Dosage compensation is a process that many multicellular organisms employ to equalize the expression of X-linked genes between males and females. In Drosophila melanogaster, it is achieved by a two-fold transcriptional activation of the single X chromosome in males. This is mediated by the male-specific lethal (MSL) complex, which is composed of at least five proteins (MSL-1, MSL-2, MSL-3, MOF, MLE) and two non-coding roXRNAs (RNA on X). In a two-step model, MSL complex targets the male X first by binding to chromatin entry sites in a sequence-dependent manner, and then spreads to bind all active genes in a sequence-independent mechanism. In order to biochemically characterize the MSL complex, we applied an approach that combines chromatin immunoprecipitation with mass spectrometry to preserve and analyze protein-protein interactions on the chromatin template. This approach enabled us to capture interacting proteins identified through genetics, but previously not detected in mass spectrometry of soluble complexes. We also identified enriched combinations of associated histone tail modifications by mass spectrometry rather than relying on antibody-based recognition. In addition to this proof-of-principle for the ChIP-MS approach, we identified novel candidates for MSL interaction, including CG4747, a putative H3K36me3 binding protein associated with transcribed bodies of active genes. We observed that CG4747 colocalizes with H3K36me3 and when the SET2 H3K36me3 methyl-transferase is disrupted, this colocalization is lost. CG4747 acts synergistically with SET2 for robust MSL-targeting on the male X chromosome at chromatin entry sites and active genes. Taken together, we successfully adapted ChIP-MS for the study of Drosophila chromatin proteins, and characterized CG4747 as a protein that interacts with the MSL dosage compensation complex. We propose that ChIP-MS is a powerful general method that may prove particularly useful for comprehensive analyses of chromatin-bound regulatory complexes.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10385007 |
| Date | January 2012 |
| Creators | Wang, Charlotte I-Wen |
| Contributors | Kuroda, Mitzi I. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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