Hepatic gene expression variations in response to high-fat diet-induced impaired glucose tolerance using RNAseq analysis in collaborative cross mouse population.

Hepatic gene expression is known to differ between healthy and type 2 diabetes conditions. Identifying these variations will provide better knowledge to the development of gene-targeted therapies. The aim of this study is to assess diet-induced hepatic gene expression of susceptible versus resistant CC lines to T2D development. Next-generation RNA-sequencing was performed for 84 livers of diabetic and non-diabetic mice of 41 different CC lines (both sexes) following 12 weeks on high-fat diet (42% fat). Data analysis revealed significant variations of hepatic gene expression in diabetic versus non-diabetic mice with significant sex effect, where 601 genes were differentially expressed (DE) in overall population (males and females), 718 genes in female mice, and 599 genes in male mice. Top prioritized DE candidate genes were Lepr, Ins2, Mb, Ckm, Mrap2, and Ckmt2 for the overall population; for females-only group were Hdc, Serpina12, Socs1, Socs2, and Mb, while for males-only group were Serpine1, Mb, Ren1, Slc4a1, and Atp2a1. Data analysis for sex differences revealed 193 DE genes in health (Top: Lepr, Cav1, Socs2, Abcg2, and Col5a3), and 389 genes DE between diabetic females versus males (Top: Lepr, Clps, Ins2, Cav1, and Mrap2). Furthermore, integrating gene expression results with previously published QTL, we identified significant variants mapped at chromosomes at positions 36-49 Mb, 62-71 Mb, and 79-99 Mb, on chromosomes 9, 11, and 12, respectively. Our findings emphasize the complexity of T2D development and that significantly controlled by host complex genetic factors. As well, we demonstrate the significant sex differences between males and females during health and increasing to extent levels during disease/diabetes. Altogether, opening the venue for further studies targets the discovery of effective sex-specific and personalized preventions and therapies.

Effect of Linearization in a WNT Signaling Model.

A nonlinear model consisting of a system of coupled ordinary differential equations (ODE), describing a biological process linked with cancer development, is linearized using Taylor series and tested against different magnitudes of input perturbations, in order to investigate the extent to which the linearization is accurate. The canonical wingless/integrated (WNT) signaling pathway is considered. The linearization procedure is described, and special considerations for linearization validity are analyzed. The analytical properties of nonlinear and linearized systems are studied, including aspects such as existence of steady state and initial value sensitivity. Linearization is a useful tool for speeding up drug response computations or for providing analytical answers to problems such as required drug concentrations. A Monte Carlo-based error testing workflow is employed to study the errors introduced by the linearization for different input conditions and parameter vectors. The deviations between the nonlinear and the linearized system were found to increase in a polynomial fashion w.r.t. the magnitude of tested perturbations. The linearized system closely followed the original one for perturbations of magnitude within 10% of the base input vector which yielded the state-space fixed point used for the linearization.

The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response.

The epigenetic sensor BRD4 (bromodomain protein 4) is a potent target for anti-cancer therapies. To study the transcriptional impact of BRD4 in cancer, we generated an expression signature of BRD4 knockdown cells and found oxidative stress response genes significantly enriched. We integrated the RNA-Seq results with DNA-binding sites of BRD4 generated by chromatin immunoprecipitations, correlated these with gene expressions from human prostate cancers and identified 21 top BRD4 candidate genes among which the oxidative stress pathway genes KEAP1, SESN3 and HDAC6 are represented. Knock down of BRD4 or treatment with the BRD4 inhibitor JQ1 resulted in decreased reactive oxygen species (ROS) production and increased cell viability under H2O2 exposure. Consistently, a deregulation of BRD4 diminished the KEAP1/NRF2 axis and led to a disturbed regulation of the inducible heme oxygenase 1 (HMOX1). Without exogenous stress induction, we also found BRD4 directly targeting the HMOX1 promoter over the SP1-binding sites. Our findings provide insight into the transcriptional regulatory network of BRD4 and highlight BRD4 as signal transducer of the cellular response to oxidative stress.

Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced interpatient heterogeneity. To characterize RA at the molecular level and to uncover pathomechanisms, we performed genome-wide gene expression analysis. We identified a set of 1,054 genes significantly deregulated in pair-wise comparisons between RA and osteoarthritis (OA) patients, RA and normal donors (ND), or OA and ND. Correlation analysis revealed gene sets regulated identically in all three groups. As a prominent example secreted phosphoprotein 1 (SPP1) was identified to be significantly upregulated in RA compared with both OA and ND. SPP1 expression was found to correlate with genes expressed during an inflammatory response, T-cell activation and apoptosis, suggesting common underlying regulatory networks. A subclassification of RA patients was achieved on the basis of proteoglycan 4 (PRG4) expression, distinguishing PRG4 high and low expressors and reflecting the heterogeneity of the disease. In addition, we found that low PRG4 expression was associated with a more aggressive disease stage, which is in accordance with PRG4 loss-of-function mutations causing camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Altogether we provide evidence for molecular signatures of RA and RA subclasses, sets of new candidate genes as well as for candidate gene networks, which extend our understanding of disease mechanisms and may lead to an improved diagnosis.

Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo.

Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.

Stemming cancer: functional genomics of cancer stem cells in solid tumors.

Cancer stem cells (CSCs) were discovered about 15 years ago in hematopoietic cancers. Subsequently, cancer stem cells were discovered in various solid tumors. Based on parallels with normal stem cells, a developmental process of cancer stem cells follows paths of organized, hierarchical structure of cells with different degrees of maturity. While some investigators have reported particular markers as identification of cancer stem cells, these markers require further research. In this review, we focus on the functional genomics of cancer stem cells. Functional genomics provides useful information on the signaling pathways which are consecutively activated or inactivated amongst those cells. This information is of particular importance for cancer research and clinical treatment in many respects. (1) Understanding of self-renewal mechanisms crucial to tumor growth. (2) Allow the identification of new, more specific marker for CSCs, and (3) pathways that are suitable as future targets for anti-cancer drugs. This is of particular importance, because today's chemotherapy targets the proliferating cancer cells sparing the relatively slow dividing cancer stem cells. The first step on this long road therefore is to analyze genome-wide expression-profiles within the same type of cancer and then between different types of cancer, encircling those target genes and pathways, which are specific to these cells.

Molecular characterization of signalling pathways in cancer stem cells.

To avoid artefacts introduced by culturing cells for extended periods of time, it is crucial to use low-passage patient-derived tumour cells. The ability to enrich, isolate and assay sub-populations of cells that behave as cancer stem cells (CSCs) from these primary cell lines is essential before performing characterizations such as gene-expression profiling. We have isolated cells from glioblastomas which show characteristics of CSCs. Although glioblastomas contain only a relatively small amount of putative CSCs, these cells express many genes which seem to be worthy targets for future therapies.

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

High-throughput subcellular protein localization using cell arrays.

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

Establishment of T cell-specific and natural killer cell-specific unigene sets: towards high-throughput genomics of leukaemia.

We report the establishment of highly non-redundant unigene sets consisting of cDNA clones derived from T lymphocytes and natural killer cells. Each set consists of 10 506 and 13 409 clones, respectively, arrayed on nylon membranes in duplicate. The sets provide an excellent tool for genome-wide gene expression analysis studies in immunology research.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

Statistical evaluation of differential expression on cDNA nylon arrays with replicated experiments.

In this paper we focus on the detection of differentially expressed genes according to changes in hybridization signals using statistical tests. These tests were applied to 14 208 zebrafish cDNA clones that were immobilized on a nylon support and hybridized with radioactively labeled target mRNA from wild-type and lithium-treated zebrafish embryos. The methods were evaluated with respect to 16 control clones that correspond to eight different genes which are known to be involved in dorso-ventral axis specification. Moreover, 4608 Arabidopsis thaliana clones on the same array were used to judge statistical significance of expression changes and to control the false positive rates of the test decisions. Utilizing this special array design we show that differential expression of a high proportion of cDNA clones (15/16) and the respective genes (7/8) were identified, with a false positive error of <5% using the constant control data. Furthermore, we investigated the influence of the number of repetitions of experiments on the accuracy of the procedures with experimental and simulated data. Our results suggest that the detection of differential expression with repeated hybridization experiments is an accurate and sensitive way of identifying even small expression changes (1:1.5) of a large number of genes in parallel.

Advanced integrated mouse YAC map including BAC framework.

Functional characterization of the mouse genome requires the availability of a comprehensive physical map to obtain molecular access to chromosomal regions of interest. Positional cloning remains a crucial way of linking phenotype with particular genes. A key step and frequent stumbling block in positional cloning is making a contig of a genetically defined candidate region. The most efficient first step is isolating YAC (Yeast Artificial Chromosome) clones. A robust, detailed YAC contig map is thus an important tool. Employing Interspersed Repetitive Sequence (IRS)-PCR genomics, we have generated an advanced second-generation YAC contig map of the mouse genome that doubles both the depth of clones and the density of markers available. In addition to the primarily YAC-based map, we located 1942 BAC (Bacterial Artificial Chromosome) clones. This allows us to present for the first time a dense framework of BACs spanning the genome of the mouse, which, for instance, can serve as a nucleus for genomic sequencing. Four large-insert mouse YAC libraries from three different strains are included in our data, and our analysis incorporates the data of Hunter et al. and Nusbaum et al. There is a total of 20,205 markers on the final map, 12,033 from our own data, and a total of 56,093 YACs, of which 44,401 are positive for more than one marker.

Identification of nodal signaling targets by array analysis of induced complex probes.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals.

UniGene cDNA array-based monitoring of transcriptome changes during mouse placental development.

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and in situ hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive in situ hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.

ACRC codes for a novel nuclear protein with unusual acidic repeat tract and maps to DYT3 (dystonia parkinsonism) critical interval in xq13.1.

We searched for novel genes as candidates of X-linked dystonia parkinsonism (XDP) in the critical interval of Xq13.1 that harbors the disease locus (DYT3). A gene, ACRC (acidic repeat containing), was discovered by a combination of in silico and "wet" experiments. ACRC is composed of at least 12 exons and 11 introns. It is expressed in all tissues tested, including skeletal muscle, liver, kidney, pancreas, heart, lung, and brain. Highest levels of expression are found in skeletal muscle. The ACRC protein is characterized by a previously undescribed acidic repeat tract of 21 units of 8-10 amino acids. The N-terminal portion of the protein is highly acidic (pI=3.2), and the C-terminal region is basic (pI=10.2). There are nuclear localization signals in its C-terminal portion. Extensive mutation analysis of the transcribed region of the gene, including intron-exon boundaries and the 5' and 3' untranslated intervals, did not reveal a mutation in XDP patients. Exclusion of a mutation in the transcribed portion of this and all other known genes within the DYT3 critical interval suggests that XDP is most likely caused by a mutation in a regulatory region of a gene within the critical interval, or by a structural rearrangement.

A human cDNA expression library in yeast enriched for open reading frames.

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.