MAPPING THE ROLE OF CHIFFON AND GCN5 HISTONE ACETYLTRANSFERASE COMPLEX IN DROSOPHILA
The histone acetyltransferase (HAT) Gcn5 was first characterized in yeast, and is conserved throughout eukaryotes where it functions as part of large multi-subunit transcriptional coactivator complexes. Drosophila melanogaster contains four Gcn5-containing complexes: the Spt-Ada-Gcn5 Acetyltransferase (SAGA), Ada2a-containing (ATAC), Ada2/Gcn5/Ada3 transcription activator (ADA), and Chiffon Histone Acetyltransferase (CHAT) complexes. Each of these Gcn5 complexes is nucleated by unique Ada2 homologs (Ada2a or Ada2b) or splice isoforms (Ada2b-PB or Ada2b-PA) that share conserved N-terminal domains, and differ only in their C-terminal domains. Whereas the SAGA and ADA complexes are also present in the yeast Saccharomyces cerevisiae, ATAC has only been identified in other metazoa such as humans, and the CHAT complex appears to be unique to insects.
I describe the first identification and characterization of the insect-specific Gcn5-containing complex, CHAT. Drosophila has two splice isoforms of Ada2b paralog. Mass spectrometry proteomic studies reveal that only the Ada2b-PB isoform is present in SAGA; in contrast, the Ada2b-PA isoform associates with the Gcn5 HAT core subunits and Chiffon, the fly ortholog of Dbf4, forming the CHAT complex. Our findings showed that CHAT is essential for both histone acetylation and fly viability through its CHAT-specific subunit, Chiffon. Chiffon is required for the specialized form of DNA replication, endoreplication, however it is not required for mitotic replication. Our mass spectrometry and genetics studies demonstrate that Chiffon interacts in a mutually exclusive manner with Cdc7 and Gcn5. Whereas the N-terminal domain of Chiffon interacts with Cdc7 (cell division cycle 7) forming the DDK complex, the C-terminal domain binds to Gcn5 nucleating the formation of CHAT. This studies also demonstrate that both complexes function independently in DNA replication and histone acetylation, respectively. Expression of the C-terminal domain, which partially rescues histone acetylation, also restores fly viability, suggesting that the essential function of Chiffon relates to its histone acetyltransferase activity rather than Cdc7 activation. I present data that supports the hypothesis that chiffon is a dicistronic gene that encodes two distinct polypeptides from alternative translation start sites, generating separate DDK and CHAT complexes. Finally, I explore the role of CHAT in regulating gene expression in Drosophila embryos. I show that the Drosophila Gcn5-containing complexes, SAGA/ADA and CHAT, have largely redundant roles in embryonic gene expression. However, when comparing RNA-sequencing (RNA-seq) of chiffon mutant and ada2b mutant embryos, we show that there is a little overlap between the genes disrupted when the Ada2b or Chiffon subunits of CHAT are disrupted. Moreover, our findings show that Chiffon is required for global H3K14ac in embryos far beyond that deposited by other Gcn5/Ada2b-containing complexes. This data suggests a model in which CHAT functions as a pioneer coactivator complex during embryogenesis that is necessary for the recruitment or activity of other HAT complexes. Finally, immunostaining studies revealed that there is a temporal switch between the expression of the chiffon gene products during a short window during early embryogenesis, in which the polypeptide product that nucleates formation of CHAT is expressed just prior to the wave of RNA polymerase II recruitment to chromatin in the early embryo. Overall my research provide further insight into the biological function of CHAT, and suggest that CHAT could have a key role in triggering the wave of early histone acetylation that stimulates zygotic genome activation.
- Doctor of Philosophy
- West Lafayette