Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells

BACKGROUND: Mucoepidermoid carcinoma (MEC) arises from multiple organs and accounts for the most common types of salivary gland malignancies. Currently, patients with unresectable and metastatic MEC have poor long-term clinical outcomes and no targeted therapies are available. The majority of MEC tumors contain a t(11;19) chromosomal translocation that fuses two genes, CRTC1 and MAML2, to generate the chimeric protein CRTC1-MAML2. CRTC1-MAML2 displays transforming activity in vitro and is required for human MEC cell growth and survival, partially due to its ability to constitutively activate CREB-mediated transcription. Consequently, CRTC1-MAML2 is implicated as a major etiologic molecular event and a therapeutic target for MEC. However, the molecular mechanisms underlying CRTC1-MAML2 oncogenic action in MEC have not yet been systematically analyzed. Elucidation of the CRTC1-MAML2-regulated transcriptional program and its underlying mechanisms will provide important insights into MEC pathogenesis that are essential for the development of targeted therapeutics. METHODS: Transcriptional profiling was performed on human MEC cells with the depletion of endogenous CRTC1-MAML2 fusion or its interacting partner CREB via shRNA-mediated gene knockdown. A subset of target genes was validated via real-time RT-PCR assays. CRTC1-MAML2-perturbed molecular pathways in MEC were identified through pathway analyses. Finally, comparative analysis of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles was carried out to assess the contribution of CREB in mediating CRTC1-MAML2-induced transcription. RESULTS: A total of 808 differentially expressed genes were identified in human MEC cells after CRTC1-MAML2 knockdown and a subset of known and novel fusion target genes was confirmed by real-time RT-PCR. Pathway Analysis revealed that CRTC1-MAML2-regulated genes were associated with network functions that are important for cell growth, proliferation, survival, migration, and metabolism. Comparison of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles revealed common and distinct genes regulated by CRTC1-MAML2 and CREB, respectively. CONCLUSION: This study identified a specific CRTC1-MAML2-induced transcriptional program in human MEC cells and demonstrated that CRTC1-MAML2 regulates gene expression in CREB-dependent and independent manners. Our data provide the molecular basis underlying CRTC1-MAML2 oncogenic functions and lay a foundation for further functional investigation of CRTC1-MAML2-induced signaling in MEC initiation and maintenance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1827-3) contains supplementary material, which is available to authorized users

Associations of HLA-DP Variants with Hepatitis B Virus Infection in Southern and Northern Han Chinese Populations: A Multicenter Case-Control Study

) locus has been reported to be associated with hepatitis B virus (HBV) infection in populations of Japan and Thailand. We aimed to examine whether the association can be replicated in Han Chinese populations. = 0.097∼0.697 and 0.198∼0.615 in northern Chinese population, respectively). loci were strongly associated with HBV infection in southern and northern Han Chinese populations, but not with HBV progression

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Diverse Amine-Acid Coupling Reactions Modulate the Potency of BRD4 PROTACs

Protein degradation with proteolysis targeting chimeras (PROTACs) has emerged as a powerful therapeutic strate-gy. PROTACs are heterobifunctional molecules consisting of a target-binding moiety, a linker and an E3 ligase-binding moiety that are commonly linked together using amide bonds. Although experimentally facile, this bond choice may impart sub-optimal drug properties to the overall PROTAC molecule. Here, we show that a suite of BRD4 PROTAC degraders were prepared via diverse amine-acid couplings beyond the amide bond, where the bromo-domain inhibitor JQ1 carboxylic acid was used as the acid and the E3-derived (Cereblon or Von Hippel Lindau) par-tial PROTACs served as the amine. Four new amine–acid coupling reactions were developed to generate the ester, ketone, alkane, or amine bond connection using miniaturized high-throughput experimentation (HTE). Subtle reac-tion condition effects in nickel and platinum catalyzed couplings on complex molecule substrates were uncovered, and the toolbox of linkage methods to synthesize heterobifunctional degraders was expanded. Calculated physico-chemical properties for these ten diversly linked PROTACs showed a wide range of lipophilicity, polar surface area, and hydrogen bonding characteristics. Ultimately, when these compounds were evaluated for BRD4 degradation, several of them showed improved efficacy. The ester, ketone, and alkane linked degraders outperform the classic amide-linked compound. This study highlights the impact that slight changes to the linkage between the protein of interest (POI) and E3 ligase binders can be used to modulate degradation and drug properties

Abstract 3026: Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma.

Abstract Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 gene fusion, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a highly specific genetic alteration in more than 50% of mucoepidermoid carcinoma (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth ad survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and the in vivo. CRTC1-MAML2 induced the up-regulation of the EGFR ligand AREG by co-activating the transcription factor CREB and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with unresectable CRTC1-MAML2-positive MEC. Citation Format: Zirong Chen, Yumei Gu, Chengbin Hu, Jian-Liang Li, Shuibin Lin, Huangxuan Shen, Chunxia Cao, Jie Chen, Jiancheng Li, Patick K. Ha, Federic J. Kaye, James D. Griffin, Lizi Wu. Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2013-3026</jats:p