A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan

Arabinoxylans (AXs) are major components of graminaceous plant cell walls, including those in the grain and straw of economically important cereals. Despite some recent advances in identifying the genes encoding biosynthetic enzymes for a number of other plant cell wall polysaccharides, the genes encoding enzymes of the final stages of AX synthesis have not been identified. We have therefore adopted a novel bioinformatics approach based on estimation of differential expression of orthologous genes between taxonomic divisions of species. Over 3 million public domain cereal and dicot expressed sequence tags were mapped onto the complete sets of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) genes, respectively. It was assumed that genes in cereals involved in AX biosynthesis would be expressed at high levels and that their orthologs in dicotyledonous plants would be expressed at much lower levels. Considering all rice genes encoding putative glycosyl transferases (GTs) predicted to be integral membrane proteins, genes in the GT43, GT47, and GT61 families emerged as much the strongest candidates. When the search was widened to all other rice or Arabidopsis genes predicted to encode integral membrane proteins, cereal genes in Pfam family PF02458 emerged as candidates for the feruloylation of AX. Our analysis, known activities, and recent findings elsewhere are most consistent with genes in the GT43 families encoding beta-1,4-xylan synthases, genes in the GT47 family encoding xylan alpha-1,2- or alpha-1,3-arabinosyl transferases, and genes in the GT61 family encoding feruloyl-AX beta-1,2-xylosyl transferases.     

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.     

Computational and experimental analysis identifies many novel human genes

Because of advances in automation, human genomic sequences are being deposited in public databases at a dramatic rate. However, the process of detecting genes in these sequences is still something of an art. Here we describe the implementation and testing of a relatively straightforward computational approach, the Virtual Transcribed Sequence project, which analyzes their gene content using the gene prediction program GENSCAN (GENSCAN 1.0 1,2) in combination with similarity-based methods. This approach identifies many novel human genes not found even in EST databases.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5⁺ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Cochlear supporting cells are glia-like cells essential for hearing. Genetic ablation of Lgr5+ supporting cells reveals a population of damage inducible, mitotically activated progenitors in the greater epithelial ridge, which can divide and migrate into the organ of Corti to replenish ablated supporting cells. Translatomic analyses provide insights into the genes that may regulate this regenerative potential.     

A pan-cancer transcriptome analysis of exitron splicing identifies novel cancer driver genes and neoepitopes

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Microarray analysis of genes differentially expressed in the liver of lean and fat chickens

Excessive accumulation of lipids in the adipose tissue is one of the main problems faced by the broiler industry nowadays. In chicken, lipogenesis occurs essentially in the liver, in which much of the triglycerides that accumulate in avian adipose tissue are synthesized. In order to better understand the gene expression and its regulation in chicken liver, the gene expression profiles of liver at developmental stages of chicken (1 week, 4 weeks and 7 weeks of age) were investigated and differentially expressed genes between lean and fat chicken lines divergently selected for abdominal fat content for eight generations were screened. Our data indicated that 4 weeks of age was a very important stage on chicken liver lipogenesis compared to 1 week and 7 weeks of age, and the glycometabolism in chicken liver could be related to lipid metabolism and the difference of glycometabolism could be another potential reason for the fat and lean phenotype occurrence besides the difference of lipogenesis in chicken liver. Our result have established groundwork for further study of the basic genetic control of chicken obesity and will benefit chicken research communities as well as researches that use chicken as a model organism for developmental biology and human therapeutics.     

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.     

Regulation of lypolysis in white adipose tissues of lean and obese Zucker rats

Obese Zucker rat is often used as a model of genetic obesity to understand the mechanism of the development of obesity. In the present work, in order to better understand the regulation of lipolysis in the Zucker rat, the lipolytic activities of adipocytes isolated from different adipose depots of lean and obese Zucker rats, in the basal state or after catecholamine stimulation have been measured. The obese Zucker rat presents hyperinsulinemia without hyperglycemia and with elevated plasma free fatty acids, suggesting a dyslipidemia. Morphological studies of three adipose deposits show a marked hypertrophic and hyperplastic type of obesity, much pronounced in the subcutaneous depot. In the current study we show that the basal lipolytic rate is higher in adipocytes from each deposit of obese rats (when results are corrected for cell surface area). This finding, associated with the increase of all deposits, could contribute to the elevated plasma FFA observed. Investigation of the responsiveness of dibutyril cAMP (DBcAMP) points out that the defect in the NE responsiveness is essentially located at post-receptor level. Nevertheless, a receptor defect could not be excluded as suggested by a decrease of the beta-ARs observed in all deposits. Our study points out that the lipolytic resistance to catecholamines in adipose tissue of obese Zucker rats appears to counteract the increase in the lipolytic rate, in order to moderate the increase in plasma FFA levels that may contribute to the hyperinsulinemia observed, characteristic of an insulino-resistant state.     

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.