Project description:FoxA transcription factors play major roles in organ-specific gene expression. How FoxA proteins achieve specificity is unclear, given their broad expression patterns and requirements in multiple cell types. Here, we characterize Sage, a basic helix-loop-helix (bHLH) transcription factor expressed exclusively in the Drosophila salivary gland (SG). We identify Sage targets and show that not only are both Sage and the single Drosophila FoxA protein, Fork head (Fkh), required for expression of these genes, but coexpression of Sage and Fkh is sufficient to drive target gene expression in multiple other cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage/Fkh targets. Importantly, Sage, Fkh and Sens colocalize on salivary gland polytene chromosomes. Thus, Fkh drives cell-type specific gene expression as part of a tissue-specific transcription module that includes Sage and Sens, providing a new paradigm for how mammalian FoxA proteins acheive specificity. Three wild type (reference) samples were obtained from the Oregon R strain, age matched and treated the same as the three experimental samples isolated from Stages 11-16 embryos expressing UAS-Sage controlled by the tub-Gal4 driver.
Project description:Quantitative comparation of the tagmata of body and head using iTRAQ based on HCD Fragmentation. The 8-plex iTRAQ Multiplex Buffer Kit (AB SCIEX, Foster City, CA) was used to label different peptide fractions, in which iTRAQ reagents dissolved in isopropyl alcohol. Another internal standard of D.melanogaster body and head mixed with 1:1 (B+H) was also used when doing iTRAQ labeling. Therefore the same amount of B (body), H1 (head), H2 (head), B+H (internal standard) peptide fractions prepared as described above were labeled by equal but different iTRAQ reagents and incubated for 5 h at room temperature. The four different labeled peptide fractions (B:113, H1:114, H2:115, B+H:116 ) were then mixed with 1:1 and dried in a speedvac followed by desalting purification using stage tip. All prepared peptides were further analyzed on an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Electron, San Jose, CA) coupled with UPLC (nano Acquity Ultra Performance LC, Waters). For HCD raw files, the profile data was firstly centralized by ReAdW.exe in TPP and then deisotoped and deconvoluted using in-house made scripts to improve the identification rate of spectra. All MGF files were searched using Mascot 2.3 against a Drosophila melanogaster database with 24,043 entries (http://flybase.org/, release 5.4, 24,043 entries). The target-decoy based strategy was used to control the peptide false discovery rate (FDR).
Project description:Nucleus is a highly structured organelle and contains many functional compartments. While the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we find structural proteins, chaperones related, DNA/RNA binding, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the 2D profile of NuMat proteome from different developmental stages of Drosophila embryos shows that less than half of the NuMat proteome is constant and rest of the proteins are stage specific dynamic components. This NuMat dynamics suggests a possible functional link between NuMat and the embryonic development. Finally, we also show that a subset of NuMat proteins remain associated with the mitotic chromosomes implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.
Project description:FoxA transcription factors play major roles in organ-specific gene expression. How FoxA proteins achieve specificity is unclear, given their broad expression patterns and requirements in multiple cell types. Here, we characterize Sage, a basic helix-loop-helix (bHLH) transcription factor expressed exclusively in the Drosophila salivary gland (SG). We identify Sage targets and show that not only are both Sage and the single Drosophila FoxA protein, Fork head (Fkh), required for expression of these genes, but coexpression of Sage and Fkh is sufficient to drive target gene expression in multiple other cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage/Fkh targets. Importantly, Sage, Fkh and Sens colocalize on salivary gland polytene chromosomes. Thus, Fkh drives cell-type specific gene expression as part of a tissue-specific transcription module that includes Sage and Sens, providing a new paradigm for how mammalian FoxA proteins acheive specificity. Three control samples were obtained from the tub-Gal4 strain, age matched and treated the same as the three experimental samples isolated from Stages 11-16 embryos expressing UAS-Sage controlled by the tub-Gal4 driver.
Project description:We report the analysis of the transcriptome in Drosophila embryos with two genotypes (1: wild type, 2: embryos from germline clones of a SHMT mutant (allele X238)) and two developmental stages (1: pre-blastoderm, stage 1 and stage 2, 0–1h after egg lay, 2: late blastoderm/cellularisation stage 5, 1.5–2.5 h after egg lay)