HOPX-associated molecular programs control cardiomyocyte cell states underpinning cardiac structure and function.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

Analysis of steric effects in DamID profiling of transcription factor target genes.

DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion.

Prediction and validation of protein-protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach.

The ability to accurately predict the DNA targets and interacting cofactors of transcriptional regulators from genome-wide data can significantly advance our understanding of gene regulatory networks. NKX2-5 is a homeodomain transcription factor that sits high in the cardiac gene regulatory network and is essential for normal heart development. We previously identified genomic targets for NKX2-5 in mouse HL-1 atrial cardiomyocytes using DNA-adenine methyltransferase identification (DamID). Here, we apply machine learning algorithms and propose a knowledge-based feature selection method for predicting NKX2-5 protein : protein interactions based on motif grammar in genome-wide DNA-binding data. We assessed model performance using leave-one-out cross-validation and a completely independent DamID experiment performed with replicates. In addition to identifying previously described NKX2-5-interacting proteins, including GATA, HAND and TBX family members, a number of novel interactors were identified, with direct protein : protein interactions between NKX2-5 and retinoid X receptor (RXR), paired-related homeobox (PRRX) and Ikaros zinc fingers (IKZF) validated using the yeast two-hybrid assay. We also found that the interaction of RXRα with NKX2-5 mutations found in congenital heart disease (Q187H, R189G and R190H) was altered. These findings highlight an intuitive approach to accessing protein-protein interaction information of transcription factors in DNA-binding experiments.

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels.

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.

CompGO: an R package for comparing and visualizing Gene Ontology enrichment differences between DNA binding experiments.

BACKGROUND: Gene ontology (GO) enrichment is commonly used for inferring biological meaning from systems biology experiments. However, determining differential GO and pathway enrichment between DNA-binding experiments or using the GO structure to classify experiments has received little attention. RESULTS: Herein, we present a bioinformatics tool, CompGO, for identifying Differentially Enriched Gene Ontologies, called DiEGOs, and pathways, through the use of a z-score derivation of log odds ratios, and visualizing these differences at GO and pathway level. Through public experimental data focused on the cardiac transcription factor NKX2-5, we illustrate the problems associated with comparing GO enrichments between experiments using a simple overlap approach. CONCLUSIONS: We have developed an R/Bioconductor package, CompGO, which implements a new statistic normally used in epidemiological studies for performing comparative GO analyses and visualizing comparisons from . BED data containing genomic coordinates as well as gene lists as inputs. We justify the statistic through inclusion of experimental data and compare to the commonly used overlap method. CompGO is freely available as a R/Bioconductor package enabling easy integration into existing pipelines and is available at: http://www.bioconductor.org/packages/release/bioc/html/CompGO.html packages/release/bioc/html/CompGO.html.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Genetic networks governing heart development.

Animal genomes contain a code for construction of the body plan from a fertilized egg. Understanding how genome information is deciphered to create the complex multilayered regulatory systems that drive organismal development, and which become altered in disease, is one of the greatest challenges in the biological sciences. The development of methods that effectively represent and communicate the complexity inherent in gene regulatory networks remains a major barrier. This review introduces the philosophy of systems biology and discusses recent progress in understanding the development of the heart at a systems biology level.

Selective inhibition of human group IIA-secreted phospholipase A2 (hGIIA) signaling reveals arachidonic acid metabolism is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes.

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris.

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

Polycomb-group proteins repress the floral activator AGL19 in the FLC-independent vernalization pathway.

Polycomb-group (PcG) proteins form a cellular memory by maintaining developmental regulators in a transcriptionally repressed state. We identified a novel flowering gene that is under PcG control in Arabidopsis--the MADS-box gene AGL19. AGL19 expression is maintained at very low levels by the PcG proteins MSI1, CLF, and EMF2, and AGL19 is partly responsible for the early flowering phenotype of clf mutants. AGL19 chromatin is strongly enriched in trimethylation of Lys 27 on histone H3 (H3K27me3) but not in H3K9me2. Repressive H3K27me3 marks were reduced by decreased CLF or MSI1 levels and by prolonged cold, suggesting that the PcG proteins MSI1 and CLF repress AGL19 in the absence of cold. Ectopic expression of AGL19 strongly accelerates flowering, and agl19 mutants have a decreased response to vernalization, the promotion of flowering by prolonged cold. Epistasis analyses revealed that AGL19 works in the poorly characterized FLC-independent vernalization pathway and does not require SOC1 to function. In this pathway, prolonged cold relieves AGL19 from PcG repression by a mechanism that requires VIN3 but not VRN2. Elevated AGL19 levels activate LFY and AP1 and eventually cause flowering.

Regulation of flowering time by Arabidopsis MSI1.

The transition to flowering is tightly controlled by endogenous programs and environmental signals. We found that MSI1 is a novel flowering-time gene in Arabidopsis. Both partially complemented msi1 mutants and MSI1 antisense plants were late flowering, whereas ectopic expression of MSI1 accelerated flowering. Physiological experiments revealed that MSI1 is similar to genes from the autonomous promotion of flowering pathway. Expression of most known flowering-time genes did not depend on MSI1, but the induction of SOC1 was delayed in partially complemented msi1 mutants. Delayed activation of SOC1 is often caused by increased expression of the floral repressor FLC. However, MSI1 function is independent of FLC. MSI1 is needed to establish epigenetic H3K4 di-methylation and H3K9 acetylation marks in SOC1 chromatin. The presence of these modifications correlates with the high levels of SOC1 expression that induce flowering in Arabidopsis. Together, the control of flowering time depends on epigenetic mechanisms for the correct expression of not only the floral repressor FLC, but also the floral activator SOC1.

MSI1-like proteins: an escort service for chromatin assembly and remodeling complexes.

MSI1-like WD40 repeat proteins are subunits of many protein complexes controlling chromatin dynamics. These proteins do not have any catalytic activity, but several recent studies using loss-of-function mutants established specific functions during development. Here, we review the current knowledge of MSI1-like proteins, including their phylogenetic history, expression patterns, biochemical interactions and mutant phenotypes. MSI1-like proteins, which are often targets or partners of tumor-suppressor proteins, are required during cell proliferation and differentiation in flies, nematodes and plants. We discuss the possibility that MSI1-like proteins could function to maintain epigenetic memory during development by targeting silencing complexes to chromatin during nucleosome assembly.

Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development.

Seed development in angiosperms initiates after double fertilization, leading to the formation of a diploid embryo and a triploid endosperm. The active repression of precocious initiation of certain aspects of seed development in the absence of fertilization requires the Polycomb group proteins MEDEA (MEA), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and FERTILIZATION-INDEPENDENT SEED2. Here we show that the Arabidopsis WD-40 domain protein MSI1 is present together with MEA and FIE in a 600 kDa complex and interacts directly with FIE. Mutant plants heterozygous for msi1 show a seed abortion ratio of 50% with seeds aborting when the mutant allele is maternally inherited, irrespective of a paternal wild-type or mutant MSI1 allele. Further more, msi1 mutant gametophytes initiate endosperm development in the absence of fertilization at a high penetrance. After pollination, only the egg cell becomes fertilized, the central cell starts dividing prior to fertilization, resulting in the formation of seeds containing embryos surrounded by diploid endosperm. Our results establish that MSI1 has an essential function in the correct initiation and progression of seed development.