Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes.

Inter-individual differences in gene expression are likely to account for an important fraction of phenotypic differences, including susceptibility to common disorders. Recent studies have shown extensive variation in gene expression levels in humans and other organisms, and that a fraction of this variation is under genetic control. We investigated the patterns of gene expression variation in a 25 Mb region of human chromosome 21, which has been associated with many Down syndrome (DS) phenotypes. Taqman real-time PCR was used to measure expression variation of 41 genes in lymphoblastoid cells of 40 unrelated individuals. For 25 genes found to be differentially expressed, additional analysis was performed in 10 CEPH families to determine heritabilities and map loci harboring regulatory variation. Seventy-six percent of the differentially expressed genes had significant heritabilities, and genomewide linkage analysis led to the identification of significant eQTLs for nine genes. Most eQTLs were in trans, with the best result (P=7.46 x 10(-8)) obtained for TMEM1 on chromosome 12q24.33. A cis-eQTL identified for CCT8 was validated by performing an association study in 60 individuals from the HapMap project. SNP rs965951 located within CCT8 was found to be significantly associated with its expression levels (P=2.5 x 10(-5)) confirming cis-regulatory variation. The results of our study provide a representative view of expression variation of chromosome 21 genes, identify loci involved in their regulation and suggest that genes, for which expression differences are significantly larger than 1.5-fold in control samples, are unlikely to be involved in DS-phenotypes present in all affected individuals.

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

Identification of corticosteroid-regulated genes in cardiomyocytes by serial analysis of gene expression.

Corticosteroids (aldosterone, cortisol/corticosterone) exert direct functional effects on cardiomyocytes. However, gene networks activated by corticosteroids in cardiomyocytes, as well as the involvement of the mineralocorticoid receptor (MR) vs the glucocorticoid receptor (GR) in these effects, remain largely unknown. Here we characterized the corticosteroid-dependent transcriptome in primary culture of neonatal mouse cardiomyocytes treated with 10(-6) M aldosterone, a concentration predicted to occupy both MR and GR. Serial analysis of gene expression revealed 101 aldosterone-regulated genes. The MR/GR specificity was characterized for one regulated transcript, namely ecto-ADP-ribosyltransferase-3 (Art3). Using cardiomyocytes from GR(null/null) or MR(null/null) mice we demonstrate that in GR(null/null) cardiomyocytes the response is abrogated, but it is fully maintained in MR(null/null) cardiomyocytes. We conclude that Art3 expression is regulated exclusively via the GR. Our study identifies a new set of corticosteroid-regulated genes in cardiomyocytes and demonstrates a new approach to studying the selectivity of MR- vs GR-dependent effects.

Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis.

Background: Sulfate and phosphate are both vital macronutrients required for plant growth and development. Despite evidence for interaction between sulfate and phosphate homeostasis, no transcriptional factor has yet been identified in higher plants that affects, at the gene expression and physiological levels, the response to both elements. This work was aimed at examining whether PHR1, a transcription factor previously shown to participate in the regulation of genes involved in phosphate homeostasis, also contributed to the regulation and activity of genes involved in sulfate inter-organ transport. Results: Among the genes implicated in sulfate transport in Arabidopsis thaliana, SULTR1;3 and SULTR3;4 showed up-regulation of transcripts in plants grown under phosphate-deficient conditions. The promoter of SULTR1;3 contains a motif that is potentially recognizable by PHR1. Using the phr1 mutant, we showed that SULTR1;3 up regulation following phosphate deficiency was dependent on PHR1. Furthermore, transcript up regulation was found in phosphate-deficient shoots of the phr1 mutant for SULTR2;1 and SULTR3;4, indicating that PHR1 played both a positive and negative role on the expression of genes encoding sulfate transporters. Importantly, both phr1 and sultr1;3 mutants displayed a reduction in their sulfate shoot-to-root transfer capacity compared to wild-type plants under phosphate-deficient conditions. Conclusions: This study reveals that PHR1 plays an important role in sulfate inter-organ transport, in particular on the regulation of the SULTR1;3 gene and its impact on shoot-to-root sulfate transport in phosphate-deficient plants. PHR1 thus contributes to the homeostasis of both sulfate and phosphate in plants under phosphate deficiency. Such a function is also conserved in Chlamydomonas reinhardtii via the PHR1 ortholog PSR1.

Steroid hormone pulsing drives cyclic gene expression.

Transcriptional cycling of activated glucocorticoid receptor (GR) and ultradian glucocorticoid secretion are well established processes. Ultradian hormone release is now shown to result in pulsatile gene transcription through dynamic exchange of GR with the target-gene promoter and GR cycling through the chaperone machinery.

Plant defense in the absence of jasmonic acid: the role of cyclopentenones.

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

Gene expression profiling in breast cancer: understanding the molecular basis of histologic grade to improve prognosis.

BACKGROUND: Histologic grade in breast cancer provides clinically important prognostic information. However, 30%-60% of tumors are classified as histologic grade 2. This grade is associated with an intermediate risk of recurrence and is thus not informative for clinical decision making. We examined whether histologic grade was associated with gene expression profiles of breast cancers and whether such profiles could be used to improve histologic grading. METHODS: We analyzed microarray data from 189 invasive breast carcinomas and from three published gene expression datasets from breast carcinomas. We identified differentially expressed genes in a training set of 64 estrogen receptor (ER)-positive tumor samples by comparing expression profiles between histologic grade 3 tumors and histologic grade 1 tumors and used the expression of these genes to define the gene expression grade index. Data from 597 independent tumors were used to evaluate the association between relapse-free survival and the gene expression grade index in a Kaplan-Meier analysis. All statistical tests were two-sided. RESULTS: We identified 97 genes in our training set that were associated with histologic grade; most of these genes were involved in cell cycle regulation and proliferation. In validation datasets, the gene expression grade index was strongly associated with histologic grade 1 and 3 status; however, among histologic grade 2 tumors, the index spanned the values for histologic grade 1-3 tumors. Among patients with histologic grade 2 tumors, a high gene expression grade index was associated with a higher risk of recurrence than a low gene expression grade index (hazard ratio = 3.61, 95% confidence interval = 2.25 to 5.78; P < .001, log-rank test). CONCLUSIONS: Gene expression grade index appeared to reclassify patients with histologic grade 2 tumors into two groups with high versus low risks of recurrence. This approach may improve the accuracy of tumor grading and thus its prognostic value.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Two waves of recombinase gene expression in developing thymocytes.

During T cell development in the thymus, T cell receptor (TCR) alpha, beta, gamma, and delta genes are rearranged and expressed. TCR rearrangement strictly depends upon the coordinate activity of two recombinase activating genes, Rag-1 and Rag-2. In this study we have followed the expression of these genes at different stages of intrathymic development. The results indicate that there are two periods of high Rag-1 and Rag-2 mRNA expression. The first wave peaks early at the CD25+CD4-CD8-CD3- stage of development and coincides with the initial appearance of transcripts derived from fully rearranged TCR beta, gamma, and delta genes, whereas the second wave occurs later at the CD4+CD8+ stage coincident with full-length TCR alpha mRNA expression. Active downregulation of Rag-1 and Rag-2 mRNA expression appears to occur in vivo between the two peaks of recombinase activity. This phenomenon can be mimicked in vitro in response to artificial stimuli such as phorbol myristate acetate and calcium ionophore. Collectively our data suggest that recombinase expression is actively regulated during early thymus development independently of cell surface expression of a mature heterodimeric TCR protein complex.

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance.

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5' regulatory sequence variation in the corresponding genes is indeed increased. However, approximately 42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a &gt;4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.

Genomic organization and gene expression in a chromosomal region of Leishmania major.

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.