The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Molecular mechanisms of hearing loss in Nager syndrome.

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.

Spliceosomopathies: Diseases and mechanisms.

The spliceosome is a complex of RNA and proteins that function together to identify intron-exon junctions in precursor messenger-RNAs, splice out the introns, and join the flanking exons. Mutations in any one of the genes encoding the proteins that make up the spliceosome may result in diseases known as spliceosomopathies. While the spliceosome is active in all cell types, with the majority of the proteins presumably expressed ubiquitously, spliceosomopathies tend to be tissue-specific as a result of germ line or somatic mutations, with phenotypes affecting primarily the retina in retinitis pigmentosa, hematopoietic lineages in myelodysplastic syndromes, or the craniofacial skeleton in mandibulofacial dysostosis. Here we describe the major spliceosomopathies, review the proposed mechanisms underlying retinitis pigmentosa and myelodysplastic syndromes, and discuss how this knowledge may inform our understanding of craniofacial spliceosomopathies.

Proteasomal inhibition attenuates craniofacial malformations in a zebrafish model of Treacher Collins Syndrome.

Treacher Collins Syndrome (TCS) is a congenital disease characterized by defects in the craniofacial skeleton and absence of mental alterations. Recently we modelled TCS in zebrafish (Danio rerio) embryos through the microinjection of Morpholino® oligonucleotides blocking the translation of the ortholog of the main causative gene (TCOF1). We showed that Cnbp, a key cytoprotective protein involved in normal rostral head development, was detected in lower levels (without changes in its mRNA expression) in TCS-like embryos. As previous reports suggested that Cnbp is degraded through the proteasomal pathway, we tested whether proteasome inhibitors (MG132 and Bortezomib (Velcade®, Millennium laboratories)) were able to ameliorate cranial skeleton malformations in TCS. Here we show that treatment with both proteasome inhibitors produced a robust craniofacial cartilage phenotype recovery. This recovery seems to be consequence of a decreased degradation of Cnbp in TCS-like embryos. Critical TCS manifestations, such as neuroepithelial cell death and cell redox imbalance were attenuated. Thus, proteasome inhibitors may offer an opportunity for TCS molecular and phenotypic manifestation's prevention. Although further development of new safe inhibitors compatible with administration during pregnancy is required, our results encourage this therapeutic approach.

tp53-dependent and independent signaling underlies the pathogenesis and possible prevention of Acrofacial Dysostosis-Cincinnati type.

Ribosome biogenesis is a global process required for growth and proliferation in all cells, but disruptions in this process surprisingly lead to tissue-specific phenotypic disorders termed ribosomopathies. Pathogenic variants in the RNA Polymerase (Pol) I subunit POLR1A cause Acrofacial Dysostosis-Cincinnati type, which is characterized by craniofacial and limb anomalies. In a zebrafish model of Acrofacial Dysostosis-Cincinnati type, we demonstrate that polr1a-/- mutants exhibit deficient 47S rRNA transcription, reduced monosomes and polysomes and, consequently, defects in protein translation. This results in Tp53-dependent neuroepithelial apoptosis, diminished neural crest cell proliferation and cranioskeletal anomalies. This indicates that POLR1A is critical for rRNA transcription, which is considered a rate limiting step in ribosome biogenesis, underpinning its requirement for neuroepithelial cell and neural crest cell proliferation and survival. To understand the contribution of the Tp53 pathway to the pathogenesis of Acrofacial Dysostosis-Cincinnati type, we genetically inhibited tp53 in polr1a-/- mutant embryos. Tp53 inhibition suppresses neuroepithelial apoptosis and partially ameliorates the polr1a mutant phenotype. However, complete rescue of cartilage development is not observed due to the failure to improve rDNA transcription and neural crest cell proliferation. Altogether, these data reveal specific functions for both Tp53-dependent and independent signaling downstream of polr1a in ribosome biogenesis during neural crest cell and craniofacial development, in the pathogenesis of Acrofacial Dysostosis-Cincinnati type. Furthermore, our work sets the stage for identifying Tp53-independent therapies to potentially prevent Acrofacial dysostosis-Cincinnati type and other similar ribosomopathies.

Treacher Collins syndrome 3 (TCS3)-associated POLR1C mutants are localized in the lysosome and inhibits chondrogenic differentiation.

Treacher Collins syndrome (TCS) is a craniofacial developmental disorder whose key feature is a combination of symptoms. For example, a patient could have bilateral downward slanting of the palpebral fissures, colobomas of the lower eyelids, hypoplasia of the facial bones, cleft palate, malformation of the external ears, and atresia of the external auditory canals. TCS3 is caused by mutations of the polr1c gene, which encodes RNA polymerase I and III subunit C (POLR1C). There have been two known missense mutations (Arg279-to-Gln [R279Q] and Arg279-to-Trp [R279W]) at the Arg-279 position. However, it remains to be clarified whether or how both or each individual mutation affects the cellular properties of POLR1C. Here we show that TCS3-associated missense mutations cause aberrant intracellular localization of POLR1C, inhibiting chondrogenic differentiation. The wild type POLR1C is normally localized in the nuclei. The R279Q or R279W mutant is primarily found to be localized in the lysosome. Expression of the R279Q or R279W mutant in mouse chondrogenic ATDC5 cells decreases phosphorylation of 4E-BP1 and ribosomal S6 proteins, which belong to the mammalian target of rapamycin (mTOR) signaling involved in critical roles in the lysosome. Furthermore, expression of the R279Q or R279W mutant inhibits chondrogenic differentiation in ATDC5 cells. Taken together, TCS3-associated mutation leads to the localization of POLR1C into the lysosome and inhibits chondrogenic differentiation, possibly explaining a portion of the pathological molecular basis underlying Treacher Collins syndrome.

Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilize RNA polymerase I and III complex integrity.

Treacher Collins syndrome (TCS) is a craniofacial disorder that is characterized by the malformation of the facial bones. Mutations in three genes (TCOF1, POLR1C and POLR1D) involved in RNA polymerase I (Pol I) transcription account for more than 90% of disease cases. Two of these TCS-associated genes, POLR1C and POLR1D, encode for essential Pol I/III subunits that form a heterodimer necessary for Pol I/III assembly, and many TCS mutations lie along their evolutionarily conserved dimerization interface. Here we elucidate the molecular basis of TCS mutations in Saccharomyces cerevisiae, and present a new model for how TCS mutations may disrupt Pol I and III complex integrity.

Mitochondrial nucleic acid binding proteins associated with diseases.

Mammalian mitochondrial DNA (mtDNA) exists in structures called nucleoids, which correspond to the configuration of nuclear DNA. Mitochondrial transcription factor A (TFAM), first cloned as an mtDNA transcription factor, is critical for packaging and maintaining mtDNA. To investigate functional aspects of TFAM, we identified many RNA-binding proteins as candidate TFAM interactors, including ERAL1 and p32. In this review, we first describe the functions of TFAM, replication proteins such as polymerase gamma and Twinkle, and mitochondrial RNA binding proteins. We describe the role of mitochondrial nucleic acid binding proteins within the mitochondrial matrix and two oxidative phosphorylation-related proteins within the mitochondrial intermembrane space. We then discuss how mitochondrial dysfunction is related to several diseases, including mitochondrial respiratory disease, Miller syndrome and cancer. We also describe p32 knockout mice, which are embryonic lethal and exhibit respiratory chain defects. Miller syndrome is a recessive disorder characterized by postaxial acrofacial dysostosis and caused by a mutation in DHODH. Finally, we explain that p32 and mitochondrial creatine kinase may be novel markers for the progression of prostate cancer.

Treacher Collins syndrome: New insights from animal models.

Treacher Collins syndrome (TCS, OMIM: 154500), an autosomal-dominant craniofacial developmental syndrome that occurs in 1 out of every 50,000 live births, is characterized by craniofacial malformation. Mutations in TCOF1, POLR1C, or POLR1D have been identified in affected individuals. In addition to established mouse models, zebrafish models have recently emerged as an valuable method to study facial disease. In this report, we summarized the two updated articles working on the pathogenesis of the newly identified polr1c and polr1d TCS mutations (Lau et al., 2016; Noack Watt et al., 2016) and discussed the possibility of using the anti-oxidants to prevent or rescue the TCS facial phenotype (Sakai et al., 2016). Taken together, this article provides an update on the disease from basic information to pathogenesis, and further summarizes the suggested therapies from recent laboratory research.

Probing the mechanisms underlying human diseases in making ribosomes.

Ribosomes are essential, highly complex machines responsible for protein synthesis in all growing cells. Because of their importance, the process of building these machines is intricately regulated. Although the proteins involved in regulating ribosome biogenesis are just beginning to be understood, especially in human cells, the consequences for dysregulating this process have been even less studied. Such interruptions in ribosome synthesis result in a collection of human disorders known as ribosomopathies. Ribosomopathies, which occur due to mutations in proteins involved in the global process of ribosome biogenesis, result in tissue-specific defects. The questions posed by this dichotomy and the steps taken to address these questions are therefore the focus of this review: How can tissue-specific disorders result from alterations in global processes? Could ribosome specialization account for this difference?

Tcof1-Related Molecular Networks in Treacher Collins Syndrome.

Treacher Collins syndrome (TCS) is a rare, autosomal-dominant disorder characterized by craniofacial deformities, and is primarily caused by mutations in the Tcof1 gene. This article was aimed to perform a comprehensive literature review and systematic bioinformatic analysis of Tcof1-related molecular networks in TCS. First, the up- and down-regulated genes in Tcof1 heterozygous haploinsufficient mutant mice embryos and Tcof1 knockdown and Tcof1 over-expressed neuroblastoma N1E-115 cells were obtained from the Gene Expression Omnibus database. The GeneDecks database was used to calculate the 500 genes most closely related to Tcof1. Then, the relationships between 4 gene sets (a predicted set and sets comparing the wildtype with the 3 Gene Expression Omnibus datasets) were analyzed using the DAVID, GeneMANIA and STRING databases. The analysis results showed that the Tcof1-related genes were enriched in various biological processes, including cell proliferation, apoptosis, cell cycle, differentiation, and migration. They were also enriched in several signaling pathways, such as the ribosome, p53, cell cycle, and WNT signaling pathways. Additionally, these genes clearly had direct or indirect interactions with Tcof1 and between each other. Literature review and bioinformatic analysis finds imply that special attention should be given to these pathways, as they may offer target points for TCS therapies.

Prevention of Treacher Collins syndrome craniofacial anomalies in mouse models via maternal antioxidant supplementation.

Craniofacial anomalies account for approximately one-third of all birth defects and are a significant cause of infant mortality. Since the majority of the bones, cartilage and connective tissues that comprise the head and face are derived from a multipotent migratory progenitor cell population called the neural crest, craniofacial disorders are typically attributed to defects in neural crest cell development. Treacher Collins syndrome (TCS) is a disorder of craniofacial development and although TCS arises primarily through autosomal dominant mutations in TCOF1, no clear genotype-phenotype correlation has been documented. Here we show that Tcof1 haploinsufficiency results in oxidative stress-induced DNA damage and neuroepithelial cell death. Consistent with this discovery, maternal treatment with antioxidants minimizes cell death in the neuroepithelium and substantially ameliorates or prevents the pathogenesis of craniofacial anomalies in Tcof1(+/-) mice. Thus maternal antioxidant dietary supplementation may provide an avenue for protection against the pathogenesis of TCS and similar neurocristopathies.

A review of craniofacial disorders caused by spliceosomal defects.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders.

The ribosome biogenesis factor Nol11 is required for optimal rDNA transcription and craniofacial development in Xenopus.

The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.

Intrafamilial variability of ZRS-associated syndrome: characterization of a mosaic ZRS mutation by pyrosequencing.

During limb development, the spatio-temporal expression of sonic hedgehog (SHH) is driven by the Zone of polarizing activity Regulatory Sequence (ZRS), located 1 megabase upstream from SHH. Gain-of-function mutations of this enhancer, which cause ectopic expression of SHH, are known to be responsible for congenital limb malformations with variable expressivity, ranging from preaxial polydactyly or triphalangeal thumbs to polysyndactyly, which may also be associated with mesomelic deficiency. In this report, we describe a patient affected with mirror-image polydactyly of the four extremities and bilateral tibial deficiency. The proband's father had isolated preaxial polydactyly type II (PPD2). Using Sanger sequencing, a ZRS point mutation (NC_000007.14, g.156584153A>G, UCSC, Build hg.19) was only identified in the patient. However, pyrosequencing analysis enabled the detection of a 10% somatic mosaic in the blood and saliva from the father. To our knowledge, this is the first description of a ZRS mosaic mutation. This report highlights the complexity of genotype-phenotype correlation in ZRS-associated syndromes and the importance of detecting somatic mosaicism for accurate genetic counselling.

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients.

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.

Fishing the molecular bases of Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development.

Guarding the 'translation apparatus': defective ribosome biogenesis and the p53 signaling pathway.

Ribosomes, the molecular factories that carry out protein synthesis, are essential for every living cell. Ribosome biogenesis, the process of ribosome synthesis, is highly complex and energy consuming. Over the last decade, many exciting and novel findings have linked various aspects of ribosome biogenesis to cell growth and cell cycle control. Defects in ribosome biogenesis have also been linked to human diseases. It is now clear that disruption of ribosome biogenesis causes nucleolar stress that triggers a p53 signaling pathway, thus providing cells with a surveillance mechanism for monitoring ribosomal integrity. Although the exact mechanisms of p53 induction in response to nucleolar stress are still unknown, several ribosomal proteins have been identified as key players in this ribosome-p53 signaling pathway. Recent studies of human ribosomal pathologies in a variety of animal models have also highlighted the role of this pathway in the pathophysiology of these diseases. However, it remains to be understood why the effect of ribosomal malfunction is not a universal response in all cell types but is restricted to particular tissues, causing the specific phenotypes seen in ribosomal diseases. A challenge for future studies will be to identify additional players in this signaling pathway and to elucidate the underlying molecular mechanisms that link defective ribosome synthesis to p53.

The role of p53 in ribosomopathies.

Impaired ribosome biogenesis is the underlying cause of the pathological conditions collectively known as ribosomopathies. Several hypotheses have been advanced to explain the mechanisms by which deficiencies in ribosome biogenesis interfere with developmental processes leading eventually to the emergence of these diseases. In recent years it has become clear that perturbation of this process triggers a cell-cycle checkpoint that, through activation of the tumor-suppressor p53, leads to cell-cycle arrest and apoptosis. Indeed, evidence is accumulating from studies in animal models that the unscheduled activation of p53 is responsible for perturbations in tissue homeostasis that cause the development of ribosomopathies such as Treacher-Collins syndrome (TCS) and 5q(-) syndrome. These findings imply that inhibition of p53, or better, of mechanisms that specifically lead to p53 activation in response to inhibition of ribosome biogenesis, could be targeted in the treatment of ribosomopathies where activation of p53 is shown to play a pathogenic role.

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients.

BACKGROUND: Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in post-embryonic human cells. METHODS: We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively. RESULTS: All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells. CONCLUSIONS: This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.