Person:

Wang, Charlotte

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.

Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs) of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET. We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS) of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.