A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3. We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Centriolar Protein C2cd3 Is Required for Craniofacial Development.

The primary cilium is a ubiquitous, microtubule-based cellular organelle. Primary cilia dysfunction results in a group of disorders termed ciliopathies. C2 domain containing 3 centriole elongation regulator (C2cd3), encodes a centriolar protein essential for ciliogenesis. Mutations in human C2CD3 are associated with the human ciliopathy Oral-Facial-Digital syndrome type 14 (OFD14). In order to better understand the etiology of ciliopathies including OFD14, we generated numerous murine models targeting C2cd3. Initial analysis revealed several tissue-specific isoforms of C2cd3, and while the loss of C2cd3 has previously been reported to result in exencephaly, tight mesencephalic flexure, pericardial edema, abnormal heart looping and a twisted body axis, further analysis revealed that genetic background may also contribute to phenotypic variation. Additional analyses of a conditional allelic series targeting C-terminal PKC-C2 domains or the N-terminal C2CD3N-C2 domain of C2cd3 revealed a variable degree of phenotypic severity, suggesting that while the N-terminal C2CD3N-C2 domain was critical for early embryonic development as a whole, there was also a craniofacial specific role for the C2CD3N-C2 domains. Together, through generation of novel models and evaluation of C2cd3 expression, these data provide valuable insight into mechanisms of pathology for craniofacial ciliopathies that can be further explored in the future.

Alveolar injury and regeneration following deletion of ABCA3.

Adaptation to air breathing after birth is dependent upon the synthesis and secretion of pulmonary surfactant by alveolar type 2 (AT2) cells. Surfactant, a complex mixture of phospholipids and proteins, is secreted into the alveolus, where it reduces collapsing forces at the air-liquid interface to maintain lung volumes during the ventilatory cycle. ABCA3, an ATP-dependent Walker domain containing transport protein, is required for surfactant synthesis and lung function at birth. Mutations in ABCA3 cause severe surfactant deficiency and respiratory failure in newborn infants. We conditionally deleted the Abca3 gene in AT2 cells in the mature mouse lung. Loss of ABCA3 caused alveolar cell injury and respiratory failure. ABCA3-related lung dysfunction was associated with surfactant deficiency, inflammation, and alveolar-capillary leak. Extensive but incomplete deletion of ABCA3 caused alveolar injury and inflammation, and it initiated proliferation of progenitor cells, restoring ABCA3 expression, lung structure, and function. M2-like macrophages were recruited to sites of AT2 cell proliferation during the regenerative process and were present in lung tissue from patients with severe lung disease caused by mutations in ABCA3. The remarkable and selective regeneration of ABCA3-sufficient AT2 progenitor cells provides plausible approaches for future correction of ABCA3 and other genetic disorders associated with surfactant deficiency and acute interstitial lung disease.

Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment.

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.

Complex haplotypes derived from noncoding polymorphisms of the intronless alpha2A-adrenergic gene diversify receptor expression.

Alpha(2A)-adrenergic receptors (alpha(2A)AR) regulate multiple central nervous system, cardiovascular, and metabolic processes including neurotransmitter release, platelet aggregation, blood pressure, insulin secretion, and lipolysis. Complex diseases associated with alpha(2A)AR dysfunction display familial clustering, phenotypic heterogeneity, and interindividual variability in response to therapy targeted to alpha(2A)ARs, suggesting common, functional polymorphisms. In a multiethnic discovery cohort we identified 16 single-nucleotide polymorphisms (SNPs) in the alpha(2A)AR gene organized into 17 haplotypes of two major phylogenetic clades. In contrast to other adrenergic genes, variability of the alpha(2A)AR was primarily due to SNPs in the promoter, 5' UTR and 3' UTR, as opposed to the coding block. Marked ethnic variability in the frequency of SNPs and haplotypes was observed: one haplotype represented 70% of Caucasians, whereas Africans and Asians had a wide distribution of less common haplotypes, with the highest haplotype frequencies being 16% and 35%, respectively. Despite the compact nature of this intronless gene, local linkage disequilibrium between a number of SNPs was low and ethnic-dependent. Whole-gene transfections into BE(2)-C human neuronal cells using vectors containing the entire approximately 5.3-kb gene without exogenous promoters were used to ascertain the effects of haplotypes on alpha(2A)AR expression. Substantial differences (P < 0.001) in transcript and cell-surface protein expression, by as much as approximately 5-fold, was observed between haplotypes, including those with common frequencies. Thus, signaling by this virtually ubiquitous receptor is under major genetic influence, which may be the basis for highly divergent phenotypes in complex diseases such as systemic and pulmonary hypertension, heart failure, diabetes, and obesity.

Polymorphisms of cardiac presynaptic alpha2C adrenergic receptors: Diverse intragenic variability with haplotype-specific functional effects.

The presynaptic alpha2C adrenergic receptors (AR) act to inhibit norepinephrine release in cardiac and other presynaptic nerves. We have recently shown that a genetic variant in the alpha2CAR coding region (Del322-325), which renders the receptor partially uncoupled from Gi, is a risk factor for heart failure. However, variability of heart failure phenotypes and a dominance of Del322-325 in those of African descent led us to hypothesize that other regions of this gene have functional polymorphisms. In a multiethnic population, we found 20 polymorphisms within 4,625 bp of contiguous sequence of this intronless gene encompassing the promoter, 5' UTR, coding, and 3' UTR. These polymorphisms occur in 24 distinct haplotypes with complex organizations, including multiple 5'-upstream polymorphisms in regions known to direct expression, a 3' UTR substitution polymorphism within an insertion/deletion sequence, and the radical coding polymorphism that deletes four amino acids. Relatively low linkage disequilibrium between many polymorphisms, few cosmopolitan haplotypes, prevalent ethnic-specific haplotypes, and substantial genetic divergence among haplotypes was noted. The dysfunctional Del322-325 allele was partitioned into multiple haplotypes, with frequencies of 48% to 2%. The functional implications of the haplotypes were ascertained by whole-gene transfections of human neuronal cells, where haplotype was significantly related (P < 0.001) to expression levels of receptor transcript and protein. Expression varied by as much as approximately 50% by haplotype, and such studies enabled haplotype clustering by phenotypic, rather than genotypic, similarities. Thus, depending on phenotype, expression-specific haplotypes may amplify, attenuate, or dominate the cardiomyopathic effect attributed to the alpha2CDel322-325 marker.

An Ile to Met polymorphism in the catalytic domain of adenylyl cyclase type 9 confers reduced beta2-adrenergic receptor stimulation.

Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled receptors such as the beta2-adrenergic receptor (beta2AR). Genetic variation in the receptor component of this pathway can alter signal transduction and the response to beta-agonists in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an Ile to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile772) and polymorphic (Met772) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met772 AC9 has lower basal and beta2AR-mediated adenylyl cyclase activities compared to Ile772 AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met772 membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta2AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma.

Hierarchy of polymorphic variation and desensitization permutations relative to beta 1- and beta 2-adrenergic receptor signaling.

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta(1)-adrenergic receptor (beta(1)AR) or a gain-of-function polymorphic variant (beta(1)AR-Arg(389)), and the beta(2)-adrenergic receptor (beta(2)AR) or a loss-of-function polymorphic receptor (beta(2)AR-Ile(164)). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta(1)AR, the enhanced function of the Arg(389) receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta(1)AR-Arg(389) signaling was equivalent to non-desensitized wild-type beta(1)AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta(2)AR underwent less desensitization compared with beta(2)AR-Ile(164), thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.

False positive non-synonymous polymorphisms of G-protein coupled receptor genes.

Polymorphisms of G-protein coupled receptor (GPCR) genes are associated with disease risk and modification, and the response to receptor-directed therapy. Genomic sequencing ( approximately 1700 automated runs) from as many as 120 chromosomes from 60 multiethnic individuals was performed to confirm non-synonymous coding polymorphisms reported in the dbSNP database from 25 randomly selected GPCR genes. These polymorphisms were in regions of the receptors responsible for structural integrity, ligand binding, G-protein coupling and phosphoregulation. However, most of these putative polymorphisms could not be confirmed (false positive rate of 68%). Based on these results, we suggest that the variability of the superfamily is not well defined, and we caution against exclusive reliance on databases for selection of candidate GPCR polymorphisms for disease association and pharmacogenetic studies.

Amino acid 49 polymorphisms of the human beta1-adrenergic receptor affect agonist-promoted trafficking.

The signaling impact of a human beta1-adrenergic receptor (beta1 AR) polymorphism at residue 49 of the aminoterminus (Ser-to-Gly substitution) was studied by recombinantly expressing each receptor. The two receptors displayed identical agonist and antagonist binding affinities. Furthermore, basal and agonist-stimulated adenylyl cyclase activities were the same for these receptors as assessed in both cell types. Although short-term agonist exposure resulted in similar degrees of receptor internalization, long-term agonist-promoted downregulation was greater for Gly49 compared with Ser49. The Gly49 receptor underwent a 24 +/- 3% loss of receptor density after exposure to isoproterenol for 18 h, whereas Ser49 underwent no such loss. In studies in which receptor synthesis was inhibited, agonist-promoted downregulation for Gly49 was 55 +/- 3% compared with 36 +/- 5% for Ser49. In the absence of agonist, degradative turnover of each receptor was the same. Immunoblotting revealed that some of the Ser49 receptor exists as a highly N-glycosylated form (approximately 105-kD molecular mass), which is not present with Gly49. Thus the phenotype of the Gly49 polymorphic receptor is that of wild-type coupling with enhanced agonist-promoted downregulation, which is associated with altered N-glycosylation. Based on this cellular phenotype, the beta1 AR Gly49 polymorphism may impart a beneficial effect in chronic heart failure.