Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Effect of intermittent hypoxic training on HIF gene expression in human skeletal muscle and leukocytes

Intermittent hypoxic exposure with exercise training is based on the assumption that brief exposure to hypoxia is sufficient to induce beneficial muscular adaptations mediated via hypoxia-inducible transcription factors (HIF). We previously demonstrated (Mounier et al. Med Sci Sports Exerc 38:1410-1417, 2006) that leukocytes respond to hypoxia with a marked inter-individual variability in HIF-1alpha mRNA. This study compared the effects of 3 weeks of intermittent hypoxic training on hif gene expression in both skeletal muscle and leukocytes. Male endurance athletes (n = 19) were divided into an Intermittent Hypoxic Exposure group (IHE) and a Normoxic Training group (NT) with each group following a similar 3-week exercise training program. After training, the amount of HIF-1alpha mRNA in muscle decreased only in IHE group (-24.7%, P < 0.05) whereas it remained unchanged in leukocytes in both groups. The levels of vEGF(121) and vEGF(165) mRNA in skeletal muscle increased significantly after training only in the NT group (+82.5%, P < 0.05 for vEGF(121); +41.2%, P < 0.05 for vEGF(165)). In leukocytes, only the IHE group showed a significant change in vEGF(165) (-28.2%, P < 0.05). The significant decrease in HIF-1alpha mRNA in skeletal muscle after hypoxic training suggests that transcriptional and post-transcriptional regulations of the hif-1alpha gene are different in muscle and leukocytes.

A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing.

AbstractBACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells.

Ewing's sarcoma is a member of Ewing's family tumors (EFTs) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWS gene with the 3' segment of the ETS family gene FLI-1. The EWS-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to EFT development. However, EWS-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are permissive for its putative oncogenic properties have not been discovered, hampering basic understanding of EFT biology. Here, we show that EWS-FLI-1 alone can transform primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of EFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWS-FLI-1 target genes. These observations provide the first identification of candidate primary cells from which EFTs originate and suggest that EWS-FLI-1 expression may constitute the initiating event in EFT pathogenesis.

The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

Lipoxin A4 is a novel estrogen receptor modulator.

Inflammation is intimately linked with naturally occurring remodeling events in the endometrium. Lipoxins comprise a group of short-lived, nonclassic eicosanoids possessing potent anti-inflammatory and proresolution properties. In the present study, we investigated the role of lipoxin A(4) (LXA(4)) in the endometrium and demonstrated that 15-LOX-2, an enzyme necessary for LX biosynthesis, is expressed in this tissue. Our results establish that LXA(4) possesses robust estrogenic activity through its capacity to alter ERE transcriptional activity, as well as expression of estrogen-regulated genes, alkaline phosphatase activity, and proliferation in human endometrial epithelial cells. Interestingly, LXA(4) also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. This estrogenic activity was directly mediated through estrogen receptors (ERs). Subsequent investigations determined that the actions of LXA(4) are exclusively mediated through ERα and closely mimic those of the potent estrogen 17β-estradiol (E2). In binding assays, LXA(4) competed with E2 for ER binding, with an IC(50) of 46 nM. Furthermore, LXA(4) exhibited estrogenic activity in vivo, increasing uterine wet weight and modulating E2-regulated gene expression. These findings reveal a previously unappreciated facet of LXA(4) bioactions, implicating this lipid mediator in novel immunoendocrine crosstalk mechanisms.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Regulated expression of GLUT2 in diabetes studied in transplanted pancreatic beta cells.

GLUT2 disappearance is a marker of the beta cell glucose-unresponsiveness associated with diabetes. Understanding the factor(s) leading to this dysfunction may shed light on pathogenesis of diabetes. Since the regulation of GLUT2 expression in diabetes can so far only be studied in in vivo experiments, we developed a novel experimental approach to study the genetic regulation of GLUT2 in diabetes. By encapsulating islets or cell lines in semi-permeable membranes, these cells can be exposed to the diabetic environment of rats or mice and can be retrieved for analysis of GLUT2 expression and for the change in the secretory response to glucose. Immunocytochemical analysis of transporter expression reveals changes in protein expression while transcriptional analysis of GLUT2 gene expression could be performed in cells transfected with promoter-reporter gene constructs. Using this last approach we hope to be able to characterize the promoter regions involved in the beta cell- and diabetes-specific regulation of GLUT2 expression and possibly to determine which factors are responsible for this regulation.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules.

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation.

An activation-independent role of transcription factors in insulator function.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Genes and Chromosomes of Leishmania infantum

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes