Variations in COL15A1 and COL18A1 influence age of onset of primary open angle glaucoma.

Primary open angle glaucoma (POAG) is a genetically and phenotypically complex disease that is a leading cause of blindness worldwide. Previously we completed a genome-wide scan for early-onset POAG that identified a locus on 9q22 (GLC1J). To identify potential causative variants underlying GLC1J, we used targeted DNA capture followed by high throughput sequencing of individuals from four GLC1J pedigrees, followed by Sanger sequencing to screen candidate variants in additional pedigrees. A mutation likely to cause early-onset glaucoma was not identified, however COL15A1 variants were found in the youngest affected members of 7 of 15 pedigrees with variable disease onset. In addition, the most common COL15A1 variant, R163H, influenced the age of onset in adult POAG cases. RNA in situ hybridization of mouse eyes shows that Col15a1 is expressed in the multiple ocular structures including ciliary body, astrocytes of the optic nerve and cells in the ganglion cell layer. Sanger sequencing of COL18A1, a related multiplexin collagen, identified a rare variant, A1381T, in members of three additional pedigrees with early-onset disease. These results suggest genetic variation in COL15A1 and COL18A1 can modify the age of onset of both early and late onset POAG.

Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection.

The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.

Molecular evolution of piRNA and transposon control pathways in Drosophila.

The mere prevalence and potential mobilization of transposable elements in eukaryotic genomes present challenges at both the organismal and population levels. Not only is transposition able to alter gene function and chromosomal structure, but loss of control over even a single active element in the germline can create an evolutionary dead end. Despite the dangers of coexistence, transposons and their activity have been shown to drive the evolution of gene function, chromosomal organization, and even population dynamics (Kazazian 2004). This implies that organisms have adopted elaborate means to balance both the positive and detrimental consequences of transposon activity. In this chapter, we focus on the fruit fly to explore some of the molecular clues into the long- and short-term adaptation to transposon colonization and persistence within eukaryotic genomes.

Analysis of large-scale sequencing of small RNAs.

The advent of large-scale sequencing has opened up new areas of research, such as the study of Piwi-interacting small RNAs (piRNAs). piRNAs are longer than miRNAs, close to 30 nucleotides in length, involved in various functions, such as the suppression of transposons in germline. Since a large number of them (many tens of thousands) are generated from a wide range of positions in the genome, large-scale sequencing is the only way to study them. The key to understanding their genesis and biological roles is efficient analysis, which is complicated by the large volumes of sequence data. Taking account of the underlying biology is also important. We describe here novel analyses techniques and tools applied to small RNAs from germ cells in D. melanogaster, that allowed us to infer mechanism and biological function.

Small RNA silencing pathways in germ and stem cells.

During the past several years, it has become clear that small RNAs guard germ cell genomes from the activity of mobile genetic elements. Indeed, in mammals, a class of small RNAs, known as Piwi-interacting RNAs (piRNAs), forms an innate immune system that discriminates transposons from endogenous genes and selectively silences the former. piRNAs enforce silencing by directing transposon DNA methylation during male germ cell development. As such, piRNAs represent perhaps the only currently known sequence-specific factor for deposition of methylcytosine in mammals. The three mammalian Piwi proteins Miwi2, Mili, and Miwi are required at different stages of germ cell development. Moreover, distinct classes of piRNAs are expressed in developmental waves, with particular generative loci and different sequence content distinguishing piRNAs populations in embryonic germ cells from those that appear during meiosis. Although our understanding of Piwi proteins and piRNA biology have deepened substantially during the last several years, major gaps still exist in our understanding of these enigmatic RNA species.

The expanding universe of noncoding RNAs.

The 71st Cold Spring Harbor Symposium on Quantitative Biology celebrated the numerous and expanding roles of regulatory RNAs in systems ranging from bacteria to mammals. It was clearly evident that noncoding RNAs are undergoing a renaissance, with reports of their involvement in nearly every cellular process. Previously known classes of longer noncoding RNAs were shown to function by every possible means-acting catalytically, sensing physiological states through adoption of complex secondary and tertiary structures, or using their primary sequences for recognition of target sites. The many recently discovered classes of small noncoding RNAs, generally less than 35 nucleotides in length, most often exert their effects by guiding regulatory complexes to targets via base-pairing. With the ability to analyze the RNA products of the genome in ever greater depth, it has become clear that the universe of noncoding RNAs may extend far beyond the boundaries we had previously imagined. Thus, as much as the Symposium highlighted exciting progress in the field, it also revealed how much farther we must go to understand fully the biological impact of noncoding RNAs.

Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors.

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

A survey of canine expressed sequence tags and a display of their annotations through a flexible web-based interface.

We have initially sequenced approximately 8,000 canine expressed sequence tags (ESTs) from several complementary DNA (cDNA) libraries: testes, whole brain, and Madin-Darby canine kidney (MDCK) cells. Analysis of these sequences shows that they provide partial sequence information for about 5%-10% of the canine genes. An analysis pipeline has been created to cluster the ESTs and to map individual ESTs as well as clustered ESTs to both the human genome and the human proteome. Gene ontology (GO) terms have been assigned to the ESTs and clusters based on their top matches to the International Protein Index (IPI) set of human proteins. The data generated is stored in a MySQL relational database for analysis and display. A Web-based Perl script has been written to display the analyzed data to the scientific community.

Design of a retroviral-mediated ecdysone-inducible system and its application to the expression profiling of the PTEN tumor suppressor.

We have engineered the ecdysone-inducible mammalian expression system for general retroviral delivery to cultured mammalian cells. We inducibly expressed PTEN in the glioblastoma cell line, U87MG, lacking this gene. Because nearly all cells are recruited on induction, we find both up- and down-regulated genes by cDNA microarray analysis. The changes we see are similar to those observed after treatment with LY294002, an inhibitor of phosphatidylinositol 3-OH kinase, fully consistent with the model that PTEN antagonizes phosphatidylinositol 3-OH kinase. Both treatments result in suppressed expression of the transforming growth factor (TGF)-beta gene and the genes of the cholesterol biosynthesis pathway. Our results illustrate the power of using a fully inducible expression system in conjunction with cDNA microarray analysis for exploring gene function.

The rest is silence.

Over the past several years, RNAi and its related phenomena have emerged not only as a powerful experimental tool but also as a new mode of gene regulation. Through a combination of genetic and biochemical approaches we have learned much about the mechanisms underlying dsRNA responses. However, many of the most intriguing aspects of dsRNA-induced gene silencing have yet to be illuminated. What has become abundantly clear is that the complex and highly conserved biology underlying RNA interference is critical both for genome maintenance and for the development of complex organisms. However, it seems probable that we have only begun to reveal the diversity of biological roles played by RNAi-related processes.

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).

Argonaute2, a link between genetic and biochemical analyses of RNAi.

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.

Post-transcriptional gene silencing by double-stranded RNA.

Imagine being able to knock out your favourite gene with only a day's work. Not just in one model system, but in virtually any organism: plants, flies, mice or cultured cells. This sort of experimental dream might one day become reality as we learn to harness the power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes. How this phenomenon works is slowly becoming clear, and might help us to develop an effortless tool to probe gene function in cells and animals.

Role for a bidentate ribonuclease in the initiation step of RNA interference.

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model.

Using an improved system for the functional identification of active antisense fragments, we have isolated antisense fragments which inactivate the p53 tumour suppressor gene. These antisense fragments map in two small regions between nt 350 and 700 and nt 800 and 950 of the coding sequence. These antisense fragments appear to act by inhibition of p53 mRNA translation both in vivo and in vitro. Expression of these antisense fragments overcame the p53-induced growth arrest in a cell line which expresses a thermolabile mutant of p53 and extended the in vitro lifespan of primary mouse embryonic fibroblasts. Continued expression of the p53 antisense fragment contributed to immortalisation of primary mouse fibroblasts. Subsequent elimination of the antisense fragment in these immortalised cells led to restoration of p53 expression and growth arrest, indicating that immortal cells continuously require inactivation of p53. Expression of MDM2 or SV40 large T antigen, but not E7 nor oncogenic ras, overcomes the arrest induced by restoration of p53 expression. Functional inactivation of both p21 and bax (by overexpression of Bcl2), but not either alone, allowed some bypass of p53-induced growth arrest, indicating that multiple transcriptional targets of p53 may mediate its antiproliferative action. The ability to conditionally inactivate and subsequently restore normal gene function may be extremely valuable for genetic analysis of genes for which loss-of-function is involved in specific phenotypes.

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

A proinflammatory cytokine inhibits p53 tumor suppressor activity.

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.

Twist is a potential oncogene that inhibits apoptosis.

Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.