Emerging Field of Cardiomics: High-Throughput Investigations into Transcriptional Regulation of Cardiovascular Development and Disease

Congenital heart defects remain a leading cause of infant mortality in the western world, despite decades of research focusing on cardiovascular development and disease,. With the recent emergence of several high-throughput technologies including RNA sequencing, chromatin immunoprecipitation-coupled sequencing, mass spectrometry-based proteomics analyses, and the numerous variations of these strategies, investigations into cardiac development have been transformed from candidate-based studies into whole-genome, -transcriptome, and -proteome undertakings. In this review, we discuss several reports that have emerged from our lab and others over the last five years that emphasize the versatility of large dataset-based investigations of cardiogenic transcription factors, from phenotypic validations and new gene implications to the identification of novel roles of well-studied transcriptional regulators

Global profiling of stimulus-induced polyadenylation in cells using a poly(A) trap

Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry-compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation

A Gro/TLE-NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression

The cardiac transcription factor Tbx20 has a critical role in the proper morphogenetic development of the vertebrate heart, and its misregulation has been implicated in human congenital heart disease. Although it is established that Tbx20 exerts its function in the embryonic heart through positive and negative regulation of distinct gene programs, it is unclear how Tbx20 mediates proper transcriptional regulation of its target genes. Here, using a combinatorial proteomic and bioinformatic approach, we present the first characterization of Tbx20 transcriptional protein complexes. We have systematically investigated Tbx20 protein-protein interactions by immunoaffinity purification of tagged Tbx20 followed by proteomic analysis using GeLC-MS/MS, gene ontology classification, and functional network analysis. We demonstrate that Tbx20 is associated with a chromatin remodeling network composed of TLE/Groucho co-repressors, members of the Nucleosome Remodeling and Deacetylase (NuRD) complex, the chromatin remodeling ATPases RUVBL1/RUVBL2, and the T-box repressor Tbx18. We determined that the interaction with TLE co-repressors is mediated via an eh1 binding motif in Tbx20. Moreover, we demonstrated that ablation of this motif results in a failure to properly assemble the repression network and disrupts Tbx20 function in vivo. Importantly, we validated Tbx20-TLE interactions in the mouse embryonic heart, and identified developmental genes regulated by Tbx20:TLE binding, thereby confirming a primary role for a Tbx20-TLE repressor complex in embryonic heart development. Together, these studies suggest a model in which Tbx20 associates with a Gro/TLE-NuRD repressor complex to prevent inappropriate gene activation within the forming heart

Nodal-Dependent Mesendoderm Specification Requires the Combinatorial Activities of FoxH1 and Eomesodermin

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA–binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4)

Yeast Rmi1/Nce4 Controls Genome Stability as a Subunit of the Sgs1-Top3 Complex

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates

A Gro/TLE-NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression

The cardiac transcription factor Tbx20 has a critical role in the proper morphogenetic development of the vertebrate heart, and its misregulation has been implicated in human congenital heart disease. Although it is established that Tbx20 exerts its function in the embryonic heart through positive and negative regulation of distinct gene programs, it is unclear how Tbx20 mediates proper transcriptional regulation of its target genes. Here, using a combinatorial proteomic and bioinformatic approach, we present the first characterization of Tbx20 transcriptional protein complexes. We have systematically investigated Tbx20 protein–protein interactions by immunoaffinity purification of tagged Tbx20 followed by proteomic analysis using GeLC-MS/MS, gene ontology classification, and functional network analysis. We demonstrate that Tbx20 is associated with a chromatin remodeling network composed of TLE/Groucho corepressors, members of the Nucleosome Remodeling and Deacetylase (NuRD) complex, the chromatin remodeling ATPases RUVBL1/RUVBL2, and the T-box repressor Tbx18. We determined that the interaction with TLE corepressors is mediated via an eh1 binding motif in Tbx20. Moreover, we demonstrated that ablation of this motif results in a failure to properly assemble the repression network and disrupts Tbx20 function <i>in vivo</i>. Importantly, we validated Tbx20–TLE interactions in the mouse embryonic heart, and identified developmental genes regulated by Tbx20–TLE binding, thereby confirming a primary role for a Tbx20-TLE repressor complex in embryonic heart development. Together, these studies suggest a model in which Tbx20 associates with a Gro/TLE-NuRD repressor complex to prevent inappropriate gene activation within the forming heart