The Gcn5 Complexes in Drosophila As A Model for Metazoa

The histone acetyltransferase Gcn5 is conserved throughout eukaryotes where it functions as part of large multi-subunit transcriptional coactivator complexes that stimulate gene expression. Here, we describe how studies in the model insect Drosophila melanogaster have provided insight into the essential roles played by Gcn5 in the development of multicellular organisms. We outline the composition and activity of the four different Gcn5 complexes in Drosophila: the Spt-Ada-Gcn5 Acetyltransferase (SAGA), Ada2a-containing (ATAC), Ada2/Gcn5/Ada3 transcription activator (ADA), and Chiffon Histone Acetyltransferase (CHAT) complexes. 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. Each of these Gcn5 complexes is nucleated by unique Ada2 homologs or splice isoforms that share conserved N-terminal domains, and differ only in their C-terminal domains. We describe the common and specialized developmental functions of each Gcn5 complex based on phenotypic analysis of mutant flies. In addition, we outline how gene expression studies in mutant flies have shed light on the different biological roles of each complex. Together, these studies highlight the key role that Drosophila has played in understanding the expanded biological function of Gcn5 in multicellular eukaryotes

The Drosophila Dbf4 Ortholog Chiffon Forms A Complex with Gcn5 That Is Necessary for Histone Acetylation and Viability

Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. Here we show only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon forming the Chiffon Histone Acetyltransferase (CHAT) complex. Chiffon is theDrosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, here we find that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, we show that chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, Chiffon’s CHAT-binding domain is essential for viability but is not required for gene amplification, arguing against a role in DNA replication

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

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. 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