Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo^{1, 2}. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.     

Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor

Neb-LFamide or AYRKPPFNGSLFamide was originally purified from the grey flesh fly Neobellieria bullata as a myotropic neuropeptide. We studied the occurrence of this peptide and its isoforms in the central nervous system of different insect species by means of whole mount fluorescence immunohistochemistry, mass spectrometry, and data mining. We found that both sequence and immunoreactive distribution pattern are very conserved in the studied insects. In all species and stages we counted two pairs of immunoreactive cells in the pars intercerebralis. These cells projected axons throughout the ventral nerve cord. In the adult CNSs they formed a large number of immunoreactive varicosities as well. Mass spectrometry and data mining revealed that SIFamide exists in two isoforms: [G1]-SIFamide and [A1]-SIFamide. In addition, the SIFamide joining peptide is relatively well conserved throughout arthropod species. The conserved presence of two cysteine residues, separated by six amino acid residues, allows the formation of disulphide bridges.     

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.     

Flexibility of the DNA-binding domains of trp repressor

An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.     

Targeted Overexpression of Drosophila Transglutaminase-B Induced Characteristic Phenotypes in a Manner Similar to Transglutaminase-A

The Drosophila transglutaminase gene (CG7356) encodes two transglutaminases, dTG-A and dTG-B. To understand the roles of dTG-B during the development of the fly, we examined phenotypes induced through ectopic expression of dTG-B. Overexpression of dTG-B induced rough eye and extra wing crossvein phenotypes. These phenotypes were similar to those observed in the case of targeted overexpression of dTG-A.     

The 50th anniversary of the publication of the operon theory in the Journal of Molecular Biology: past, present and future

A series of eight review articles that appear in the present issue of the Journal of Molecular Biology celebrates the 50th anniversary for the landmark publication of François Jacob and Jacques Monod entitled “Genetic Regulatory Mechanisms in the Synthesis of Proteins”. In this publication, the authors presented a model for the regulation of gene expression deduced from genetic and biochemical studies. They proposed that a new class of genes, regulatory genes, would code for repressors that bind to operator sequences upstream of operons consisting of a group of catabolic or biosynthetic genes with related functions. Binding is controlled by small metabolites, substrates or end products. The repressors control the transmission of information from genes to mRNA that is translated into proteins. The present review articles demonstrate how this publication influenced our thinking and how it stimulated the studies on the regulation of gene expression all the way to present day epigenetics and systems biology.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Chromatin: constructing the big picture

Chromatin is the ensemble of genomic DNA and a large number of proteins. Various genome-wide mapping techniques have begun to reveal that, despite the tremendous complexity, chromatin organization is governed by simple principles. This review discusses the principles that drive the spatial architecture of chromatin, as well as genome-wide-binding patterns of chromatin proteins.     

Pax基因家族在果蝇发育过程中的调控作用

Pax是一个在进化上相当保守的基因家族, 它们编码的产物是一组极为重要的转录调控因子, 并存在于从果蝇到人类的各种生物体中, 参与细胞内信号传导通路的调控, 在胚胎发育过程中对细胞分化、更新、凋亡起重要的调控作用, 影响器官和组织的形成。果蝇中已发现10个Pax基因家族成员, 它们对果蝇胚胎发育及成虫组织器官的分化有非常重要的调控作用。文章结合最新的研究进展, 就果蝇中Pax基因的结构、表达模式和主要功能做一简要综述。