Mapping the Role of Chiffon and GCN5 Histone Acetyltransferase Complex in Drosophila

Eliana F Torres-Zelada, Purdue University

Abstract

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. In Chapter 1 I highlight key studies in fruit flies that have provided insight into the essential roles played by Gcn5 in the development of multicellular organisms. I outline the composition and activity of the four different Gcn5- containing complexes in Drosophila, describing key subunits that convey differences to these complexes in terms of their targeting, activity, and biological roles. In Chapter 2, 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. I present our findings 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 reveal that both Chiffon FL that contain premature stop codons on either position 174 (Chiffon FLWF24) or 376 (Chiffon FL* ), can restore fly viability, and surprisingly a ~48kDa product can be detected when both Chiffon FLWF24 and Chiffon FL* were immunoprecipitated via their C-terminal FLAG epitopes tags. This data supports the hypothesis that chiffonis a dicistronic gene that encodes two distinct polypeptides from alternative translation start sites, generating separate DDK and CHAT complexes. In Chapter 3, I explore the role of CHAT in regulating gene expression in Drosophila embryos. I show that the DrosophilaGcn5-containing complexes, SAGA/ADA and CHAT, have largely redundant roles in embryonic gene expression. However, when comparing RNAsequencing (RNA-seq) of chiffon mutant and ada2b mutant embryos, the studies 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.

Degree

Ph.D.

Advisors

Weake, Purdue University.

Subject Area

Analytical chemistry|Biochemistry|Bioinformatics|Cellular biology|Chemistry|Developmental biology|Genetics

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