Abstract
Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (WT) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic downregulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated cell-enriched ba g of m arbles (bam) mutant testis, but downregulated upon differentiation in WT testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in WT testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are coenriched in undifferentiated cells in testis, suggesting that these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960.
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Acknowledgements
We would like to thank Drs Allan Spradling (Carnegie Institution, USA), Karen Beemon and Mark Van Doren (The Johns Hopkins University, USA), and Chen lab members for critical readings and suggestions on the manuscript. We thank Dr Dustin E Schones (National Institutes of Health, USA) for help to set up the initial data analysis pipeline, and Caitlin Choi and Ankit Vartak for technical assistance with the PCR experiments. This work is supported in part by Research Grant No. 05-FY09-88 from the March of Dimes Foundation, the R00HD055052 NIH Pathway to Independence Award from NICHD, the 49th Mallinckrodt Scholar Award from the Edward Mallinckrodt, Jr. Foundation, support from the Johns Hopkins University (XC) and the Division of Intramural Research, the National Heart, Lung and Blood Institute, NIH (KZ).
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(Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Figure S1
Methods of RNA-seq. (PDF 69 kb)
Figure S2
Comparison of RNA-seq data with microarray data. (PDF 83 kb)
Figure S3
More examples of genes that have sex-specific isoforms. (PDF 107 kb)
Figure S4
Venn diagrams of pair-wise comparisons of CFTRs. (PDF 96 kb)
Table S1
The RPKM values for expressed genes in at least one of the four samples. (XLS 2071 kb)
Table S2
Expression of genes involved in splicing in bam vs. wt testis. (XLS 89 kb)
Table S3
Meta table of multi-isoform genes in all four samples. (XLS 511 kb)
Table S4-1
Genes with different isoforms in bam testis vs. bam ovary samples. (XLS 260 kb)
Table S4-2
Genes with different isoforms in wt testis vs. wt ovary samples (XLS 260 kb)
Table S4-3
Genes with different isoforms in wt testis vs. bam testis samples (XLS 260 kb)
Table S4-4
Genes with different isoforms in wt ovary vs. bam ovary samples (XLS 260 kb)
Table S5
Genes that exhibit stage-specific or stage-biased isoforms in bam vs. wt gonads. (XLS 34 kb)
Table S6
Detection of CFTRs in all four samples. (XLS 1564 kb)
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Gan, Q., Chepelev, I., Wei, G. et al. Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq. Cell Res 20, 763–783 (2010). https://doi.org/10.1038/cr.2010.64
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DOI: https://doi.org/10.1038/cr.2010.64
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