Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRζ or CD3ɛ chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice

The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death

The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis.

Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor α, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon γ (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-κB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor α, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-κB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Regulation of c-maf gene expression by Pax6 in cultured cells

c-Maf is a bZip transcription factor expressed in developmental and cellular differentiation processes. Recently, a c-maf knockout mouse model, showing abnormal lens development, has been reported. In order to study the regulation mechanisms of c-maf gene expression during the differentiation process we have cloned and functionally characterized the rat c-maf (maf-2) gene. The rat c-maf gene is an intronless gene, covering a length of 3.5 kb. Transient transfection analysis of the 5′-flanking region of the c-maf gene using luciferase as the reporter gene shows that Pax6, a master transcription factor for lens development, strongly activates the c-maf promoter construct. Endogenous c-maf is also activated by the Pax6 expression vector. Electrophoresis mobility shift assay and DNase I footprinting analysis show that at least three Pax6-binding sites are located in the 5′-flanking and 5′-non-coding regions of the rat c-maf gene. The c-maf gene was also markedly activated by its own product, c-Maf, through the MARE (Maf recognition element), suggesting that a positive autoregulatory mechanism controls this gene. In situ hybridization histochemical detection of Pax6 and c-Maf in the E14 lens showed that both mRNAs are expressed in the lens equator where lens epithelial cells are differentiating to lens fiber cells. These results suggest that a Pax6/c-Maf transcription factor cascade is working in lens development.

Triplex forming oligonucleotide targeted to 3′UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3′UTR together with a TFO targeted to the 3′UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3′UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3′UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2−/− mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2−/− mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2−/− mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.

Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAs. Twelve semi-randomised RNA–DNA–RNA chimeric oligonucleotide probes, known as ‘gapmers’, were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37°C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.