Membrane mu poly(A) signal and 3' flanking sequences function as a transcription terminator for immunoglobulin-encoding genes.

Developmentally regulated mechanisms involving alternative RNA splicing and/or polyadenylation, as well as transcription termination, are implicated in controlling the levels of secreted mu (mu s), membrane mu (mu m) and delta immunoglobulin (Ig) heavy chain mRNAs during B cell differentiation (mu gene encodes the mu heavy chain). Using expression vectors constructed with genomic DNA segments composed of the mu m polyadenylation signal region, we analyzed poly(A) site utilization and termination of transcription in stably transfected myeloma cells and in murine fibroblast L cells. We found that the gene segment containing the mu m poly(A) signals, along with 536 bp of downstream flanking sequence, acted as a transcription terminator in both myeloma cells and L cell fibroblasts. Neither a 141-bp DNA fragment (which directed efficient polyadenylation at the mu m site), nor the 536-bp flanking nucleotide sequence alone, were sufficient to obtain a similar regulation. This shows that the mu m poly(A) region plays a central role in controlling developmentally regulated transcription termination by blocking downstream delta gene expression. Because this gene segment exhibited the same RNA processing and termination activities in fibroblasts, it appears that these processes are not tissue-specific.

A Y-like social chromosome causes alternative colony organization in fire ants.

Intraspecific variability in social organization is common, yet the underlying causes are rarely known. In the fire ant Solenopsis invicta, the existence of two divergent forms of social organization is under the control of a single Mendelian genomic element marked by two variants of an odorant-binding protein gene. Here we characterize the genomic region responsible for this important social polymorphism, and show that it is part of a pair of heteromorphic chromosomes that have many of the key properties of sex chromosomes. The two variants, hereafter referred to as the social B and social b (SB and Sb) chromosomes, are characterized by a large region of approximately 13 megabases (55% of the chromosome) in which recombination is completely suppressed between SB and Sb. Recombination seems to occur normally between the SB chromosomes but not between Sb chromosomes because Sb/Sb individuals are non-viable. Genomic comparisons revealed limited differentiation between SB and Sb, and the vast majority of the 616 genes identified in the non-recombining region are present in the two variants. The lack of recombination over more than half of the two heteromorphic social chromosomes can be explained by at least one large inversion of around 9 megabases, and this absence of recombination has led to the accumulation of deleterious mutations, including repetitive elements in the non-recombining region of Sb compared with the homologous region of SB. Importantly, most of the genes with demonstrated expression differences between individuals of the two social forms reside in the non-recombining region. These findings highlight how genomic rearrangements can maintain divergent adaptive social phenotypes involving many genes acting together by locally limiting recombination.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

Relationship between estrogen receptor alpha location and gene induction reveals the importance of downstream sites and cofactors.

BACKGROUND: To understand cancer-related modifications to transcriptional programs requires detailed knowledge about the activation of signal-transduction pathways and gene expression programs. To investigate the mechanisms of target gene regulation by human estrogen receptor alpha (hERalpha), we combine extensive location and expression datasets with genomic sequence analysis. In particular, we study the influence of patterns of DNA occupancy by hERalpha on expression phenotypes. RESULTS: We find that strong ChIP-chip sites co-localize with strong hERalpha consensus sites and detect nucleotide bias near hERalpha sites. The localization of ChIP-chip sites relative to annotated genes shows that weak sites are enriched near transcription start sites, while stronger sites show no positional bias. Assessing the relationship between binding configurations and expression phenotypes, we find binding sites downstream of the transcription start site (TSS) to be equally good or better predictors of hERalpha-mediated expression as upstream sites. The study of FOX and SP1 cofactor sites near hERalpha ChIP sites shows that induced genes frequently have FOX or SP1 sites. Finally we integrate these multiple datasets to define a high confidence set of primary hERalpha target genes. CONCLUSION: Our results support the model of long-range interactions of hERalpha with the promoter-bound cofactor SP1 residing at the promoter of hERalpha target genes. FOX motifs co-occur with hERalpha motifs along responsive genes. Importantly we show that the spatial arrangement of sites near the start sites and within the full transcript is important in determining response to estrogen signaling.

Silencing of both beta-TrCP1 and HOS (beta-TrCP2) is required to suppress human immunodeficiency virus type 1 Vpu-mediated CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interacts with CD4 within the endoplasmic reticula of infected cells and targets CD4 for degradation through interaction with beta-TrCP1. Mammals possess a homologue of beta-TrCP1, HOS, which is also named beta-TrCP2. We show by coimmunoprecipitation experiments that beta-TrCP2 binds Vpu and is able to induce CD4 down-modulation as efficiently as beta-TrCP1. In two different cell lines, HeLa CD4+ and Jurkat, Vpu-mediated CD4 down-modulation could not be reversed through the individual silencing of endogenous beta-TrCP1 or beta-TrCP2 but instead required the two genes to be silenced simultaneously.

Protective role of early aquaporin 4 induction against postischemic edema formation.

Aquaporin 4 (AQP4) is a water channel involved in water movements across the cell membrane and is spatially organized on the cell surface in orthogonal array particles (OAPs). Its role in edema formation or resolution after stroke onset has been studied mainly at late time points. We have shown recently that its expression is rapidly induced after ischemia coinciding in time with an early swelling of the ischemic hemisphere. There are two isoforms of AQP4: AQP4-M1 and AQP4-M23. The ratio of these isoforms influences the size of the OAPs but the functional impact is not known. The role of the early induction of AQP4 is not yet known. Thrombin preconditioning in mice provides a useful model to study endogenous protective mechanisms. Using this model, we provide evidence for the first time that the early induction of AQP4 may contribute to limit the formation of edema and that the AQP4-M1 isoform is predominantly induced in the ischemic tissue at this time point. Although it prevents edema formation, the early induction of the AQP4 expression does not prevent the blood-brain barrier disruption, suggesting an effect limited to the prevention of edema formation possibly by removing of water from the tissue.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Disturbed local auxin homeostasis enhances cellular anisotropy and reveals alternative wiring of auxin-ethylene crosstalk in Brachypodium distachyon seminal roots.

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2l(hypo) ). Counterintuitive however, Bdtar2l(hypo) mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2l(hypo) root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase.

The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

PPARs: transcriptional effectors of fatty acids and their derivatives.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. These receptors stimulate transcription after activation by their cognate ligand and binding to the promoter of target genes. In this review, we discuss how fatty acids affect PPAR functions in the cell. We first describe the structural features of the ligand binding domains of PPARs, as defined by crystallographic analyses. We then present the ligand-binding characteristics of each of the three PPARs (alpha, beta/delta, gamma) and relate ligand activation to various cellular processes: (i) fatty acid catabolism and modulation of the inflammatory response for PPARalpha, (ii) embryo implantation, cell proliferation and apoptosis for PPARbeta, and (iii) adipocytic differentiation, monocytic differentiation and cell cycle withdrawal for PPARgamma. Finally, we present possible cross-talk between the PPAR pathway and different endocrine routes within the cell, including the thyroid hormone and retinoid pathways.

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation.

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that in Arabidopsis thaliana, two paralogous auxin response transcription factors, ARF6 and ARF8, regulate both stamen and gynoecium maturation. arf6 arf8 double-null mutant flowers arrested as infertile closed buds with short petals, short stamen filaments, undehisced anthers that did not release pollen and immature gynoecia. Numerous developmentally regulated genes failed to be induced. ARF6 and ARF8 thus coordinate the transition from immature to mature fertile flowers. Jasmonic acid (JA) measurements and JA feeding experiments showed that decreased jasmonate production caused the block in pollen release, but not the gynoecium arrest. The double mutant had altered auxin responsive gene expression. However, whole flower auxin levels did not change during flower maturation, suggesting that auxin might regulate flower maturation only under specific environmental conditions, or in localized organs or tissues of flowers. arf6 and arf8 single mutants and sesquimutants (homozygous for one mutation and heterozygous for the other) had delayed stamen development and decreased fecundity, indicating that ARF6 and ARF8 gene dosage affects timing of flower maturation quantitatively.