CF2 Represses Actin 88F Gene Expression and Maintains Filament Balance during Indirect Flight Muscle Development in Drosophila

The zinc finger protein CF2 is a characterized activator of muscle structural genes in the body wall muscles of the Drosophila larva. To investigate the function of CF2 in the indirect flight muscle (IFM), we examined the phenotypes of flies bearing five homozygous viable mutations. The gross structure of the IFM was not affected, but the stronger hypomorphic alleles caused an increase of up to 1.5X in the diameter of the myofibrils. This size increase did not cause any disruption of the hexameric arrangement of thick and thin filaments. RT-PCR analysis revealed an increase in the transcription of several structural genes. Ectopic overexpression of CF2 in the developing IFM disrupts muscle formation. While our results indicate a role for CF2 as a direct negative regulator of the thin filament protein gene Actin 88F (Act88F), effects on levels of transcripts of myosin heavy chain (mhc) appear to be indirect. This role is in direct contrast to that described in the larval muscles, where CF2 activates structural gene expression. The variation in myofibril phenotypes of CF2 mutants suggest the CF2 may have separate functions in fine-tuning expression of structural genes to insure proper filament stoichiometry, and monitoring and/or controlling the final myofibril size

The quantification of COMT mRNA in post mortem cerebellum tissue: diagnosis, genotype, methylation and expression

BACKGROUND: The COMT gene is located on chromosome 22q11, a region strongly implicated in the aetiology of several psychiatric disorders, in particular schizophrenia. Previous research has suggested that activity and expression of COMT is altered in schizophrenia, and is mediated by one or more polymorphisms within the gene, including the functional Val(158)Met polymorphism. METHOD: In this study we examined the expression levels of COMT mRNA using quantitative RT-PCR in 60 post mortem cerebellum samples derived from individuals with schizophrenia, bipolar disorder, depression, and no history of psychopathology. Furthermore, we have examined the methylation status of two CpG sites in the promoter region of the gene. RESULTS: We found no evidence of altered COMT expression or methylation in any of the psychiatric diagnoses examined. We did, however, find evidence to suggest that genotype is related to COMT gene expression, replicating the findings of two previous studies. Specifically, val(158)met (rs165688; Val allele) rs737865 (G allele) and rs165599 (G allele) all showed reduced expression (P < 0.05). Finally, we observe a strong sexual dimorphism in COMT expression, with females exhibiting significantly greater levels of COMT mRNA. CONCLUSION: The expression of COMT does not appear to be altered in the cerebellum of individuals suffering from schizophrenia, bipolar disorder or depression, but does appear to be influenced by single nucleotide polymorphisms within the gene

Transcription Factor SP4 Is a Susceptibility Gene for Bipolar Disorder

The Sp4 transcription factor plays a critical role for both development and function of mouse hippocampus. Reduced expression of the mouse Sp4 gene results in a variety of behavioral abnormalities relevant to human psychiatric disorders. The human SP4 gene is therefore examined for its association with both bipolar disorder and schizophrenia in European Caucasian and Chinese populations respectively. Out of ten SNPs selected from human SP4 genomic locus, four displayed significant association with bipolar disorder in European Caucasian families (rs12668354, p = 0.022; rs12673091, p = 0.0005; rs3735440, p = 0.019; rs11974306, p = 0.018). To replicate the genetic association, the same set of SNPs was examined in a Chinese bipolar case control sample. Four SNPs displayed significant association (rs40245, p = 0.009; rs12673091, p = 0.002; rs1018954, p = 0.001; rs3735440, p = 0.029), and two of them (rs12673091, rs3735440) were shared with positive SNPs from European Caucasian families. Considering the genetic overlap between bipolar disorder and schizophrenia, we extended our studies in Chinese trios families for schizophrenia. The SNP7 (rs12673091, p = 0.012) also displayed a significant association. The SNP7 (rs12673091) was therefore significantly associated in all three samples, and shared the same susceptibility allele (A) across all three samples. On the other hand, we found a gene dosage effect for mouse Sp4 gene in the modulation of sensorimotor gating, a putative endophenotype for both schizophrenia and bipolar disorder. The deficient sensorimotor gating in Sp4 hypomorphic mice was partially reversed by the administration of dopamine D2 antagonist or mood stabilizers. Both human genetic and mouse pharmacogenetic studies support Sp4 gene as a susceptibility gene for bipolar disorder or schizophrenia. The studies on the role of Sp4 gene in hippocampal development may provide novel insights for the contribution of hippocampal abnormalities in these psychiatric disorders

A Transposon in Comt Generates mRNA Variants and Causes Widespread Expression and Behavioral Differences among Mice

Background: Catechol-O-methyltransferase (COMT) is a key enzyme responsible for the degradation of dopamine and norepinephrine. COMT activity influences cognitive and emotional states in humans and aggression and drug responses in mice. This study identifies the key sequence variant that leads to differences in Comt mRNA and protein levels among mice, and that modulates synaptic function and pharmacological and behavioral traits. Methodology/Principal Findings: We examined Comt expression in multiple tissues in over 100 diverse strains and several genetic crosses. Differences in expression map back to Comt and are generated by a 230 nt insertion of a B2 short interspersed element (B2 SINE) in the proximal 39 UTR of Comt in C57BL/6J. This transposon introduces a premature polyadenylation signal and creates a short 39 UTR isoform. The B2 SINE is shared by a subset of strains, including C57BL/6J

Catechol-O-Methyltransferase (COMT) Val(108/158 )Met polymorphism does not modulate executive function in children with ADHD

BACKGROUND: An association has been observed between the catechol-O-methyltransferase (COMT) gene, the predominant means of catecholamine catabolism within the prefrontal cortex (PFC), and neuropsychological task performance in healthy and schizophrenic adults. Since several of the cognitive functions typically deficient in children with Attention Deficit Hyperactivity Disorder (ADHD) are mediated by prefrontal dopamine (DA) mechanisms, we investigated the relationship between a functional polymorphism of the COMT gene and neuropsychological task performance in these children. METHODS: The Val(108/158 )Met polymorphism of the COMT gene was genotyped in 118 children with ADHD (DSM-IV). The Wisconsin Card Sorting Test (WCST), Tower of London (TOL), and Self-Ordered Pointing Task (SOPT) were employed to evaluate executive functions. Neuropsychological task performance was compared across genotype groups using analysis of variance. RESULTS: ADHD children with the Val/Val, Val/Met and Met/Met genotypes were similar with regard to demographic and clinical characteristics. No genotype effects were observed for WCST standardized perseverative error scores [F(2,97 )= 0.67; p > 0.05], TOL standardized scores [F(2,99 )= 0.97; p > 0.05], and SOPT error scores [F(2,108 )= 0.62; p > 0.05]. CONCLUSIONS: Contrary to the observed association between WCST performance and the Val(108/158 )Met polymorphism of the COMT gene in both healthy and schizophrenic adults, this polymorphism does not appear to modulate executive functions in children with ADHD

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Association Analysis of 94 Candidate Genes and Schizophrenia-Related Endophenotypes

While it is clear that schizophrenia is highly heritable, the genetic basis of this heritability is complex. Human genetic, brain imaging, and model organism studies have met with only modest gains. A complementary research tactic is to evaluate the genetic substrates of quantitative endophenotypes with demonstrated deficits in schizophrenia patients. We used an Illumina custom 1,536-SNP array to interrogate 94 functionally relevant candidate genes for schizophrenia and evaluate association with both the qualitative diagnosis of schizophrenia and quantitative endophenotypes for schizophrenia. Subjects included 219 schizophrenia patients and normal comparison subjects of European ancestry and 76 schizophrenia patients and normal comparison subjects of African ancestry, all ascertained by the UCSD Schizophrenia Research Program. Six neurophysiological and neurocognitive endophenotype test paradigms were assessed: prepulse inhibition (PPI), P50 suppression, the antisaccade oculomotor task, the Letter-Number Span Test, the California Verbal Learning Test-II, and the Wisconsin Card Sorting Test-64 Card Version. These endophenotype test paradigms yielded six primary endophenotypes with prior evidence of heritability and demonstrated schizophrenia-related impairments, as well as eight secondary measures investigated as candidate endophenotypes. Schizophrenia patients showed significant deficits on ten of the endophenotypic measures, replicating prior studies and facilitating genetic analyses of these phenotypes. A total of 38 genes were found to be associated with at least one endophenotypic measure or schizophrenia with an empirical p-value<0.01. Many of these genes have been shown to interact on a molecular level, and eleven genes displayed evidence for pleiotropy, revealing associations with three or more endophenotypic measures. Among these genes were ERBB4 and NRG1, providing further support for a role of these genes in schizophrenia susceptibility. The observation of extensive pleiotropy for some genes and singular associations for others in our data may suggest both converging and independent genetic (and neural) pathways mediating schizophrenia risk and pathogenesis

The Complex Spatio-Temporal Regulation of the Drosophila Myoblast Attractant Gene duf/kirre

A key early player in the regulation of myoblast fusion is the gene dumbfounded (duf, also known as kirre). Duf must be expressed, and function, in founder cells (FCs). A fixed number of FCs are chosen from a pool of equivalent myoblasts and serve to attract fusion-competent myoblasts (FCMs) to fuse with them to form a multinucleate muscle-fibre. The spatial and temporal regulation of duf expression and function are important and play a deciding role in choice of fibre number, location and perhaps size. We have used a combination of bioinformatics and functional enhancer deletion approaches to understand the regulation of duf. By transgenic enhancer-reporter deletion analysis of the duf regulatory region, we found that several distinct enhancer modules regulate duf expression in specific muscle founders of the embryo and the adult. In addition to existing bioinformatics tools, we used a new program for analysis of regulatory sequence, PhyloGibbs-MP, whose development was largely motivated by the requirements of this work. The results complement our deletion analysis by identifying transcription factors whose predicted binding regions match with our deletion constructs. Experimental evidence for the relevance of some of these TF binding sites comes from available ChIP-on-chip from the literature, and from our analysis of localization of myogenic transcription factors with duf enhancer reporter gene expression. Our results demonstrate the complex regulation in each founder cell of a gene that is expressed in all founder cells. They provide evidence for transcriptional control—both activation and repression—as an important player in the regulation of myoblast fusion. The set of enhancer constructs generated will be valuable in identifying novel trans-acting factor-binding sites and chromatin regulation during myoblast fusion in Drosophila. Our results and the bioinformatics tools developed provide a basis for the study of the transcriptional regulation of other complex genes

Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood

Co-regulation of a large and rapidly evolving repertoire of odorant receptor genes

The olfactory system meets niche- and species-specific demands by an accelerated evolution of its odorant receptor repertoires. In this review, we describe evolutionary processes that have shaped olfactory and vomeronasal receptor gene families in vertebrate genomes. We emphasize three important periods in the evolution of the olfactory system evident by comparative genomics: the adaptation to land in amphibian ancestors, the decline of olfaction in primates, and the delineation of putative pheromone receptors concurrent with rodent speciation. The rapid evolution of odorant receptor genes, the sheer size of the repertoire, as well as their wide distribution in the genome, presents a developmental challenge: how are these ever-changing odorant receptor repertoires coordinated within the olfactory system? A central organizing principle in olfaction is the specialization of sensory neurons resulting from each sensory neuron expressing only ~one odorant receptor allele. In this review, we also discuss this mutually exclusive expression of odorant receptor genes. We have considered several models to account for co-regulation of odorant receptor repertoires, as well as discussed a new hypothesis that invokes important epigenetic properties of the system