Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

Survey of the irp2 gene among Yersinia pestis strains isolated during several plague outbreaks in northeast Brazil

The irp2 gene codes for a 190 kDa protein (HMWP2) synthesidez when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil wasstudied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to bepositive with the A13 probe, indicating that the locus was lost after subculturein vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcorRV or AvaII, indicatingthat the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host.

Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Isolation of a distally located gene possibly correlated with gametocyte production ability

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

Attempted isolation of the gene encoding the 21 Kd Plasmodium berghei ookinete transmission blocking antigen from Plasmodium yoelli and Plasmodium vivax

The 21kD ookinete antigen of Plasmodium berghei (Pbs 21) has been shown to elicit an effective and long lasting transmission blocking immune response in mice. Having cloned and sequenced this antigen (Paton et al. 1993) the sequence was compared to the genes of the same family previously identified in P. falciparum, P. gallinaceum (Kaslow et al. 1989) and P. reichenowi (Lal et al. 1990). Four conserved areas were identified in this comparison, to which degenerate oligonucleotides were designed. PCR amplification and screening of genomic libraries was then carried out using these oligonucleotides. The P. yoelii gene was successfully cloned and a number of novel P. vivax genes identified but the P. vivax homologue of Pbs21 remains elusive.

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression.

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.

Mutation in the gene for connexin 30.3 in a family with erythrokeratodermia variabilis.

Erythrokeratodermia variabilis (EKV) is an autosomal dominant keratinization disorder characterized by migratory erythematous lesions and fixed keratotic plaques. All families with EKV show mapping to chromosome 1p34-p35, and mutations in the gene for connexin 31 (Cx31) have been reported in some but not all families. We studied eight affected and three healthy subjects in an Israeli family, of Kurdish origin, with EKV. After having mapped the disorder to chromosome 1p34-p35, we found no mutations in the genes for Cx31, Cx31.1, and Cx37. Further investigation revealed a heterozygous T-->C transition leading to the missense mutation (F137L) in the human gene for Cx30.3 that colocalizes on chromosome 1p34-p35. This nucleotide change cosegregated with the disease and was not found in 200 alleles from normal individuals. This mutation concerns a highly conserved phenylalanine, in the third transmembrane region of the Cx30.3 molecule, known to be implicated in the wall formation of the gap-junction pore. Our results show that mutations in the gene for Cx30.3 can be causally involved in EKV and point to genetic heterogeneity of this disorder. Furthermore, we suggest that our family presents a new type of EKV because of the hitherto unreported association with erythema gyratum repens.

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.

Autosomal-Recessive Posterior Microphthalmos Is Caused by Mutations in PRSS56, a Gene Encoding a Trypsin-Like Serine Protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

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.