Dominant Repressor Research Articles

The Arabidopsis B3 Domain Protein VERNALIZATION1 (VRN1) Is Involved in Processes Essential for Development, with Structural and Mutational Studies Revealing Its DNA-binding Surface

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease

Liver fatty acid binding protein (FABP1) prevents lipotoxicity of free fatty acids and regulates fatty acid trafficking and partition. Our objective is to investigate the transcription factors controlling the human FABP1 gene and their regulation in nonalcoholic fatty liver disease (NAFLD). Adenovirus-mediated expression of multiple transcription factors in HepG2 cells and cultured human hepatocytes demonstrated that FOXA1 and PPARα are among the most effective activators of human FABP1, whereas C/EBPα is a major dominant repressor. Moreover, FOXA1 and PPARα induced re-distribution of FABP1 protein and increased cytoplasmic expression. Reporter assays demonstrated that the major basal activity of the human FABP1 promoter locates between −96 and −229bp, where C/EBPα binds to a composite DR1-C/EBP element. Mutation of this element at −123bp diminished basal reporter activity, abolished repression by C/EBPα and reduced transactivation by HNF4α. Moreover, HNF4α gene silencing by shRNA in HepG2 cells caused a significant down-regulation of FABP1 mRNA expression. FOXA1 activated the FABP1 promoter through binding to a cluster of elements between −229 and −592bp, whereas PPARα operated through a conserved proximal element at −59bp. Finally, FABP1, FOXA1 and PPARα were concomitantly repressed in animal models of NAFLD and in human nonalcoholic fatty livers, whereas C/EBPα was induced or did not change. We conclude that human FABP1 has a complex mechanism of regulation where C/EBPα displaces HNF4α and hampers activation by FOXA1 and PPARα. Alteration of expression of these transcription factors in NAFLD leads to FABP1 gen repression and could exacerbate lipotoxicity and disease progression.

Inverse PPARβ/δ agonists suppress oncogenic signaling to the ANGPTL4 gene and inhibit cancer cell invasion

Besides its established functions in intermediary metabolism and developmental processes, the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has a less defined role in tumorigenesis. In the present study, we have identified a function for PPARβ/δ in cancer cell invasion. We show that two structurally divergent inhibitory ligands for PPARβ/δ, the inverse agonists ST247 and DG172, strongly inhibit the serum- and transforming growth factor β (TGFβ)-induced invasion of MDA-MB-231 human breast cancer cells into a three-dimensional matrigel matrix. To elucidate the molecular basis of this finding, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and microarray analyses, which identified the gene encoding angiopoietin-like 4 (ANGPTL4) as the major transcriptional PPARβ/δ target in MDA-MB-231 cells, previously implicated in TGFβ-mediated tumor progression and metastatic dissemination. We show that the induction of ANGPTL4 by TGFβ and other oncogenic signals is strongly repressed by ST247 and DG172 in a PPARβ/δ-dependent fashion, resulting in the inhibition of ANGPTL4 secretion. This effect is attributable to these ligands' ability to induce a dominant transcriptional repressor complex at the site of transcription initiation that blocks preinitiation complex formation through an histone deacetylase-independent, non-canonical mechanism. Repression of ANGPTL4 transcription by inverse PPARβ/δ agonists is functionally linked to the inhibition of cancer cell invasion into a three-dimensional matrix, as (i) invasion of MDA-MB-231 cells is critically dependent on ANGPTL4 expression, (ii) recombinant ANGPTL4 stimulates invasion, and (iii) reverses the inhibitory effect of ST247 and DG172. These findings indicate that a PPARβ/δ–ANGPTL4 pathway is involved in the regulation of tumor cell invasion and that its pharmacological manipulation by inverse PPARβ/δ agonists is feasible.

A Small Molecule Inhibitor of the CBFβ-SMMHC/RUNX Interaction Attenuates Inv(16) Leukemia in Vivo

Abstract 286Emerging literature suggests that inability to cure cancers with current therapies may be attributed to a population of so-called cancer stem cells or cancer initiating cells that have long term self-renewal potential and can fully recapitulate tumor phenotype at time of relapse. The use of targeted therapies that inhibit the activity of oncoproteins directly present the potential for specificity in ablating tumor cells, including cancer stem cells, while having minimal impact on normal tissues.The gene encoding CBFβ (CBFB) is disrupted by the chromosome 16 inversion [inv(16)(p13q22)], associated with ∼12% of acute myeloid leukemia (AML) in humans, resulting in a fusion protein containing most of CBFβ fused to the coiled-coil tail region of smooth muscle myosin heavy chain (SMMHC). The CBFβ-SMMHC fusion protein acts as a dominant repressor of CBF function, binding RUNX1 and dysregulating the expression of multiple target genes required for normal hematopoiesis. Current AML treatment utilizing cytotoxic chemotherapy results in 45–65% five year overall survival but only 20% for patients older than 60. These data clearly indicate that the development of targeted therapy that can improve the therapeutic response for inv(16) AML patients is essential.The targeting of transcription factors in cancer therapy is a relatively new approach with tremendous potential. We developed a small molecule inhibitor, AI-10-49, that binds to the aberrant transcription factor CBFβ-SMMHC and disrupts its interaction with RUNX proteins. We have taken advantage of the oligomeric nature of CBFβ-SMMHC by developing dimeric compounds to achieve selectivity toward the fusion protein. Optimization of the linker length in these dimeric compounds shows a clear linker length dependence and a dramatic enhancement in activity for the dimeric versus monomeric inhibitors, providing validation for this approach. AI-10-49 is a result of this optimization and modifications to improve pharmacokinetic properties, resulting in a potent inhibitor with a half-life of ∼3 hours in mice. This compound displays selective toxicity to human leukemia cell lines with the inv(16) at a submicromolar dose (IC50=0.4μM). We have also shown no effect on growth of normal mouse or human bone marrow mononuclear cells. Using RT-PCR, we have shown dramatic derepression of the well-validated target genes CSF1R (9-fold) and RUNX3 (18-fold) in ME-1 (inv(16)) cells and little to no effect in Kasumi-1 (t(8;21)) and U937 cells. We have also shown increased apoptosis of the mouse preleukemic myeloid progenitor cells expressing CBFβ-SMMHC upon treatment ex vivo.The toxicity of AI-10-49 was tested in mice at the 25 mg/kg dosage employed for efficacy studies. Mice were dosed twice a day at 12 hour intervals for 1 week. No change in counts of white blood cells, red blood cells, or platelets was observed compared to formulation control. No changes were observed in granulocytes, monocytes, T cell, and B cell frequency by flow cytometry. Analysis of tissues after treatment showed no defects in spleen, kidney, liver, lungs, GI tract, heart, and bone marrow. A maximum tolerated dose (MTD) study showed no toxicity at doses up to 500 mg/kg, indicating the compound is very well tolerated.The efficacy of AI-10-49 was tested in vivo using the Cbfb+/MYH11/Mx1Cre/NrasG12D mouse model of inv(16) AML. Mice were transplanted with 5×105 leukemic cells, allowed 5 days for engraftment, and treated between days 5 and 15 post transplantation with 2 doses of vehicle or of AI-10-49 per day (25mg/kg/day). The control group (n=4) developed leukemia with a median latency of 4.6 weeks and full penetrance, while the test group (n=4) developed leukemia with a median latency of 8.4 weeks and incomplete penetrance. One mouse (25%) from the test group showed progressive decrease of leukemic cells to background levels at week 12 in peripheral blood, and remains healthy with no evidence of disease to date (week 16). Current efforts are focused on testing the compound efficacy of AI-10-49 with first line therapy drug Ara-C in mice.This work presents strong evidence of the identification of a small molecule that specifically targets CBFβ-SMMHC function and ablates cells expressing the leukemia fusion protein in vitro and in mice. This study provides proof of principle that oncogenic transcription factors can be targeted and may have significant impact on the treatment of inv(16) acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

MYB103 is required for FERULATE‐5‐HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of Slignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of Slignin are dependent on MYB103.

Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA(20) and GA(8), in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.

Regulatory role of cAMP receptor protein over Escherichia coli fumarase genes

Escherichia coli expresses three fumarase genes, namely, fumA, fumB, and fumC. In the present study, catabolite repression was observed in the fumA-lacZ and fumC-lacZ fusion strains, but not in the fumB-lacZ fusion strain. The Crp-binding sites in fumA and fumC were identified using an electrophoretic mobility shift assay and footprint analysis. However, the electrophoretic mobility shift assay did not detect band shifts in fumB. Fnr and ArcA serve as transcription regulators of fumarase gene expression. In relation to this, different mutants, including Δcya, Δcrp, Δfnr, and ΔarcA, were used to explore the regulatory role of Crp over fumA and fumC. The results show that Crp is an activator of fumA and fumC gene expression under various oxygen conditions and growth rates. ArcA was identified as the dominant repressor, with the major repression occurring at 0-4% oxygen. In addition, Fnr was confirmed as a repressor of fumC for the first time. This study elucidates the effects of Crp on fumarase gene expression.

The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins

In most dicot plants, lateral root (LR) formation, which is important for the construction of the plant root system, is initiated from coordinated asymmetric cell divisions (ACD) of the primed LR founder cells in the xylem pole pericycle (XPP) of the existing roots. In Arabidopsis thaliana, two AUXIN RESPONSE FACTORs (ARFs), ARF7 and ARF19, positively regulate LR formation through activation of the plant-specific transcriptional regulators LATERAL ORGAN BOUNDARIES-DOMAIN 16/ASYMMETRIC LEAVES2-LIKE 18 (LBD16/ASL18) and the other related LBD/ASL genes. The exact biological role of these LBD/ASLs in LR formation is still unknown. Here, we demonstrate that LBD16/ASL18 is specifically expressed in the LR founder cells adjacent to the XPP before the first ACD and that it functions redundantly with the other auxin-inducible LBD/ASLs in LR initiation. The spatiotemporal expression of LBD16/ASL18 during LR initiation is dependent on the SOLITARY-ROOT (SLR)/IAA14-ARF7-ARF19 auxin signaling module. In addition, XPP-specific expression of LBD16/ASL18 in arf7 arf19 induced cell divisions at XPP, thereby restoring the LR phenotype. We also demonstrate that expression of LBD16-SRDX, a dominant repressor of LBD16/ASL18 and its related LBD/ASLs, does not interfere in the specification of LR founder cells with local activation of the auxin response, but it blocks the polar nuclear migration in LR founder cells before ACD, thereby blocking the subsequent LR initiation. Taken together, these results indicate that the localized activity of LBD16/ASL18 and its related LBD/ASLs is involved in the symmetry breaking of LR founder cells for LR initiation, a key step for constructing the plant root system.

A mechanism for robust circadian timekeeping via stoichiometric balance

Circadian (∼24 h) timekeeping is essential for the lives of many organisms. To understand the biochemical mechanisms of this timekeeping, we have developed a detailed mathematical model of the mammalian circadian clock. Our model can accurately predict diverse experimental data including the phenotypes of mutations or knockdown of clock genes as well as the time courses and relative expression of clock transcripts and proteins. Using this model, we show how a universal motif of circadian timekeeping, where repressors tightly bind activators rather than directly binding to DNA, can generate oscillations when activators and repressors are in stoichiometric balance. Furthermore, we find that an additional slow negative feedback loop preserves this stoichiometric balance and maintains timekeeping with a fixed period. The role of this mechanism in generating robust rhythms is validated by analysis of a simple and general model and a previous model of the Drosophila circadian clock. We propose a double-negative feedback loop design for biological clocks whose period needs to be tightly regulated even with large changes in gene dosage.

The role of SND2 in the regulation of Arabidopsisfibre secondary cell wall formation

Background Transcription factors (TFs) play important roles in the regulation of secondary cell wall (SCW) biosynthesis in herbaceous and woody plants. In Arabidopsis, the onset of SCW deposition is initiated by a nexus of NAC, MYB, homeodomain and several other families of TFs, which function in a transcriptional network regulating SCW biosynthetic genes. NAC family members SND1/ NST1 and VND6/VND7 have been identified as functionally redundant master regulators of SCW formation in fibres and vessels, respectively [1,2]. Arabidopsis plants overexpressing SND2, an indirect target of fibre master regulator SND1, exhibited increased SCW thickness in inflorescence stem fibres, whilst dominant repression lines exhibited a decrease in fibre SCW thickness associated with a reduction in glucose and xylose cell wall sugar content [3]. The ability of SND2 to transactivate the CesA8 promoter [3] suggested that SND2 may regulate cellulose biosynthetic genes during fibre SCW formation. The evaluation of this hypothesis necessitates the identification of all downstream genes potentially regulated by SND2, and the analysis of SCW chemistry and morphology in overexpression lines. The aim of this ongoing study is to further elucidate the role of SND2 in fibre SCW formation through microarray analysis of overexpression lines and by independent confirmation of the effect of SND2 overexpression on fibre SCW chemistry and morphology in Arabidopsis plants.

Dissection of the Transcriptional Program Regulating Secondary Wall Biosynthesis during Wood Formation in Poplar

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.

Tcf3 function is required for the inhibition of oligodendroglial fate specification in the spinal cord of zebrafish embryos.

The generation of various subtypes of neurons and glial cells at the right time and place is crucial for the proper development of the vertebrate CNS. Although the mechanisms and factors for the regulation of neuronal diversity in the CNS have been well studied, the mechanisms regulating the sequential production of neuronal and glial cells from neural precursors remain poorly understood. This study shows that Tcf3, a member of the Lef/Tcf family of proteins, is required to inhibit the premature oligodendroglial fate specification of spinal cord precursors using the transgenic zebrafish, which expresses a dominant repressor form of Tcf3 under the control of a heat-shock inducible promoter. In addition, the data revealed that Tcf3 function in oligodendroglial fate specification is mediated independently of canonical Wnt signaling. Altogether, these results show a novel function for Tcf3 in regulating the timing of oligodendroglial fate specification in the spinal cord.

Transcription factor TEAD4 regulates expression of Myogenin and the unfolded protein response genes during C2C12 cell differentiation

The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes.

The Requirement of GLI Transcriptional Activity in KRAS-Induced Pancreatic Neoplasia

Background and Aim: Kras mutations are detected in 90% of human pancreatic ductal adenocarcinoma (PDAC) and are believed to be the critical oncogenic event during pancreatic tumorigenesis. However, the downstream transcriptional mechanisms by Kras in pancreatic cancer still remain poorly understood. Recent In Vitro studies suggested that Gli transcriptional factors, the intracellular mediator of Hedgehog signaling, are important for Kras activity in human PDAC cell lines. However, a requirement for Gli activation in pancreatic malignancy has not been demonstrated In Vivo. Here we carried out both loss-of-function and gain-offunction genetic analyses to establish the functional significance of Gli transcriptional activation in Kras-Induced pancreatic tumorigenesis using genetically modified mouse models. Methods: We utilized a conditional Rosa26 allele of Gli3T, R26-Gli3T. Gli3T is a dominant repressor of Gli3 that blocks Gli transcriptional activation. The R26-Gli3T mice were crossed to the Ptf1a-Cre;LDL-KrasG12D mice, a mouse model that allows pancreatic-specific Kras activation and tumor formation. A cohort of Ptf1a-Cre;LDL-KrasG12D;R26-Gli3T mice was generated and monitored for pancreatic tumor development. For Gli gain of function studies, we ectopically expressed Gli1 along with oncogenic Kras in mouse pancreas to determine whether Gli1 cooperates with Kras to promote tumorigenesis. Results: Our results showed that Gli3T expression caused a severe growth defect and loss of tumorigenicity in human PDAC cell lines. More importantly, the mice carrying Gli3T along with oncogenic Kras failed to develop PanIN lesions, in contrast to the mice with Kras activation alone. Mice expressing Gli1 along with activated Kras in pancreas also showed marked acceleration of tumor formation. Furthermore, our gene expression profiling identified a distinct transcriptional program mediated by Gli proteins in pancreatic tumor cells. Conclusions: Gli transcriptional activation not only accelerates pancreatic tumorigenesis in cooperation with Kras, but is also required for Kras-induced tumorigenesis In Vivo.

CESTA, a positive regulator of brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroid hormones that are essential for the development of plants. A tight control of BR homeostasis is vital for modulating their impact on growth responses. Although it is recognized that the rapid adaptation of de novo synthesis has a key role in adjusting required BR levels, our knowledge of the mechanisms governing feedback control is limited. In this study, we identify the transcription factor CESTA as a regulator of BR biosynthesis. ces-D was isolated in a screen of Arabidopsis mutants by BR over-accumulation phenotypes. Loss-of-function analysis and the use of a dominant repressor version revealed functional overlap among CESTA and its homologues and confirmed the role of CESTA in the positive control of BR-biosynthetic gene expression. We provide evidence that CESTA interacts with its homologue BEE1 and can directly bind to a G-box motif in the promoter of the BR biosynthesis gene CPD. Moreover, we show that CESTA subnuclear localization is BR regulated and discuss a model, in which CESTA interplays with BEE1 to control BR biosynthesis and other BR responses.

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

AbstractAbstract 1695 Introduction:The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods:The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results:A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions:Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures:No relevant conflicts of interest to declare.

Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.

Tobacco (Nicotiana tabacum) synthesizes nicotine and related pyridine alkaloids in the root, and their synthesis increases upon herbivory on the leaf via a jasmonate-mediated signaling cascade. Regulatory NIC loci that positively regulate nicotine biosynthesis have been genetically identified, and their mutant alleles have been used to breed low-nicotine tobacco varieties. Here, we report that the NIC2 locus, originally called locus B, comprises clustered transcription factor genes of an ethylene response factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes are deleted altogether. Overexpression, suppression, and dominant repression experiments using transgenic tobacco roots showed both functional redundancy and divergence among the NIC2-locus ERF genes. These transcription factors recognized a GCC-box element in the promoter of a nicotine pathway gene and specifically activated all known structural genes in the pathway. The NIC2-locus ERF genes are expressed in the root and upregulated by jasmonate with kinetics that are distinct among the members. Thus, gene duplication events generated a cluster of highly homologous transcription factor genes with transcriptional and functional diversity. The NIC2-locus ERFs are close homologs of ORCA3, a jasmonate-responsive transcriptional activator of indole alkaloid biosynthesis in Catharanthus roseus, indicating that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages.

Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling

Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-l-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCF(COI1) ) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates the JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and seven C-terminal (X(5) PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X(5) PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre-mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Repression of Lim only protein 4-activated transcription inhibits proliferation and induces apoptosis of normal mammary epithelial cells and breast cancer cells

Lim only protein (LMO) 4 acts as a transcriptional adapter and modulates mammary gland morphogenesis as well as breast oncogenesis in transgenic mice. Yet, the molecular and cellular mechanisms of these effects remain to be fully elucidated. Engrailed LMO4 fusion protein is a powerful dominant repressor of LMO4 activated transcription that was successfully used to discover the role of LMO4 as a transcriptional activator in mammary gland development in our previous studies using mouse models. In this manuscript, we investigated the cellular effects of LMO4 in human normal mammary epithelial cells (HMECs) and breast cancer cell lines using the Engrailed-LMO4 fusion protein. HMEC cell growth was inhibited by the expression of the Engrailed-LMO4 fusion protein. The decrease in cell number was due to both decreased cell proliferation and enhanced apoptosis, suggesting that LMO4 promotes proliferation and survival of normal mammary epithelial cells. The expression of the Engrailed-LMO4 fusion protein also suppressed cell growth, and induced apoptosis in two breast cancer cell lines, MDA-MB-231 and T47D, suggesting that LMO4 contributes to oncogenesis by similar mechanisms of enhanced cell survival and proliferation. Taken together, our data indicate that LMO4 has similar cellular effects in normal mammary epithelial cells and breast cancer cells, and also provide direct evidence for the idea that normal development and carcinogenesis share conserved molecular mechanisms.

Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE2 synthesis

Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. After treatment with icariin, the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of 10(-8)M. Icariin (10(-8)M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) expression. The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-κB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin E(2) (PGE(2)). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1α (HIF-1α) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (IκBα), but increased the activation of p38. In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.