Genomic sources of regulatory variation in cis and in trans

Regulatory variation results from genetic changes with both cis and trans acting effects on gene expression. Here I describe the types of genetic variants that alter cis and trans regulation and discuss differences in the potential for cis and trans changes among different classes of genes. I argue that the molecular function of the protein encoded by each gene and how the gene is wired into the genomic regulatory network may influence its propensity for cis and trans regulatory changes.Received 15 February 2005; received after revision 12 April 2005; accepted 26 April 2005     

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins. Received 30 September 2005; received after revision 5 November 2005; accepted 28 November 2005     

Dictyostelium mobile elements: strategies to amplify in a compact genome

Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long termina l repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome. Received 10 May 2002; received after revision 10 June 2002; accepted 12 June 2002 RID="*" ID="*"Corresponding author.     

Chromatin domain boundaries in the Bithorax complex

Eukaryotic chromosomes are thought to be organized into a series of discrete higher-order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fibre, but also in the utilization of genetic information. Critical to this model are the domain boundaries that delimit and segregate the chromosomes into units of independent gene activity. In Drosophila, such domain boundaries have been identified through two different approaches. On the one hand, elements like scs/scs′ and the reiterated binding site for the SU(HW) protein have been characterized through their activity of impeding enhancer-promoter interactions when intercalated between them. Their role of chromatin insulators can protect transgenes from genomic position effects, thereby establishing in dependent functional domains within the chromosome. On the other hand, domain boundaries of the Bithorax complex (BX-C) like Fab-7 and Mcp have been identified through mutational analysis. Mcp and Fab-7, however, may represent a specific class of boundary elements; instead of separating adjacent domains that contain separate structural genes, Mcp and Fab-7 delimit adjacent cis-regulatory domains, each of which interacts independently with their target promoters. In this article, we review the genetic and molecular characteristics of the domain boundaries of the BX-C. We describe how Fab-7 functions to confine activating as well as repressive signals to the flanking regulatory domains. Although the mechanisms by which Fab-7 works as a domain boundary remain an open issue, we provide preliminary evidence that Fab-7 is not a mere insulator like scs or the reiterated binding site for the SU(HW) protein.     

CCCTC-binding factor: to loop or to bridge

Eukaryotic genomes have complex spatial organization in the nucleus. The factors and the mechanisms involved in this organization remain an enigma. Among the many proteins implicated in such a role, the ubiquitous Zn-finger protein CTCF stands out. Here we summarize the evidence placing CTCF in the enviable position of a master organizer of the genome. CTCF can form loops in cis, and can bridge sequences located on different chromosomes in trans. The thousands of CTCF binding sites, identified in recent genome-wide localization studies, and their distribution along the genome further support a crucial role of CTCF as a chromatin organizer. Received 10 October 2008; received after revision 11 December 2008; accepted 16 December 2008     

Short-range linkage relationships, genomic organisation and sequence comparisons of a cluster of five HSP70 genes in Fugu rubripes

Twelve cosmids containing sequences resembling genes encoding members of the 70-kDa heat-shock protein family, HSP70, have been isolated from Fugu rubripes. They can be broadly divided into three groups of overlapping cosmids. Restriction analysis and sequencing of one set of five cosmids have revealed five intronless Fugu HSP70 genes spanning 42 kb, arranged in a combined head-to-head, tail-to-tail and head-to-tail orientation. The levels of DNA and amino acid identity are very high with respect to one another, and are most similar to HSP70 sequences linked to the major histocompatibility complex (MHC) region in other species. Putative heat-shock consensus elements are identified. Non-HSP70 sequences with homology to known genes have been found physically linked to this Fugu HSP70 cluster: the Drosophila melanogaster SOL gene, the Drosophila melanogaster nemo gene, the Caenorhabditis elegans T17E9.1 gene and the sequence encoding the serine protease domain. The linkage relationships described here so far bear no resemblance to those of HSP70 in other organisms. Convergence of mammalian HSP70 and MHC class I and II loci probably occurred after fish had diverged. Received 17 November 1998; received after revision 25 February 1999; accepted 26 February 1999     

The BAG proteins: a ubiquitous family of chaperone regulators

The BAG (Bcl-2 associated athanogene) family is a multifunctional group of proteins that perform diverse functions ranging from apoptosis to tumorigenesis. An evolutionarily conserved group, these proteins are distinguished by a common conserved region known as the BAG domain. BAG genes have been found in yeasts, plants, and animals, and are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in carcinogenesis, HIV infection, and Parkinson’s disease. These proteins are therefore potential therapeutic targets, and their expression in cells may serve as a predictive tool for such diseases. In plants, the Arabidopsis thaliana genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. Three members contain a calmodulin-binding domain possibly reflecting differences between plant and animal programmed cell death. This review summarizes current understanding of BAG proteins in both animals and plants. Received 21 November 2007; received after revision 17 December 2007; accepted 2 January 2008     

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell–cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.     

Conserved eukaryotic transposable elements and the evolution of gene regulation

Multiple remnants of transposable elements preserved in cis-regulatory modules may represent a record of mutations that were critical to the evolution of gene regulation and speciation. Received 13 August 2007; received after revision 8 October 2007; accepted 23 October 2007     

Retrotransposable elements in the Dictyostelium discoideum genome

Repetitive DNA is a major component of any living cell. In eukaryotes retrotransposable elements make up several percent of the genome size, and consequently, retroelements are often identified in experiments aimed at establishing physical maps and whole genome sequences. In this review, recent progress in the characterization of retrotransposable elements in the genome of the eukaryotic mi croorganism Dictyostelium discoideum is summarized with a focus on retroelements which integrate near transfer RNA genes with intriguing position specificity. Received 21 November 1997; received after revision 6 January 1998; accepted 6 January 1998     

Functional diversity of melanopsins and their global expression in the teleost retina

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct “fine-tuning” of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.     

Translational control of endogenous and recoded nuclear genes in yeast mitochondria: Regulation and membrane targeting

Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.