Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function

Hand1 phosphoregulation within the distal arch neural crest is essential for craniofacial morphogenesis

In this study we examine the consequences of altering Hand1 phosphoregulation in the developing neural crest cells (NCCs) of mice. Whereas Hand1 deletion in NCCs reveals a nonessential role for Hand1 in craniofacial development and embryonic survival, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, in NCCs results in severe mid-facial clefting and neonatal death. Hand1 phosphorylation mutants exhibit a non-cell-autonomous increase in pharyngeal arch cell death accompanied by alterations in Fgf8 and Shh pathway expression. Together, our data indicate that the extreme distal pharyngeal arch expression domain of Hand1 defines a novel bHLH-dependent activity, and that disruption of established Hand1 dimer phosphoregulation within this domain disrupts normal craniofacial patterning

Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure

Nkx2.5 regulates Endothelin Converting Enzyme-1 during pharyngeal arch patterning

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediates this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders

Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis

The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prrx1-Cre reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of Irx3, Irx5, Gli3 and Alx4, all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis., Summary: Altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of anterior-proximal limb elements in mice

Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function

Rationale The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. Genome-wide association studies (GWAS) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5’ to the gene encoding the bHLH transcription factor HAND1. Objective Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS, and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify three additional SNPs, located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays (EMSA), disrupts GATA4 DNA-binding. Modeling two of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in EMSA and this enhancer in total, is required for VCS development and function in mice and perhaps humans

Partially Penetrant Cardiac Neural Crest Defects in Hand1 Phosphomutant Mice: Dimer Choice That Is Not So Critical

Hand1 is a basic Helix-loop-Helix transcription factor that exhibits post-translationally regulated dimer partner choice that allows for a diverse set of Hand1 transcriptional complexes. Indeed, when Hand1 phosphoregulation is altered, conditionally activated hypophorylation (Hand1PO4−) and phosphorylation mimic (Hand1PO4+) Hand1 alleles disrupt both craniofacial, and limb morphogenesis with 100% penetrance. Interestingly, activation of conditional Hand1 Phosphomutant alleles within post migratory neural crest cells produce heart defects that include ventricular septal defects, double outlet right ventricle, persistent truncus arteriosus with partial penetrance. Single vs double lobed thymus is a distinguishing feature between Wnt1Cre;Hand1PO4−/+ and Wnt1-Cre;Hand1PO4+/+ mice. These data show that although Hand1 dimer regulation play critical and consistent roles in disrupting craniofacial and limb morphogenesis, Hand1 dimer regulation during cardiac outflow track formation is less critical for normal morphogenesis. This review will present the OFT phenotypes observed in Hand1 Phosphomutant mice, and discuss possible mechanisms of how penetrance differences within the same tissues within the same embryos could be variable

Fluorescence Resonance Energy Transfer (FRET) as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH) proteins

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy