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At least 649 records · Page 36Linked to original sources
PubMed2004

Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.

PURPOSE: Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells. METHODS: Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-beta 1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA). RESULTS: In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of alpha-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins. CONCLUSIONS: ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-beta 1-induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.

Actins↗
PubMed1981

Effects of inhibition of RBC HCO3-/Cl- exchange on CO2 excretion and downstream pH disequilibrium in isolated rat lungs.

To determine the effects of the speed of the erythrocyte membrane chloride shift on pulmonary gas transfer, CO2 exchange and the kinetics of pH equilibration were measured with isolated rat lungs perfused with 20% suspensions of human erythrocytes. The lungs were ventilated with room air, and the inflowing perfusate blood gases were similar to those in mixed venous blood in vivo. All experiments were performed at 37 degrees C. Rates of CO2 excretion were determined by measuring the fraction of CO2 in mixed expired gas in a steady state. The time-course of extracellular pH equilibration in the effluent perfusate was measured in a downstream stopflow pH electrode apparatus. CO2 excretion was reduced by approximately 16% when the lungs were perfused with suspensions containing erythrocytes whose HCO-3/Cl- exchange rates was inhibited, compared with CO2 excretion when the lungs were perfused with normal erythrocyte suspensions. A fall of 0.06 in effluent perfusate extracellular pH was noted during perfusion with inhibited erythrocyte suspensions, in contrast to no observable downstream pH change during perfusion with normal erythrocyte suspensions. These results are in close agreement with the predictions of a theoretical model. Our observations suggest that CO2 transfer in capillary beds will be adversely affected in vivo when the rate of the erythrocyte HCO-3/Cl- exchange is abnormally low. Since a number of commonly used drugs are known to inhibit the chloride shift in human erythrocytes, these findings may have important clinical implications, especially in patients with impaired lung function.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Adenyl cyclase and interleukin 6 are downstream effectors of parathyroid hormone resulting in stimulation of bone resorption.

Parathyroid hormone and other bone resorptive agents function, at least in part, by inducing osteoblasts to secrete cytokines that stimulate both differentiation and resorptive activity of osteoclasts. We previously identified two potentially important cytokines by demonstrating that parathyroid hormone induces expression by osteoblasts of IL-6 and leukemia inhibitory factor without affecting levels of 14 other cytokines. Although parathyroid hormone activates multiple signal transduction pathways, induction of IL-6 and leukemia inhibitory factor is dependent on activation of adenyl cyclase. This study demonstrates that adenyl cyclase is also required for stimulation of osteoclast activity in cultures containing osteoclasts from rat long bones and UMR106-01 rat osteoblast-like osteosarcoma cells. Since the stimulation by parathyroid hormone of both cytokine production and bone resorption depends on the same signal transduction pathway, we hypothesized that IL-6 might be a downstream effector of parathyroid hormone. We found that addition of exogenous IL-6 mimics the ability of parathyroid hormone to stimulate bone resorption. More importantly, an antibody directed against the IL-6 receptor blocks moderate stimulation of osteoclast activity induced by the hormone. Interestingly, strong stimulation of resorption overcomes this dependence on IL-6. Thus, parathyroid hormone likely induces multiple, redundant cytokines that can overcome the IL-6 requirement associated with moderate stimulation. Taken together with studies showing that many other bone resorptive agents also stimulate IL-6 production, our results suggest that IL-6 may be a downstream effector of these agents as well as of parathyroid hormone.

Adenylyl Cyclases↗
PubMed2026

Daylight, Daily Rhythms, and Downstream Physiology: A Translational Study in Diurnal Nile Grass Rats (Arvicanthis niloticus).

The circadian system evolved under natural light-dark cycles, while modern humans spend much of their time indoors under electric lighting that differs substantially from daylight in intensity, spectral composition, and temporal structure. How such lighting environments influence the circadian system has not been systematically examined in a diurnal animal model under ecologically relevant conditions. In this study, we used the diurnal Nile grass rat (Arvicanthis niloticus) to assess daily locomotor rhythms across 4 lighting conditions designed to approximate common human exposure scenarios: rectangular daylight (D65‑R; ~5600 lux), semi‑sigmoidal daylight mimicking natural intensity dynamics (D65‑S; matched peak intensity with ~50% lower cumulative energy), fluorescent indoor light (F12; ~150 lux), and fluorescent light supplemented with a 1‑h midday daylight pulse (F12 + D65‑P). Using a within‑subject design (n = 8), male grass rats were housed under each condition for 2 weeks. D65‑R produced the highest daytime activity levels and the strongest day-night activity ratio, consistent with robust circadian entrainment. Despite matching peak intensity, D65‑S did not yield comparable circadian outcomes, indicating that cumulative photon exposure, rather than peak intensity alone, contributes to entrainment strength. Notably, the addition of a 1‑h midday daylight pulse (D65-P) partially increased circadian amplitude under otherwise fluorescent conditions, with higher periodogram amplitude relative to F12 alone. A separate cohort of males was exposed to D65-R or F12 for 6 weeks (n = 10/condition) to assess physiological outcomes, including metabolic and reproductive measures. Compared with the D65-R group, the F12 group showed a higher diabetic rate (10% vs. 40%) and reduced sperm motility (45 ± 6 vs. 19 ± 1%), consistent with potential downstream correlates of circadian rhythm disruption. Together, these findings demonstrate that lighting conditions characteristic of indoor environments produce weaker circadian organization than daylight-equivalent lighting in a diurnal rodent, which underscore the importance of light quality in shaping circadian rhythms and downstream physiological processes.

daylight↗
PubMed2005

Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells.

We tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N'-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 microM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.

Aniline Compounds↗
PubMed2005

Differential involvement of PU.1 and Id2 downstream of TGF-beta1 during Langerhans-cell commitment.

Langerhans cells (LCs) are highly abundant dendritic cells (DCs) in epidermal and mucosal tissues. The transcription factors PU.1 and Id2 have been implicated as positive regulators of LC development from hematopoietic progenitor cells. LC differentiation from progenitors is absolutely dependent on transforming growth factor beta 1 (TGF-beta1) in vitro as well as in vivo; however, downstream mechanisms are poorly defined. We found that both PU.1 and Id2 are induced by TGF-beta1 in human CD34+ monocyte/LC (M/LC) progenitor cells, and that neither ectopic PU.1 or Id2 alone, nor both together, could replace TGF-beta1 in its instructive function on LC commitment. However, both factors critically contributed to LC differentiation by acting at 2 distinct intersection points. Ectopic PU.1 strongly enhanced TGF-beta1-dependent LC development. Additionally, Notch-induced generation of interstitial-type DCs was associated with PU.1 up-regulation. Thus, PU.1 is generally increased during myeloid DC development. Ectopic Id2 inhibits the acquisition of early monocytic characteristics by cells generated in the absence of TGF-beta1 and also inhibits monocyte induction by alternative stimuli. Since TGF-beta1 represses a default monocyte pathway of common progenitor cells, PU.1 and Id2 seem to modulate lineage options of M/LC precursors, downstream of TGF-beta1.

Antigens, CD↗
PubMed2006

c-Myc acts downstream of IL-15 in the regulation of memory CD8 T-cell homeostasis.

A subset of CD8 T cells in normal mice, expressing high levels of activation markers such as CD44, shares many properties with antigen-specific memory CD8 T cells. Homeostasis of CD44(high) CD8 T cells depends upon cytokines such as interleukin-15 (IL-15); however, the downstream signaling pathways regulating IL-15-dependent homeostatic proliferation are poorly defined. Surprisingly, we show here that haploinsufficiency of the protooncogene c-myc leads to a highly selective decrease in CD44(high) CD8 T cells in mice. Although steady-state proliferation and survival of CD44(high) CD8 T cells appeared not to be dependent on c-Myc, homeostatic proliferation of c-myc(+/-) CD44(high) CD8 T cells in lymphopenic hosts was strongly reduced, and the residual homeostatic proliferation of these cells appeared to occur independently of IL-15. Moreover, c-myc(+/-) CD44(high) CD8 T cells responded very poorly to purified IL-15 in vitro. Backcrossing of c-myc(+/-) mice to IL-15(-/-) mice revealed that the number of CD44(high) CD8 T cells decreased in an additive fashion in mice heterozygous for c-myc and IL-15. Finally homeostatic proliferation of antigen-specific memory CD44(high) CD8 T cells was also impaired in c-myc(+/-) mice. Collectively, our data identify c-Myc as a novel downstream component of the IL-15-dependent pathway controlling homeostatic proliferation of memory CD44(high) CD8 T cells.

Adoptive Transfer↗
PubMed2002

Insulinlike growth factor-I signaling in multiple myeloma: downstream elements, functional correlates, and pathway cross-talk.

In multiple myeloma cells, insulinlike growth factor-I (IGF-I) activates 2 distinct signaling pathways, mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI-3K), leading to both proliferative and antiapoptotic effects. However, it is unclear through which of these cascades IGF-I regulates these different responses. The present studies identify a series of downstream targets in the PI-3K pathway, including glycogen synthase kinase-3beta, p70S6 kinase, and the 3 members of the Forkhead family of transcription factors. The contribution of the MAPK and PI-3K pathways and, where possible, individual elements to proliferation and apoptosis was evaluated by means of a series of specific kinase inhibitors. Both processes were regulated almost exclusively by the PI-3K pathway, with only minor contributions associated with the MAPK cascade. Within the PI-3K cascade, inhibition of p70S6 kinase led to significant decreases in proliferation and protection from apoptosis. Activation of p70S6 kinase could also be prevented by MAPK inhibitors, indicating regulation by both pathways. The Forkhead transcription factor FKHRL1 was observed to provide a dual effect in that phosphorylation upon IGF-I treatment resulted in a loss of ability to inhibit proliferation and induce apoptosis. The PI-3K pathway was additionally shown to exhibit cross-talk and to regulate the MAPK cascade, as inhibition of PI-3K prevented activation of Mek1/2 and other downstream MAPK elements. These results define important elements in IGF-I regulation of myeloma cell growth and provide biological correlates critical to an understanding of growth-factor modulation of proliferation and apoptosis.

Apoptosis↗
PubMed2006

A multispecies comparison of the metazoan 3'-processing downstream elements and the CstF-64 RNA recognition motif.

BACKGROUND: The Cleavage Stimulation Factor (CstF) is a required protein complex for eukaryotic mRNA 3'-processing. CstF interacts with 3'-processing downstream elements (DSEs) through its 64-kDa subunit, CstF-64; however, the exact nature of this interaction has remained unclear. We used EST-to-genome alignments to identify and extract large sets of putative 3'-processing sites for mRNA from ten metazoan species, including Homo sapiens, Canis familiaris, Rattus norvegicus, Mus musculus, Gallus gallus, Danio rerio, Takifugu rubripes, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans. In order to further delineate the details of the mRNA-protein interaction, we obtained and multiply aligned CstF-64 protein sequences from the same species. RESULTS: We characterized the sequence content and specific positioning of putative DSEs across the range of organisms studied. Our analysis characterized the downstream element (DSE) as two distinct parts - a proximal UG-rich element and a distal U-rich element. We find that while the U-rich element is largely conserved in all of the organisms studied, the UG-rich element is not. Multiple alignment of the CstF-64 RNA recognition motif revealed that, while it is highly conserved throughout metazoans, we can identify amino acid changes that correlate with observed variation in the sequence content and positioning of the DSEs. CONCLUSION: Our analysis confirms the early reports of separate U- and UG-rich DSEs. The correlated variations in protein sequence and mRNA binding sequences provide novel insights into the interactions between the precursor mRNA and the 3'-processing machinery.

Amino Acid Motifs↗
PubMed2006

A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.

BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.

Alternative Splicing↗
PubMed2005

Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast.

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1alpha and its downstream targets in phyllodes tumors and fibroadenomas of the breast. METHODS: The expression of HIF-1alpha, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium. RESULTS: Only two fibroadenomas displayed low-level stromal HIF-1alpha reactivity in the absence of CAIX expression. Stromal HIF-1alpha expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1alpha expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1alpha overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032). CONCLUSION: These results indicate that HIF-1alpha expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1alpha most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1alpha and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1alpha and CAIX seem to be of minor relevance in breast fibroadenomas.

Adult↗
PubMed2001

Identification of candidate downstream genes for the homeodomain transcription factor Labial in Drosophila through oligonucleotide-array transcript imaging.

BACKGROUND: Homeotic genes are key developmental regulators that are highly conserved throughout evolution. Their encoded homeoproteins function as transcription factors to control a wide range of developmental processes. Although much is known about homeodomain-DNA interactions, only a small number of genes acting downstream of homeoproteins have been identified. Here we use a functional genomic approach to identify candidate target genes of the Drosophila homeodomain transcription factor Labial. RESULTS: High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in Drosophila embryos. We find significant expression level changes for 96 genes belonging to all functional classes represented on the array. In accordance with our experimental procedure, we expect that these genes are either direct or indirect targets of labial gene action. Among these genes, 48 were upregulated and 48 were downregulated following labial overexpression. This corresponds to 6.3% of the genes represented on the array. For a selection of these genes, we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative RT-PCR. CONCLUSIONS: Our results identify a number of novel candidate downstream target genes for Labial, suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed Drosophila genes.

Animals↗
PubMed2003

Rescue of cells from apoptosis by antioxidants occurs downstream from GSH extrusion.

Antioxidants-that is, scavengers of free radicals and anaerobic conditions (5%CO(2), 95% N(2))-protect monocytic U937 cells from damage-induced apoptosis. Antioxidants rescue the cells acting on the apoptotic pathway at a step downstream from gluthatione extrusion. Reducing agents, such as DTT, also reduce stress-induced apoptosis. Thus, apoptotic GSH extrusion triggers the downstream events of apoptosis by leaving cells unprotected against thiol\s oxidation and radical production.

Anaerobiosis↗
PubMed2004

Evolving therapeutic strategies for Duchenne muscular dystrophy: targeting downstream events.

Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 of 3500 boys born worldwide. The disease results from mutation of the dystrophin gene that encodes a cytoskeletal protein associated with the muscle cell membrane. Although gene therapy will likely provide the cure for DMD, it remains on the distant horizon, emphasizing the need for more rapid development of palliative treatments that build on improved understanding of the complex pathology of dystrophin deficiency. In this review, we have focused on therapeutic strategies that target downstream events in the pathologic progression of DMD. Much of this work has been developed initially using the dystrophin-deficient mdx mouse to explore basic features of the pathophysiology of dystrophin deficiency and to test potential therapeutic interventions to slow, reverse, or compensate for functional losses that occur in muscular dystrophy. In some cases, the initial findings in the mdx model have led to clinical treatments for DMD boys that have produced improvements in muscle function and quality of life. Many of these investigations have concerned interventions that can affect protein balance in muscle, by inhibiting specific proteases implicated in the DMD pathology, or by providing anabolic factors or depleting catabolic factors that can contribute to muscle wasting. Other investigations have exploited the use of anti-inflammatory agents that can reduce the contribution of leukocytes to promoting secondary damage to dystrophic muscle. A third general strategy is designed to increase the regenerative capacity of dystrophic muscle and thereby help retain functional muscle mass. Each of these general approaches to slowing the pathology of dystrophin deficiency has yielded encouragement and suggests that targeting downstream events in dystrophinopathy can yield worthwhile, functional improvements in DMD.

Animals↗
PubMed2001

Nuclear forms of parathyroid hormone-related peptide are translated from non-AUG start sites downstream from the initiator methionine.

PTH-related peptide (PTHrP), normally a secreted protein, exerts at least part of its biological functions via intracrine actions at the level of the cell nucleus/nucleolus. To gain insight into the mechanism whereby this peptide accesses the cytosol for subsequent nuclear import, we characterized its nuclear forms. In transfected COS-1 cells, three nuclear PTHrP species were produced, which were larger than the mature form of the protein but smaller than prepro-PTHrP and comprised both the amino- and carboxyl-terminal regions of the peptide. This suggested that nuclear PTHrP proteins contain part of the prepropeptide and likely arise from alternate initiation of translation at downstream non-AUG codons within the signal sequence. Transient expression of two PTHrP forms, one in which the unique initiator ATG was mutated to a noninitiator ATC codon, and another encompassing an engineered N-linked glycosylation site, generated peptides of size comparable to nuclear PTHrP proteins. These were inefficiently targeted to the ER, bypassed ER transit, and exclusively localized to nucleoli. Using a polyclonal antiserum against the pro-region of PTHrP, nuclear PTHrP species were shown to harbor the propeptide sequence. Hence, our data argue that nuclear PTHrP forms result from translation initiated at alternate, internal codons. This may constitute the first example of translation initiation at downstream non-AUG codons in a mammalian protein.

Animals↗
PubMed2001

A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1).

Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

COUP Transcription Factor I↗
PubMed2003

The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp.

Dpp signals are responsible for establishing a variety of cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although we have a reasonably clear picture of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. In the present study, we describe three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, which are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. We show that these genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, we demonstrate that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, we show that the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend our knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals.

Amino Acid Sequence↗
PubMed2003

A Twist-like bHLH gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos.

Ascidian larvae develop mesenchyme cells in their trunk. A fibroblast growth factor (FGF9/16/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. We have identified two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 as downstream factors of this FGF. These two genes are phylogenetically closely related to each other, and were expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrated that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream factor of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos.

Amino Acid Sequence↗