Search

Background Transposable elements (TEs) are DNA sequences able to mobilize themselves and to increase their copy-number in the host genome. In the past, they have been considered mainly selfish DNA without evident functions. Nevertheless, currently they are believed to have been extensively involved in the evolution of primate genomes, especially from a regulatory perspective. Due to their recent activity they are also one of the primary sources of structural variants (SVs) in the human genome. By taking advantage of sequencing technologies and bioinformatics tools, recent surveys uncovered specific TE structural variants (TEVs) that gave rise to polymorphisms in human populations. When combined with RNA-seq data this information provides the opportunity to study the potential impact of TEs on gene expression in human. Results In this work, we assessed the effects of the presence of specific TEs in cis on the expression of flanking genes by producing associations between polymorphic TEs and flanking gene expression levels in human lymphoblastoid cell lines. By using public data from the 1000 Genome Project and the Geuvadis consortium, we exploited an expression quantitative trait loci (eQTL) approach integrated with additional bioinformatics data mining analyses. We uncovered human loci enriched for common, less common and rare TEVs and identified 323 significant TEV-cis-eQTL associations. SINE-R/VNTR/Alus (SVAs) resulted the TE class with the strongest effects on gene expression. We also unveiled differential functional enrichments on genes associated to TEVs, genes associated to TEV-cis-eQTLs and genes associated to the genomic regions mostly enriched in TEV-cis-eQTLs highlighting, at multiple levels, the impact of TEVs on the host genome. Finally, we also identified polymorphic TEs putatively embedded in transcriptional units, proposing a novel mechanism in which TEVs may mediate individual-specific traits. Conclusion We contributed to unveiling the effect of polymorphic TEs on transcription in lymphoblastoid cell lines.

BackgroundTransposable elements (TEs) widely contributed to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of theOctopus bimaculoidesrevealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome ofOctopus vulgaris. However a specific search for the existence of full-length transpositionally competent TEs has not been performed in this genus.ResultsHere we report the identification of LINE elements competent for retrotransposition inOctopus vulgarisandOctopus bimaculoidesand show evidence suggesting that they might be active driving germline polymorphisms among individuals and somatic polymorphisms in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of bothOctopusspecies, further testifying the crucial activity of TEs in the evolution of the octopus genomes.ConclusionsThe neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full lenght LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 &micro, M, 15 &micro, M, and 20 &micro, M. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

Minisatellites, also called variable number of tandem repeats (VNTRs), are a class of repetitive elements that may affect gene expression at multiple levels and have been correlated to disease. Their identification and role as expression quantitative trait loci (eQTL) have been limited by their absence in comparative genomic hybridization and single nucleotide polymorphisms arrays. By taking advantage of cap analysis of gene expression (CAGE), we describe a new example of a minisatellite hosting a transcription start site (TSS) which expression is dependent on the repeat number. It is located in the third intron of the gene nitrogen permease regulator like protein 3 (NPRL3). NPRL3 is a component of the GAP activity toward rags 1 protein complex that inhibits mammalian target of rapamycin complex 1 (mTORC1) activity and it is found mutated in familial focal cortical dysplasia and familial focal epilepsy. CAGE tags represent an alternative TSS identifying TAGNPRL3 messenger RNAs (mRNAs). TAGNPRL3 is expressed in red blood cells both at mRNA and protein levels, it interacts with its protein partner NPRL2 and its overexpression inhibits cell proliferation. This study provides an example of a minisatellite that is both a TSS and an eQTL as well as identifies a new VNTR that may modify mTORC1 activity.

Background Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. Results The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Conclusions Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1983-5) contains supplementary material, which is available to authorized users.

Background Phlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, including Leishmania parasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular basis of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies. Results Using comparative genomics/transcriptomics data mining and transcriptional profiling, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, the transformer (tra) homolog which exhibits both conserved and novel features. The analysis of the tra locus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit fly Ceratitis capitata and several other insect species. Conclusions Our results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotechnological applications to control natural population of sand flies, reducing their impact on public health. Electronic supplementary material The online version of this article (10.1186/s12864-019-5898-4) contains supplementary material, which is available to authorized users.

The common octopus, Octopus vulgaris, is an active marine predator known for the richness and plasticity of its behavioral repertoire, and remarkable learning and memory capabilities. Octopus and other coleoid cephalopods, cuttlefish and squid, possess the largest nervous system among invertebrates, both for cell counts and body to brain size. O. vulgaris has been at the center of a long-tradition of research into diverse aspects of its biology. To leverage research in this iconic species, we generated 270 Gb of genomic sequencing data, complementing those available for the only other sequenced congeneric octopus, Octopus bimaculoides. We show that both genomes are similar in size, but display different levels of heterozygosity and repeats. Our data give a first quantitative glimpse into the rate of coding and non-coding regions and support the view that hundreds of novel genes may have arisen independently despite the close phylogenetic distance. We furthermore describe a reference-guided assembly and an open genomic resource (CephRes-gdatabase), opening new avenues in the study of genomic novelties in cephalopods and their biology., Design Type(s)species comparison design • sequence analysis objective • sequence assembly objectiveMeasurement Type(s)whole genome sequencing assayTechnology Type(s)DNA sequencingFactor Type(s) Sample Characteristic(s)Octopus vulgaris • testis • ocean biome Machine-accessible metadata file describing the reported data (ISA-Tab format)

Connecting millions of neurons to create a functional neural circuit is a daunting challenge. Vertebrates developed a molecular system at the cell membrane to allow neurons to recognize each other by distinguishing self from non-self through homophilic protocadherin interactions. In mammals, the protocadherin gene family counts about 50 different genes. By hetero-multimerization, protocadherins are capable of generating an impressive number of molecular interfaces. Surprisingly, in the California two-spot octopus, Octopus bimaculoides, an invertebrate belonging to the Phylum Mollusca, over 160 protocadherins (PCDHs) have been identified. Here we briefly discuss the role of PCDHs in neural wiring and conduct a comparative study of the protocadherin gene family in two closely related octopus species, Octopus vulgaris and O. bimaculoides. A first glance at the expression patterns of protocadherins in O. vulgaris is also provided. Finally, we comment on PCDH evolution in the light of invertebrate nervous system plasticity. ispartof: Frontiers in Physiology vol:9 issue:JAN ispartof: location:Switzerland status: published

BackgroundPhlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, includingLeishmaniaparasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular bases of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies.ResultsUsing a comparative genomics/transcriptomics approach, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, thetransformer(tra) homolog which exhibits both conserved and novel features. The analysis of thetralocus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit flyCeratitis capitataand several other insect species.ConclusionsOur results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotech applications to control natural population of sand flies, reducing their impact on public health.

Disruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass-spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced genes IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data encloses potential for designing targeted therapies to rescue E-cadherin function. Fundação para a Ciência e Tecnologia

The transition from quiescence to proliferation is a key regulatory step that can be induced by serum stimulation in cultured fibroblasts. The transcription factor Myc is directly induced by serum mitogens and drives a secondary gene expression program that remains largely unknown. Using mRNA profiling, we identify close to 300 Myc-dependent serum response (MDSR) genes, which are induced by serum in a Myc-dependent manner in mouse fibroblasts. Mapping of genomic Myc-binding sites by ChIP-seq technology revealed that most MDSR genes were directly targeted by Myc, but represented a minor fraction (5.5%) of all Myc-bound promoters (which were 22.4% of all promoters). Other target loci were either induced by serum in a Myc-independent manner, were not significantly regulated or were negatively regulated. MDSR gene products were involved in a variety of processes, including nucleotide biosynthesis, ribosome biogenesis, DNA replication and RNA control. Of the 29 MDSR genes targeted by RNA interference, three showed a requirement for cell-cycle entry upon serum stimulation and 11 for long-term proliferation and/or survival. Hence, proper coordination of key regulatory and biosynthetic pathways following mitogenic stimulation relies upon the concerted regulation of multiple Myc-dependent genes. Oncogene (2012) 31, 1695-1709; doi:10.1038/onc.2011.359; published online 22 August 2011

We report gene profiling data on genomic processes underlying the progression towards recurrent seizures after injection of kainic acid (KA) into the mouse hippocampus. Focal injection enabled us to separate the effects of proepileptic stimuli initiated by KA injection. Both the injected and contralateral hippocampus participated in the status epilepticus. However, neuronal death induced by KA treatment was restricted to the injected hippocampus, although there was some contralateral axonal degeneration. We profiled gene expression changes in dorsal and ventral regions of both the injected and contralateral hippocampus. Changes were detected in the expression of 1526 transcripts in samples from three time-points: (i) during the KA-induced status epilepticus, (ii) at 2 weeks, before recurrent seizures emerged, and (iii) at 6 months after seizures emerged. Grouping genes with similar spatio-temporal changes revealed an early transcriptional response, strong immune, cell death and growth responses at 2 weeks and an activation of immune and extracellular matrix genes persisting at 6 months. Immunostaining for proteins coded by genes identified from array studies provided evidence for gliogenesis and suggested that the proteoglycan biglycan is synthesized by astrocytes and contributes to a glial scar. Gene changes at 6 months after KA injection were largely restricted to tissue from the injection site. This suggests that either recurrent seizures might depend on maintained processes including immune responses and changes in extracellular matrix proteins near the injection site or alternatively might result from processes, such as growth, distant from the injection site and terminated while seizures are maintained.

Background Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. Results The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Conclusions Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

International audience; Diatoms are a key phytoplankton group in the contemporary ocean, showing extraordinary adaptation capacities to rapidly changing environments. The recent availability of whole genome sequences from representative species has revealed distinct features in their genomes, like novel combinations of genes encoding distinct metabolisms and a significant number of diatom-specific genes. However, the regulatory mechanisms driving diatom gene expression are still largely uncharacterized. Considering the wide variety of fields of study orbiting diatoms, ranging from ecology, evolutionary biology to biotechnology, it is thus essential to increase our understanding of fundamental gene regulatory processes such as transcriptional regulation. To this aim, we explored the functional properties of the 5'-flanking region of the Phaeodatylum tricornutum Lhcf2 gene, encoding a member of the Light Harvesting Complex superfamily and we showed that this region enhances transcription of a GUS reporter gene in an orientation- and distance-independent fashion. This represents the first example of a cis-regulatory sequence with enhancer-like features discovered in diatoms and it is instrumental for the generation of novel genetic tools and diatom exploitation in different areas of study. (C) 2015 Elsevier B.V. All rights reserved.

Diatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways) and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae.

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research., Italian Telethon Foundation [TGM11SB2]

Diatoms dominate productive regions in the oceans and have traditionally been regarded as sustaining the marine food chain to top consumers and fisheries. However, many of these unicellular algae produce cytotoxic oxylipins that impair reproductive and developmental processes in their main grazers, crustacean copepods. The molecular mode of action of diatoms and diatom oxylipins on copepods is still unclear. In the present study we generated two Expressed Sequence Tags (ESTs) libraries of the copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi and the cryptophyte Rhodomonas baltica as a control, using suppression subtractive hybridization (SSH). Our aim was to investigate differences in the transcriptome between females fed toxic and non-toxic food and identify differentially expressed genes and biological processes targeted by this diatom. We produced 947 high quality ESTs from both libraries, 475 of which were functionally annotated and deposited in GenBank. Clustering and assembling of ESTs resulted in 376 unique transcripts, 200 of which were functionally annotated. Functional enirchment analysis between the two SSH libraries showed that ESTs belonging to biological processes such as response to stimuli, signal transduction, and protein folding were significantly over-expressed in the S. marinoi-fed C. helgolandicus compared to R. baltica-fed C. helgolandicus library. These findings were confirmed by RT-qPCR analysis. In summary, 2 days of feeding on S. marinoi activated a generalized Cellular Stress Response (CSR) in C. helgolandicus, by over-expressing genes of molecular chaperones and signal transduction pathways that protect the copepod from the immediate effects of the diatom diet. Our results provide insights into the response of copepods to a harmful diatom diet at the transcriptome level, supporting the hypothesis that diatom oxylipins elicit a stress response in the receiving organism. They also increase the genomic resources for this copepod species, whose importance could become ever more relevant for pelagic ecosystem functioning in European waters due to global warming.

Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as 'Olfactores conserved non-coding elements'.

Cadherins are cell–cell adhesion proteins essential for the maintenance of tissue architecture and integrity, and their impairment is often associated with human cancer. Knowledge regarding regulatory mechanisms associated with cadherin misexpression in cancer is scarce. Specific features of the intronic-structure and intronic-based regulatory mechanisms in the cadherin superfamily are unidentified. This study aims at systematically characterizing the intronic portion of cadherin superfamily members and the identification of intronic regions constituting putative targets/triggers of regulation, using a bioinformatic approach and biological data mining. Our study demonstrates that the cadherin superfamily genes harbour specific characteristics in comparison to all non-cadherin genes, both from the genomic and transcriptional standpoints. Cadherin superfamily genes display higher average total intron number and significantly longer introns than other genes and across the entire vertebrate lineage. Moreover, in the human genome, we observed an uncommon high frequency of MIR (mammalian-wide interspersed repeats) and MaLR (mammalian-wide interspersed repeats, a subtype of LTR) regulatory-associated repetitive elements at 5′-located introns, concomitantly with increased de novo intronic transcription. Using this approach, we identified cadherin intronic-specific sites that may constitute novel targets/triggers of cadherin superfamily expression regulation. These findings pinpoint the need to identify mechanisms affecting particularly MIR and MaLR elements located in introns 2 and 3 of human cadherin genes, possibly important in the expression modulation of this superfamily in homeostasis and cancer.

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant G93A-SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre-mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A-SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage. © 2011 Wiley-Liss, Inc.

We report here the mapping of a chromosomal region responsible for strain-specific development of congenital hypothyroidism in mice heterozygous for null mutations in genes encoding Nkx2-1/Titf1 and Pax8. The two strains showing a differential predisposition to congenital hypothyroidism contain several single-nucleotide polymorphisms in this locus, one of which leads to a nonsynonymous amino acid change in a highly conserved region of Dnajc17, a member of the type III heat-shock protein-40 (Hsp40) family. We demonstrate that Dnajc17 is highly expressed in the thyroid bud and had an essential function in development, suggesting an important role of this protein in organogenesis and/or function of the thyroid gland.

Germline CDH1 point or small frameshift mutations can be identified in 30-50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT-PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is approximately 46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

Background The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. Results Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. Conclusions The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.