub-types, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered.

UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein–DNA interactions

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k (‘k-mers’). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org.

DNA-binding specificity changes in the evolution of forkhead transcription factors

The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood phylogenetic inference and independent, comprehensive functional assays of DNA-binding capacity to explore the evolution of DNA-binding specificity within the forkhead family. We present converging evidence that similar alternative sequence preferences have arisen repeatedly and independently in the course of forkhead evolution. The vast majority of DNA-binding specificity changes we observed are not explained by alterations in the known DNA-contacting amino acid residues conferring specificity for canonical forkhead binding sites. Intriguingly, we have found forkhead DBDs that retain the ability to bind very specifically to two completely distinct DNA sequence motifs. We propose an alternate specificity-determining mechanism whereby conformational rearrangements of the DBD broaden the spectrum of sequence motifs that a TF can recognize. DNA-binding bispecificity suggests a previously undescribed source of modularity and flexibility in gene regulation and may play an important role in the evolution of transcriptional regulatory networks.

Expression-Guided in Silico Evaluation of Candidate Cis Regulatory Codes for Drosophila Muscle Founder Cells

While combinatorial models of transcriptional regulation can be inferred for metazoan systems from a priori biological knowledge, validation requires extensive and time-consuming experimental work. Thus, there is a need for computational methods that can evaluate hypothesized cis regulatory codes before the difficult task of experimental verification is undertaken. We have developed a novel computational framework (termed “CodeFinder”) that integrates transcription factor binding site and gene expression information to evaluate whether a hypothesized transcriptional regulatory model (TRM; i.e., a set of co-regulating transcription factors) is likely to target a given set of co-expressed genes. Our basic approach is to simultaneously predict cis regulatory modules (CRMs) associated with a given gene set and quantify the enrichment for combinatorial subsets of transcription factor binding site motifs comprising the hypothesized TRM within these predicted CRMs. As a model system, we have examined a TRM experimentally demonstrated to drive the expression of two genes in a sub-population of cells in the developing Drosophila mesoderm, the somatic muscle founder cells. This TRM was previously hypothesized to be a general mode of regulation for genes expressed in this cell population. In contrast, the present analyses suggest that a modified form of this cis regulatory code applies to only a subset of founder cell genes, those whose gene expression responds to specific genetic perturbations in a similar manner to the gene on which the original model was based. We have confirmed this hypothesis by experimentally discovering six (out of 12 tested) new CRMs driving expression in the embryonic mesoderm, four of which drive expression in founder cells.

Using a structural and logics systems approach to infer bHLH–DNA binding specificity determinants

Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein–DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH–DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.

The transmembrane protein Perdido interacts with Grip and integrins to mediate myotube projection and attachment in the Drosophila embryo.

The molecular mechanisms underlying muscle guidance and formation of myotendinous junctions are poorly understood both in vertebrates and in Drosophila. We have identified a novel gene that is essential for Drosophila embryonic muscles to form proper projections and stable attachments to epidermal tendon cells. Loss-of-function of this gene - which we named perdido (perd)-results in rounded, unattached muscles. perd is expressed prior to myoblast fusion in a subset of muscle founder cells, and it encodes a conserved single-pass transmembrane cell adhesion protein that contains laminin globular extracellular domains and a small intracellular domain with a C-terminal PDZ-binding consensus sequence. Biochemical experiments revealed that the Perd intracellular domain interacts directly with one of the PDZ domains of the Glutamate receptor interacting protein (Grip), another factor required for formation of proper muscle projections. In addition, Perd is necessary to localize Grip to the plasma membrane of developing myofibers. Using a newly developed, whole-embryo RNA interference assay to analyze genetic interactions, perd was shown to interact not only with Grip but also with multiple edematous wings, which encodes one subunit of the αPS1-βPS integrin expressed in tendon cells. These experiments uncovered a previously unrecognized role for the αPS1-βPS integrin in the formation of muscle projections during early stages of myotendinous junction development. We propose that Perd regulates projection of myotube processes toward and subsequent differentiation of the myotendinous junction by priming formation of a protein complex through its intracellular interaction with Grip and its transient engagement with the tendon cell-expressed laminin-bindingα PS1-βPS integrin.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

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.