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Eric Lécuyer
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ABSTRACT: Tissue-specific gene expression is a major determinant in the elaboration of cells with distinctive phenotypes and functions, which is crucial for the development and homeostasis of multicellular organisms. Fluorescent in situ hybridization (FISH) is a powerful method for assessing the expression and localization properties of RNA at subcellular resolution in whole mount organism and tissue specimens. This chapter describes a high-resolution FISH protocol for the detection of RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster. The approach utilizes tyramide signal amplification (TSA) for enhanced sensitivity and resolution in the detection of coding and noncoding RNAs, for the codetection of different RNA species or of RNA and a protein marker of interest. Furthermore, the protocol outlines details for conducting FISH in microtiter plates, which greatly enhances the throughput, practicality, and economy of the procedure.
Methods in molecular biology (Clifton, N.J.) 01/2011; 714:31-47.
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ABSTRACT: Genome expression profiling has led to the important realization that RNA molecules are more numerous and diverse than previously expected. One aspect of RNA biology that is just beginning to be fully appreciated is the extent to which mRNAs are transported to specific subcellular destinations before being translated, an exquisite mechanism for targeting proteins where they are required in the cell. While several excellent reviews have discussed the subject of mRNA localization, it is only in recent years that high-throughput technologies have been applied to address issues such as the extent and diversity of RNA localization events and mechanisms. This review focuses on these recent functional genomic approaches, their implications, and the new tools and methods that will be needed to further elucidate mRNA localization pathways.
Current opinion in cell biology 03/2009; 21(3):409-15. · 14.15 Impact Factor
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ABSTRACT: Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.
Current opinion in genetics & development 10/2008; 18(6):506-12. · 8.99 Impact Factor
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Eric Lécuyer
Medecine sciences: M/S 05/2008; 24(4):350-1. · 0.64 Impact Factor
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ABSTRACT: Fluorescent in situ hybridization is the standard method for visualizing the spatial distribution of RNA. Although traditional histochemical RNA detection methods suffered from limitations in resolution or sensitivity, the recent development of peroxidase-mediated tyramide signal amplification provides strikingly enhanced sensitivity and subcellular resolution. In this chapter, we describe optimized fluorescent in situ hybridization protocols for Drosophila embryos and tissues utilizing tyramide signal amplification, either for single genes or in a high-throughput format, which greatly increases the sensitivity, consistency, economy, and throughput of the procedure. We also describe variations of the method for RNA-RNA and RNA-protein codetection.
Methods in molecular biology (Clifton, N.J.) 01/2008; 420:289-302.
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ABSTRACT: INTRODUCTIONFluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.
CSH protocols. 01/2008; 2008:pdb.prot5019.
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ABSTRACT: Gene expression programs are established by networks of interacting transcription factors. The basic helix-loop-helix factor SCL and the LIM-only protein LMO2 are components of transcription factor complexes that are essential for hematopoiesis. Here we show that LMO2 and SCL are predominant interaction partners in hematopoietic cells and that this interaction occurs through a conserved interface residing in the loop and helix 2 of SCL. This interaction nucleates the assembly of SCL complexes on DNA and is required for target gene induction and for the stimulation of erythroid and megakaryocytic differentiation. We also demonstrate that SCL determines LMO2 protein levels in hematopoietic cells and reveal that interaction with SCL prevents LMO2 degradation by the proteasome. We propose that the SCL-LMO2 interaction couples protein stabilization with higher order protein complex assembly, thus providing a powerful means of modulating the stoichiometry and spatiotemporal activity of SCL complexes. This interaction likely provides a rate-limiting step in the transcriptional control of hematopoiesis and leukemia, and similar mechanisms may operate to control the assembly of diverse protein modules.
Journal of Biological Chemistry 12/2007; 282(46):33649-58. · 4.77 Impact Factor
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ABSTRACT: Although subcellular mRNA trafficking has been demonstrated as a mechanism to control protein distribution, it is generally believed that most protein localization occurs subsequent to translation. To address this point, we developed and employed a high-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis. Surprisingly, of the 3370 genes analyzed, 71% of those expressed encode subcellularly localized mRNAs. Dozens of new and striking localization patterns were observed, implying an equivalent variety of localization mechanisms. Tight correlations between mRNA distribution and subsequent protein localization and function, indicate major roles for mRNA localization in nucleating localized cellular machineries. A searchable web resource documenting mRNA expression and localization dynamics has been established and will serve as an invaluable tool for dissecting localization mechanisms and for predicting gene functions and interactions.
Cell 11/2007; 131(1):174-87. · 32.40 Impact Factor
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ABSTRACT: SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.
Molecular and Cellular Biology 03/2004; 24(4):1439-52. · 5.53 Impact Factor
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ABSTRACT: In the hematopoietic system, lineage commitment and differentiation is controlled by the combinatorial action of transcription factors from diverse families. SCL is a basic helix-loop-helix transcription factor that is an essential regulator at several levels in the hematopoietic hierarchy and whose inappropriate regulation frequently contributes to the development of pediatric T-cell acute lymphoblastic leukemia. This review discusses advances that have shed important light on the functions played by SCL during normal hematopoiesis and leukemogenesis and have revealed an unexpected robustness of hematopoietic stem cell function. Molecular studies have unraveled a mechanism through which gene expression is tightly controlled, as SCL functions within multifactorial complexes that exhibit an all-or-none switch-like behavior in transcription activation, arguing for a quantal process that depends on the concurrent occupation of target loci by all members of the complex. Finally, variations in composition of SCL-containing complexes may ensure flexibility and specificity in the regulation of lineage-specific programs of gene expression, thus providing the molecular basis through which SCL exerts its essential functions at several branch points of the hematopoietic hierarchy.
Experimental Hematology 02/2004; 32(1):11-24. · 2.90 Impact Factor
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ABSTRACT: The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells.
Blood 11/2002; 100(7):2430-40. · 9.90 Impact Factor
