The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type-specific phenotypes.     

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.     

Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.     

Conserved roles for peptidases in the processing of invertebrate neuropeptides

Invertebrates use a wide range of peptides as transmitters and hormones to regulate complex behaviour, physiology and development. These animals, especially those that are amenable to genetic study and are the subject of genome-sequencing projects, provide powerful model systems for understanding the functions of peptidases in controlling the bioactivity of peptides. Neprilysin, a zinc metallopeptidase and a key enzyme in the metabolism of mammalian peptides, is also implicated in the inactivation of peptides at synapses and of circulating peptide hormones in insects and nematodes. A family of neprilysin-like genes are present in the genomes of both Drosophila melanogaster and Caenorhabditis elegans; in C. elegans it seems that individual family members have evolved to take on different physiological functions, because they are expressed in a tissue-specific manner. Angiotensin I-converting enzymes (peptidyl dipeptidase A, angiotensin-converting enzyme) are another group of zinc metallopeptidases found in some invertebrates that lack angiotensin peptides. In D. melanogaster there are two functional angiotensin-converting enzymes that are essential for normal development. One of these (Acer) is expressed in the embryonic heart, whereas the second enzyme (Ance) is expressed in several tissues at different stages of the life cycle. The accumulation of Ance within secretory vesicles of some peptide-synthesizing cells suggests a role for the enzyme in the intracellular processing of insect peptides. Ance is very efficient at cleaving pairs of basic residues from the C-terminus of partly processed peptides, suggesting a novel role for the enzyme in prohormone processing. Invertebrates will continue to provide insights into the evolutionarily conserved functions of known peptidases and of those additional family members that are expected to be identified in the future from genome-sequencing projects.     

Conserved fungal genes as potential targets for broad-spectrum antifungal drug discovery

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.     

Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila

Spinal muscular atrophy is a neurodegenerative disorder caused by mutations or deletions in the survival motor neuron (SMN) gene. We have cloned the Drosophila ortholog of SMN (DmSMN) and disrupted its function by ectopically expressing human SMN. This leads to pupal lethality caused by a dominant-negative effect, whereby human SMN may bind endogenous DmSMN resulting in non-functional DmSMN/human SMN hetero-complexes. Ectopic expression of truncated versions of DmSMN and yeast two-hybrid analysis show that the C-terminus of SMN is necessary and sufficient to replicate this effect. We have therefore generated a system which can be utilized to carry out suppressor and high-throughput screens, and provided in vivo evidence for the importance of SMN oligomerization for SMN function at the level of an organism as a whole.     

Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2

Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of-motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a C→T transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, exonic mutations that cause defective splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF–dependent exonic splicing enhancer or to the creation of an hnRNP A/B–dependent exonic splicing silencer, as a result of the C→T transition. We report the extensive testing of the enhancer-loss and silencer-gain models by mutagenesis, RNA interference, overexpression, RNA splicing, and RNA-protein interaction experiments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF–dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the C→T transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative splicing, and underscore the importance of antagonistic splicing factors and exonic elements in a disease context.     

Parental modifiers,antisense transcripts and loss of imprinting

The kinship theory of genomic imprinting has explained parent-specific gene expression as the outcome of an evolutionary conflict between the two alleles at a diploid locus of an offspring over how much to demand from parents. Previous models have predicted that maternally derived (madumnal) alleles will be silent at demand-enhancing loci, while paternally derived (padumnal) alleles will be silent at demand-suppressing loci, but these models have not considered the evolution of trans-acting modifiers that are expressed in parents and influence imprinted expression in offspring. We show that such modifiers will sometimes be selected to reactivate the silent padumnal allele at a demand-suppressing locus but will not be selected to reactivate the silent madumnal allele at a demand-enhancing locus. Therefore, imprinting of demand-suppressing loci is predicted to be less evolutionarily stable than imprinting of demand-enhancing loci.     

A loss of function screen identifies nine new radiation susceptibility genes

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G₂/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.     

Genetic modifiers of cardiovascular phenotype caused by elastin haploinsufficiency act by extrinsic noncomplementation

Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln(+/-) mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln(+/-), two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln(+/-)x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln(+/-) animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln(+/-)x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln(+/-), including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln(+/-) aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation.     

Conserved microRNA function as a basis for Chinese hamster ovary cell engineering

The use of microRNAs (miRNAs) for improving the efficiency of recombinant protein production by CHO cells is gaining considerable interest for their ability to regulate entire molecular networks. Differential miRNA expression profiling and large-scale transient screening have been the prerequisite for the selection of miRNA candidates for stable manipulation, reported in CHO cells expressing a range of recombinant products. We selected a potent and well characterised tumour suppressor miRNA, miR-34a, as a high priority candidate for CHO cell engineering based on the conservation of both its sequence and function across species and cell type. Ectopic expression of miR-34a retained its functional conservation in CHO-SEAP cells by inhibiting growth by 90 % in addition to decreasing the viable cell population by 30 % when compared to controls. When the miR-34 family was stably depleted using a miRNA sponge decoy vector, the overall product yield was enhanced by ~2-fold in both fed-batch and small scale clonal batch cultures, despite having a negative impact on cell growth. These findings further strengthen the utility of miRNAs as engineering tools to modify and improve CHO cell performance.     

Conserved deployment of genes during odontogenesis across osteichthyans

Odontogenesis has only been closely scrutinized at the molecular level in the mouse, an animal with an extremely restricted dentition of only two types and one set. However, within osteichthyans many species display complex and extensive dentitions, which questions the extent to which information from the mouse is applicable to all osteichthyans. We present novel comparative molecular and morphological data in the rainbow trout (Oncorhynchus mykiss) that show that three genes, essential for murine odontogenesis, follow identical spatial-temporal expression. Thus, at all tooth bud sites, epithelial genes Pitx-2 and Shh initiate the odontogenic cascade, resulting in dental mesenchymal Bmp-4 expression, importantly, including the previously unknown formation of replacement teeth. Significantly, this spatial-temporal sequence is the same for marginal and lingual dentitions, but we find notable differences regarding the deployment of Pitx-2 in the developing pharyngeal dentition. This difference may be highly significant in relation to the theory that dentitions may have evolved from pharyngeal tooth sets in jawless fishes. We have provided the first data on operational genes in tooth development to show that the same signalling genes choreograph this evolutionary stable event in fishes since the osteichthyan divergence 420 Myr ago, with the identical spatial-temporal expression as in mammals.     

Human tRNA genes function as chromatin insulators

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.     

Fluoxetine-resistance genes in Caenorhabditis elegans function in the intestine and may act in drug transport

Fluoxetine (Prozac) is one of the most widely prescribed pharmaceuticals, yet important aspects of its mechanism of action remain unknown. We previously reported that fluoxetine and related antidepressants induce nose muscle contraction of C. elegans. We also reported the identification and initial characterization of mutations in seven C. elegans genes that cause defects in this response (Nrf, nose resistant to fluoxetine). Here we present genetic evidence that the known nrf genes can be divided into two subgroups that confer sensitivity to fluoxetine-induced nose contraction by distinct pathways. Using both tissue-specific promoters and genetic mosaic analysis, we show that a gene from one of these classes, nrf-6, functions in the intestine to confer fluoxetine sensitivity. Finally, we molecularly identify nrf-5, another gene in the same class. The NRF-5 protein is homologous to a family of secreted lipid-binding proteins with broad ligand specificity. NRF-5 is expressed in the intestine and is likely secreted into the pseudocoelomic fluid, where it could function to transport fluoxetine. One model that explains these findings is that NRF-5 binds fluoxetine and influences its presentation or availability to in vivo targets.