Cilia Genes Research Articles

Identification of Genetic Predisposition in Noncirrhotic Portal Hypertension Patients With Multiple Renal Cysts by Integrated Analysis of Whole-Genome and Single-Cell RNA Sequencing.

Background and Aims: The multiple renal cysts (MRC) occur in some patients with noncirrhotic portal hypertension (NCPH) could be a subset of ciliopathy. However, the potential genetic influencers and/or determinants in NCPH with MRC are largely unknown. The aim of this study was to explore the potential candidate variants/genes associated with those patients. Methods: 8,295 cirrhotic patients with portal hypertension were enrolled in cohort 1 and 267 patients affected with NCPH were included in cohort 2. MRC was defined as at least two cysts in both kidneys within a patient detected by ultrasonography or computed tomography. Whole-genome sequencing (WGS) was performed in nine patients (four from cohort 1 and five from cohort 2). Then we integrated WGS and publicly available single-cell RNA sequencing (scRNA-seq) to prioritize potential candidate genes. Genes co-expressed with known pathogenic genes within same cell types were likely associated NCPH with MRC. Results: The prevalence of MRC in NCPH patients (19.5%, 52/267) was significantly higher than cirrhotic patients (6.2%, 513/8,295). Further, the clinical characteristics of NCPH patients with MRC were distinguishable from cirrhotic patients, including late-onset, more prominent portal hypertension however having preserved liver functions. In the nine whole genome sequenced patients, we identified three patients with early onset harboring compound rare putative pathogenic variants in the known disease gene PKHD1. For the remaining patients, by assessing cilia genes profile in kidney and liver scRNA-seq data, we identified CRB3 was the most co-expressed gene with PKHD1 that highly expressed in ureteric bud cell, kidney stromal cell and hepatoblasts. Moreover, we found a homozygous variant, CRB3 p.P114L, that caused conformational changes in the evolutional conserved domain, which may associate with NCPH with MRC. Conclusion: ScRNA-seq enables unravelling cell heterogeneity with cell specific gene expression across multiple tissues. With the boosting public accessible scRNA-seq data, we believe our proposed analytical strategy would effectively help disease risk gene identification.

Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface

During tumor progression, cancer cells come into contact with various non-tumor cell types, but it is unclear how tumors adapt to these new environments. Here, we integrate spatially resolved transcriptomics, single-cell RNA-seq, and single-nucleus RNA-seq to characterize tumor-microenvironment interactions at the tumor boundary. Using a zebrafish model of melanoma, we identify a distinct “interface” cell state where the tumor contacts neighboring tissues. This interface is composed of specialized tumor and microenvironment cells that upregulate a common set of cilia genes, and cilia proteins are enriched only where the tumor contacts the microenvironment. Cilia gene expression is regulated by ETS-family transcription factors, which normally act to suppress cilia genes outside of the interface. A cilia-enriched interface is conserved in human patient samples, suggesting it is a conserved feature of human melanoma. Our results demonstrate the power of spatially resolved transcriptomics in uncovering mechanisms that allow tumors to adapt to new environments.

Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8–10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a “transient ciliopathy”, as a possible cellular mechanism of prenatal alcohol pathogenesis.

Large-scale analysis reveals spatiotemporal circadian patterns of cilia transcriptomes in the primate brain.

Cilia are dynamic subcellular systems, with core structural and functional components operating in a highly coordinated manner. Since many environmental stimuli sensed by cilia are circadian in nature, it is reasonable to speculate that genes encoding cilia structural and functional components follow rhythmic circadian patterns of expression. Using computational methods and the largest spatiotemporal gene expression atlas of primates, we identified and analyzed the circadian rhythmic expression of cilia genes across 22 primate brain areas. We found that around 73% of cilia transcripts exhibited circadian rhythmicity across at least one of 22 brain regions. In 12 brain regions, cilia transcriptomes were significantly enriched with circadian oscillating transcripts, as compared to the rest of the transcriptome. The phase of the cilia circadian transcripts deviated from the phase of the majority of the background circadian transcripts, and transcripts coding for cilia basal body components accounted for the majority of cilia circadian transcripts. In addition, adjacent or functionally connected brain nuclei had large overlapping complements of circadian cilia genes. Most remarkably, cilia circadian transcripts shared across the basal ganglia nuclei and the prefrontal cortex peaked in these structures in sequential fashion that is similar to the sequential order of activation of the basal ganglia-cortical circuitry in connection with movement coordination, albeit on completely different timescales. These findings support a role for the circadian spatiotemporal orchestration of cilia gene expression in the normal physiology of the basal ganglia-cortical circuit and motor control. Studying orchestrated cilia rhythmicity in the basal ganglia-cortical circuits and other brain circuits may help develop better functional models, and shed light on the causal effects cilia functions have on these circuits and on the regulation of movement and other behaviors.

Ciliopathy genes are required for apical secretion of Cochlin, an otolith crystallization factor

Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.

Molecular Genetics and Complex Inheritance of Congenital Heart Disease.

Congenital heart disease (CHD) is the most common congenital malformation and the leading cause of mortality therein. Genetic etiologies contribute to an estimated 90% of CHD cases, but so far, a molecular diagnosis remains unsolved in up to 55% of patients. Copy number variations and aneuploidy account for ~23% of cases overall, and high-throughput genomic technologies have revealed additional types of genetic variation in CHD. The first CHD risk genotypes identified through high-throughput sequencing were de novo mutations, many of which occur in chromatin modifying genes. Murine models of cardiogenesis further support the damaging nature of chromatin modifying CHD mutations. Transmitted mutations have also been identified through sequencing of population scale CHD cohorts, and many transmitted mutations are enriched in cilia genes and Notch or VEGF pathway genes. While we have come a long way in identifying the causes of CHD, more work is required to end the diagnostic odyssey for all CHD families. Complex genetic explanations of CHD are emerging but will require increasingly sophisticated analysis strategies applied to very large CHD cohorts before they can come to fruition in providing molecular diagnoses to genetically unsolved patients. In this review, we discuss the genetic architecture of CHD and biological pathways involved in its pathogenesis.

A mouse model of Bardet-Biedl Syndrome has impaired fear memory, which is rescued by lithium treatment.

Primary cilia are microtubule-based organelles present on most cells that regulate many physiological processes, ranging from maintaining energy homeostasis to renal function. However, the role of these structures in the regulation of behavior remains unknown. To study the role of cilia in behavior, we employ mouse models of the human ciliopathy, Bardet-Biedl Syndrome (BBS). Here, we demonstrate that BBS mice have significant impairments in context fear conditioning, a form of associative learning. Moreover, we show that postnatal deletion of BBS gene function, as well as congenital deletion, specifically in the forebrain, impairs context fear conditioning. Analyses indicated that these behavioral impairments are not the result of impaired hippocampal long-term potentiation. However, our results indicate that these behavioral impairments are the result of impaired hippocampal neurogenesis. Two-week treatment with lithium chloride partially restores the proliferation of hippocampal neurons which leads to a rescue of context fear conditioning. Overall, our results identify a novel role of cilia genes in hippocampal neurogenesis and long-term context fear conditioning.

SOX9 modulates cancer biomarker and cilia genes in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown; however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC, and both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1s, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop and SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1s to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top 10 downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.

Subcellular localization of the voltage-gated K+ channel EGL-36 , a member of the KV3 subfamily, in the ciliated sensory neurons in C. elegans.

Delineated as the first cellular organelle in 1675 by Antonie van Leeuwenhoek, cilia did not receive much attention until the 2000s, when it became apparent that cilia played a key role in the development of embryos, a variety of signaling pathways. Therefore, collective efforts by many scientists have led to the identification of many novel ciliopathy and cilia genes, while we are still far from disclosing the complete components of cilia.Here we used the ciliated sensory neurons in C. elegans as a model system that revealed the voltage-gated K+ channel EGL-36 (a member of the Shaw subfamily) as a new component associated with cilia. The confocal microscopy examination of fluorescence tagged EGL-36 together with ciliary (IFT-140) or transition zone (MKS-6) markers reveal that EGL-36 is only expressed in subsets of the ciliated sensory neurons, where it partially overlaps with the basal body signals and predominantly localizes to the periciliary membrane compartment. This expression pattern along with studies of egl-36 gain-of-function variants indicates that egl-36 is not essential for ciliogenesis in C. elegans. Our data identify the voltage-gated K+ channel EGL-36 as a new cilia-associated protein, and future studies should reveal the functional significance of EGL-36 in cilia biogenesis.

Reply: Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating

Study question How does exposure to a testosterone rich environment affect the function and gene expression of human fallopian tube epithelium (hFTE)? Summary answer Elevated testosterone level alters several gene transcripts that regulate cilia expression and negatively impacts the rate of cilia beating. What is known already The presence of estrogen in the follicular phase of the menstrual cycle increases the human fallopian tube ciliary beating frequency. The luteal phase, triggered by ovulation and increasing progesterone, is marked by a decrease in ciliary beating. Women with polycystic ovarian syndrome (PCOS) may have twice the serum level of testosterone than ovulatory women. To date, the effect of elevated androgens on the function of the human fallopian tube is not well-understood. We chose to examine the impact of elevated testosterone on hFTE. Study design, size, duration A prospective basic science study of human fallopian tube specimens from reproductive-aged women undergoing benign gynecologic surgery was performed. Fallopian tube removal at a large US academic center was collected and provided to us to continue with epithelium isolation and culturing. A total of 12 patients were analyzed in the study. Participants/materials, setting, methods Fallopian tube epithelium was isolated and exposed to two different conditions: normal with low testosterone concentration of 0.8 nM and PCOS-like, with high testosterone concentration of 2 nM. The study was conducted in both static and dynamic conditions in microfluidic devices for a total of 14 days, after which the tissue was collected for processing including RNA extraction, quantitative PCR and immunohistochemistry. After the first 7 days of each experiment, a sample of tissue from each condition was imaged to quantify cilia beating frequency. Main results and the role of chance hFTE exposed to the 2 nM testosterone displayed slower cilia beating, inhibited estrogen signaling and decreased expression of the ciliary marker FOXJ1 when compared to stimulation with 0.8 nM testosterone. Large scale data N/A. Limitations, reasons for caution The in vivo response to elevated testosterone may differ from in vitro studies. RNA amount was limited from tissue cultured in the microfluidic devices as compared to static culture. Wider implications of the findings Understanding elevated testosterone in tubal function may explain an additional contribution to subfertility in women with PCOS and other hyper-androgen disorders, aside from oligo-ovulation. Furthermore, this adds to the body of literature of fallopian tube function using a microfluidic device. Study funding/competing interest(s) NIH grants: UH3 ES029073 and R01 CA240301. There are no competing interests.

Patterns of cilia gene dysregulations in major psychiatric disorders

Primary cilia function as cells' antennas to detect and transduce external stimuli and play crucial roles in cell signaling and communication. The vast majority of cilia genes that are causally linked with ciliopathies are also associated with neurological deficits, such as cognitive impairments. Yet, the roles of cilia dysfunctions in the pathogenesis of psychiatric disorders have not been studied. Our aim is to identify patterns of cilia gene dysregulation in the four major psychiatric disorders: schizophrenia (SCZ), autism spectrum disorder (ASD), bipolar disorder (BP), and major depressive disorder (MDD). For this purpose, we acquired differentially expressed genes (DEGs) from the largest and most recent publicly available databases. We found that 42%, 24%, 17%, and 15% of brain-expressed cilia genes were significantly differentially expressed in SCZ, ASD, BP, and MDD, respectively. Several genes exhibited cross-disorder overlap, suggesting that typical cilia signaling pathways' dysfunctions determine susceptibility to more than one psychiatric disorder or may partially underlie their pathophysiology. Our study revealed that genes encoding proteins of almost all sub-cilia structural and functional compartments were dysregulated in the four psychiatric disorders. Strikingly, the genes of 75% of cilia GPCRs and 50% of the transition zone proteins were differentially expressed in SCZ.The present study is the first to draw associations between cilia and major psychiatric disorders, and is the first step toward understanding the role that cilia components play in their pathophysiological processes, which may lead to novel therapeutic targets for these disorders.

Cilia interactome with predicted protein\u2013protein interactions reveals connections to Alzheimer\u2019s disease, aging and other neuropsychiatric processes

Cilia are dynamic microtubule-based organelles present on the surface of many eukaryotic cell types and can be motile or non-motile primary cilia. Cilia defects underlie a growing list of human disorders, collectively called ciliopathies, with overlapping phenotypes such as developmental delays and cognitive and memory deficits. Consistent with this, cilia play an important role in brain development, particularly in neurogenesis and neuronal migration. These findings suggest that a deeper systems-level understanding of how ciliary proteins function together may provide new mechanistic insights into the molecular etiologies of nervous system defects. Towards this end, we performed a protein–protein interaction (PPI) network analysis of known intraflagellar transport, BBSome, transition zone, ciliary membrane and motile cilia proteins. Known PPIs of ciliary proteins were assembled from online databases. Novel PPIs were predicted for each ciliary protein using a computational method we developed, called High-precision PPI Prediction (HiPPIP) model. The resulting cilia “interactome” consists of 165 ciliary proteins, 1,011 known PPIs, and 765 novel PPIs. The cilia interactome revealed interconnections between ciliary proteins, and their relation to several pathways related to neuropsychiatric processes, and to drug targets. Approximately 184 genes in the cilia interactome are targeted by 548 currently approved drugs, of which 103 are used to treat various diseases of nervous system origin. Taken together, the cilia interactome presented here provides novel insights into the relationship between ciliary protein dysfunction and neuropsychiatric disorders, for e.g. interconnections of Alzheimer’s disease, aging and cilia genes. These results provide the framework for the rational design of new therapeutic agents for treatment of ciliopathies and neuropsychiatric disorders.

Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia.

Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.

Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating

How does exposure to a testosterone rich environment affect the function and gene expression of human fallopian tube epithelium (hFTE)? Elevated testosterone level alters several gene transcripts that regulate cilia expression and negatively impacts the rate of cilia beating. The presence of estrogen in the follicular phase of the menstrual cycle increases the human fallopian tube ciliary beating frequency. The luteal phase, triggered by ovulation and increasing progesterone, is marked by a decrease in ciliary beating. Women with polycystic ovarian syndrome (PCOS) may have twice the serum level of testosterone than ovulatory women. To date, the effect of elevated androgens on the function of the human fallopian tube is not well-understood. We chose to examine the impact of elevated testosterone on hFTE. A prospective basic science study of human fallopian tube specimens from reproductive-aged women undergoing benign gynecologic surgery was performed. Fallopian tube removal at a large US academic center was collected and provided to us to continue with epithelium isolation and culturing. A total of 12 patients were analyzed in the study. Fallopian tube epithelium was isolated and exposed to two different conditions: normal with low testosterone concentration of 0.8 nM and PCOS-like, with high testosterone concentration of 2 nM. The study was conducted in both static and dynamic conditions in microfluidic devices for a total of 14 days, after which the tissue was collected for processing including RNA extraction, quantitative PCR and immunohistochemistry. After the first 7 days of each experiment, a sample of tissue from each condition was imaged to quantify cilia beating frequency. hFTE exposed to the 2 nM testosterone displayed slower cilia beating, inhibited estrogen signaling and decreased expression of the ciliary marker FOXJ1 when compared to stimulation with 0.8 nM testosterone. N/A. The in vivo response to elevated testosterone may differ from in vitro studies. RNA amount was limited from tissue cultured in the microfluidic devices as compared to static culture. Understanding elevated testosterone in tubal function may explain an additional contribution to subfertility in women with PCOS and other hyper-androgen disorders, aside from oligo-ovulation. Furthermore, this adds to the body of literature of fallopian tube function using a microfluidic device. NIH grants: UH3 ES029073 and R01 CA240301. There are no competing interests.

Multifaceted Roles of Primary Cilia in Polycystic Kidney Disease and Obesity

The primary cilium is a small, antenna‐like sensory organelle that protrudes from most mammalian cells and mediates signaling pathways. Cilia are built by intraflagellar transport (IFT) protein complexes B and A, which have separate roles. Dysfunction of IFT and cilia results in ciliopathies, disease syndromes which commonly cause renal cystic disease and can also lead to obesity.Intriguingly, in mice, deletion of Ift‐B genes, which causes loss of primary cilia, can markedly attenuate Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most common renal cystic disease and a leading cause of renal failure. These data indicate that targeting primary cilia genes can have immense therapeutic potential in ADPKD through mechanisms that remain elusive. To help elucidate these mechanisms, we have ablated an Ift‐A gene in ADPKD mouse models. IFT‐A dysfunction partially rescues the ADPKD elongated renal ciliary phenotype and attenuates the disease in a renal tubular‐ and maturation‐dependent manner.In adult mice, Ift gene deletion causes hyperphagia‐induced obesity. We have found that prior to an obese phenotype, IFT‐A dysfunction misregulates expression of appetite‐controlling neuropeptides in the hypothalamus. Additionally, in a pre‐obese state and through a non‐cell autonomous mechanism, hyperphagia induces hepatic insulin resistance, which drives systemic insulin resistance. These data uncouple obesity and insulin resistance, and indicate that hyperphagia is a major driver of both phenotypes. Thus, we are examining methods to counter hyperphagia. In collaboration with Zafgen Inc., we have found that pharmacological inhibition of MetAP2, which cleaves the N‐terminal methionine of nascent proteins, reduces food intake and body weight, and substantially improves metabolic parameters in an Ift‐A mutant mouse. To our knowledge, this is the first demonstration of rescue of an obese phenotype in a ciliopathy mouse model.Support or Funding InformationThis research is supported by NIH/NIDDK R01DK103033

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Combinatorial action of NF–Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF–Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF–Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF–Y or TALE defines genes in specific developmental processes, such that NF–Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF–Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF–Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.

TRPPing up bronchiectasis.

TRPPing up bronchiectasis.

Defects in the Exocyst-Cilia Machinery Cause Bicuspid Aortic Valve Disease and Aortic Stenosis.

Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities. We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography. Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models. These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.

Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility

Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD is unknown. Histone H2B monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40, and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment 6.01, P = 1.67 × 10-03), some of whom had abnormal laterality associated with ciliary dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right (LR) asymmetry, and impaired cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and nonciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3 Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.