Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression in Aneuploid Drosophila S2 Cells

ABSTRACT: Chromosomal and segmental aneuploidies are usually lethal or common features of cancer cells and variety of disease, but little is known about how copy number relates to gene expression. Drosophila males have a single X chromosome and two sets of autosomes, but X chromosome and autosome transcripts are equally abundant. This 2-fold increase in X chromosome gene expression requires a dosage compensation complex (MSL) that acetylates histone 4 at lysine 16 (H4AcK16). To determine the contribution of general buffering and MSL to X chromosome dosage compensation, we analyzed genome-wide copy number, expression and histone modification pattern in male Drosophila S2 cells with and without MSL. Keywords: Gene Regulation Study, genomic analyses RNA-seq and microarray were performed in Drosophila non-treated or RNAi treated S2 cells. Two biological replicates were used for expression profile. DNA-seq and CGH were performed (CGH data forthcoming) to check the copy number variation in S2 cells. Chromatin immunoprecipitations were performed in Drosophila non-treated or RNAi treated S2 cells with different histone modification antibodies. At least three biological replicates were performed for each antibody and treatment.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Brian Oliver

PROVIDER: E-GEOD-16344 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Expression in aneuploid Drosophila S2 cells.

Zhang Yu Y Malone John H JH Powell Sara K SK Periwal Vipul V Spana Eric E Macalpine David M DM Oliver Brian B

PLoS biology 20100223 2

Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets ...[more]

PMID: 20186269

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Project description:Dosage compensation refers to the equalization of most X-linked gene products between males and females. In Drosophila, it is mediated by the MSL complex that preferentially associates with numerous sites on the X chromosome in somatic cells of males, and is responsible for an enhancement of the transcriptional rate of a substantial number of X-linked genes. Here we show that topoisomerase II (Topo II) is an integral part of the mechanistic basis of dosage compensation and we highlight a novel function for this enzyme. A widely accepted model of transcription postulates that the moving bubble generated by an RNA polymerase elongating complex induces positive DNA supercoiling in the region ahead of the complex and negative supercoiling in its wake. These transitory changes in supercoiling are resolved by the action of topoisomerases. We have investigated the role of Topo II in dosage compensation by RNAi-mediated depletion, and we have used chromatin immunoprecipitation to determine its genomic distribution and relative abundance on X-linked genes. Topo II is enriched on dosage compensated genes and this enrichment is independent from the approximate two-fold enhancement in transcription of these genes. We have demonstrated an RNA-dependent association of Topo II with MLE, the ATPase-helicase subunit of the MSL complex. Our results indicate a role for Topo II that is additional to and different from its function in restoring normal DNA superhelicity during the transcription process. We suggest that the enhanced level of Topo II alters the DNA supercoiling of compensated gene units to facilitate transcription and could provide a basis for the recent report that the MSL complex enhances transcription by increasing the rate of elongation of RNA polymerase II. Investigation of dosage compensated gene expression levels in S2 and S2 Topo II RNAi knockdown cells. mRNA was isolated from S2 cells and S2 cells with RNAi knockdown of Topo II. The mRNA was then converted to cDNA and hybridized to a NimbleGen D. melanogaster gene expression array. 2 replicates each.

Project description:The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at a subset of sites. However, the consensus sequence motif of entry sites (“MSL recognition element” or MRE) is only slightly enriched on the X (~2 fold), and only a fraction of them is utilized by the MSL complex. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the NHGRI modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells, which contain MSL complex, and female Kc cells, which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in surrounding sequence, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites, and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our finding serves as a model to understand how chromatin and local sequence features are involved in the selection of functional protein binding sites in the genome. The key Drosophila female sex determinant protein, SXL, represses dosage compensation by inhibiting MSL2 translation. Loss of SXL results in the expression, stabilization, and targetting of the MSL complex in female cells. Therefore, depletion of SXL by RNA interference (RNAi) in female Kc cells will lead to a MSL2-dependent increase in transcription from the female X chromosomes, consistent with the induction of dosage compensation. In this experiment, we generated gene expression profiles of Kc cells of control (GFP), Sxl RNAi and Sxl-Msl2 RNAi experiments.

Dataset Information

Expression in Aneuploid Drosophila S2 Cells

Publications

Expression in aneuploid Drosophila S2 cells.

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