ChIP-mass spectrometry captures protein interactions and modified histones associated with dosage compensation in Drosophila
| Autor: | Wang, C. I., Alekseyenko, A. A., LeRoy, G., Elia, A. E. H., Gorchakov, A. A., Britton, L-M. P., Elledge, S. J., Kharchenko, P. V., Garcia, B. A., Kuroda, M. I. |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Male
endocrine system Chromatin Immunoprecipitation fungi Blotting Western Nuclear Proteins Acetylation Histone-Lysine N-Methyltransferase Methylation Article Mass Spectrometry Animals Genetically Modified Histones Drosophila melanogaster Dosage Compensation Genetic Animals Drosophila Proteins Female RNA Interference Oxidoreductases |
| Popis: | X chromosome dosage compensation is required in male Drosophila to increase gene expression from the single X to equal that of both female X chromosomes. Although this is a sex-specific process, the MSL dosage compensation complex is thought to interface with non-sex-specific factors for both targeting and function. Therefore, a biochemical approach to MSL-associated factors is needed to complement genetic studies based on male-specific lethality. Here, we applied chromatin interacting protein-mass spectrometry (ChIP-MS) to identify MSL interactions on crosslinked chromatin, rather than focusing solely on complexes released from the DNA. Using this approach we identified MSL-enriched histone modifications, CG1832, a zinc finger protein implicated in initial MSL localization, and CG4747, a putative H3K36me3 binding protein. We found that CG4747 is associated with the bodies of active genes, coincident with H3K36me3, and is mis-localized in the set2 mutant lacking H3K36me3. CG4747 loss-of-function in vivo results in partial mis-localization of MSL complex to autosomes, and RNAi in cell culture confirms that CG4747 and SET2 function together to facilitate targeting of MSL complex to active genes. Our results demonstrate that the combination of crosslinking, affinity-purification, and mass spectrometry is a promising avenue for discovery of functional interactions on the chromatin template. |
| Databáze: | OpenAIRE |
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