Expression profile of CREB knockdown in myeloid leukemia cells.

BackgroundThe cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.MethodsTo understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA.ResultsBy combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA.ConclusionWe have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

A Myb Transcription Factor of Phytophthora sojae, Regulated by MAP Kinase PsSAK1, Is Required for Zoospore Development

PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae

CREB is a critical regulator of normal hematopoiesis and leukemogenesis

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.</p

Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections

Copyright: © 2013 Baron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by ANR (ANR-07-BLAN-0214 and ANR-12-EMMA-00O7-01), CNRS and INRA. PvW was financially supported by the BBSRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells

Sex differences in risk factors for coronary heart disease: a study in a Brazilian population

BACKGROUND: In Brazil coronary heart disease (CHD) constitutes the most important cause of death in both sexes in all the regions of the country and interestingly, the difference between the sexes in the CHD mortality rates is one of the smallest in the world because of high rates among women. Since a question has been raised about whether or how the incidence of several CHD risk factors differs between the sexes in Brazil the prevalence of various risk factors for CHD such as high blood cholesterol, diabetes mellitus, hypertension, obesity, sedentary lifestyle and cigarette smoking was compared between the sexes in a Brazilian population; also the relationships between blood cholesterol and the other risk factors were evaluated. RESULTS: The population presented high frequencies of all the risk factors evaluated. High blood cholesterol (CHOL) and hypertension were more prevalent among women as compared to men. Hypertension, diabetes and smoking showed equal or higher prevalence in women in pre-menopausal ages as compared to men. Obesity and physical inactivity were equally prevalent in both sexes respectively in the postmenopausal age group and at all ages. CHOL was associated with BMI, sex, age, hypertension and physical inactivity. CONCLUSIONS: In this population the high prevalence of the CHD risk factors indicated that there is an urgent need for its control; the higher or equal prevalences of several risk factors in women could in part explain the high rates of mortality from CHD in females as compared to males

Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants

Sequence Variants of the Phytophthora sojae RXLR Effector Avr3a/5 Are Differentially Recognized by Rps3a and Rps5 in Soybean

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5