Gcn5: The Quintessential Histone Acetyltransferase

In this Special Issue, we bring together many of the original researchers involved in the initial studies to identify and characterize Gcn5, together with leaders from the field who have contributed to our understanding of this quintessential histone acetyltransferase. First, Jim Brownell and David Allis describe the discovery of Gcn5 [8], followed by Brittany Albaugh and John Denu who highlight key structural and catalytic attributes of Gcn5 as the defining member of the Gcn5-related N-acetyltransferase (GNAT) protein superfamily [15]. Next, Michael Sack and colleagues describe a protein that is closely related to Gcn5, Gcn5L1, which lacks intrinsic histone acetylation activity but is still involved in protein acetylation as part of multi-subunit complexes that regulate aspects of vacuolar organelle function [16]. Gcn5, like Gcn5L1, is also found as part of large multi-subunit complexes, and in [17], Shelley Berger, Patrick Grant, and Fred Winston describe the genetic and biochemical studies that led to the identification of the most famous of these Gcn5 complexes, the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex in the yeast S. cerevisiae. Next, Jose M. Espinola Lopez and Song Tan describe the close interactions between Gcn5 and its immediate binding partners, the Ada2, Ada3 and Sgf29 proteins, that influence Gcn5 activity and control its incorporation into different complexes [18] . A recent series of cryo-EM studies have provided a window into SAGA structure and function, and our current understanding of the structure of the Gcn5 complexes and their function is outlined in the Special Issue by Dominique Helmlinger, Gabor Papai, Didier Devys, and László Tora [19]. The discussion of SAGA’s role in transcription is elaborated on by Brian Strahl and Scott Briggs in [20], who also discuss the interplay between histone modifications catalyzed by SAGA and other chromatin marks including histone phosphorylation, ubiquitination, and methylation

The non-dosage compensated Lsp1α gene of Drosophila melanogaster escapes acetylation by MOF in larval fat body nuclei, but is flanked by two dosage compensated genes

<p>Abstract</p> <p>Background</p> <p>In <it>Drosophila melanogaster </it>dosage compensation of most X-linked genes is mediated by the male-specific lethal (MSL) complex, which includes MOF. MOF acetylates histone H4 at lysine 16 (H4K16ac). The X-linked <it>Larval serum protein one </it>α (<it>Lsp1</it>α) gene has long been known to be not dosage compensated. Here we have examined possible explanations for why the <it>Lsp1</it>α gene is not dosage compensated.</p> <p>Results</p> <p>Quantitative RNase protection analysis showed that the genes flanking <it>Lsp1</it>α are expressed equally in males and females and confirmed that <it>Lsp1</it>α is not dosage compensated. Unlike control X-linked genes, <it>Lsp1</it>α was not enriched for H4K16ac in the third instar larval fat body, the tissue in which the gene is actively expressed. X-linked <it>Lsp1α promoter-lacZ </it>reporter transgenes are enriched for H4K16ac in third instar larval fat body. An X-linked reporter gene bracketed by <it>Lsp1</it>α flanking regions was dosage compensated. One of the genes flanking <it>Lsp1</it>α is expressed in the same tissue. This gene shows a modest enrichment for H4K16ac but only at the part of the gene most distant from <it>Lsp1</it>α. Phylogenetic analyses of the sequences of the genomes of 12 <it>Drosophila </it>species shows that <it>Lsp1</it>α is only present within the <it>melanogaster </it>subgroup of species.</p> <p>Conclusion</p> <p><it>Lsp1</it>α is not modified by the MSL complex but is in a region of the X chromosome that is regulated by the MSL complex. The high activity or tissue-specificity of the <it>Lsp1</it>α promoter does not prevent regulation by the MSL complex. The regions flanking <it>Lsp1</it>α do not appear to block access by the MSL complex. <it>Lsp1</it>α appears to have recently evolved within the <it>melanogaster </it>subgroup of <it>Drosophila </it>species. The most likely explanation for why <it>Lsp1</it>α is not dosage compensated is that the gene has not evolved a mechanism to independently recruit the MSL complex, possibly because of its recent evolutionary origin, and because there appears to be a low level of bound MSL complex in a nearby gene that is active in the same tissue.</p

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

Saccharomyces Cerevisiae Cdc7 Homology in Drosophila Melanogaster

Saccharomyces cerevisiae Dbf4(Dumbbell former 4) and Cdc7(Cell Division Cycle 7) form a complex that phosphorylates Mcm2 (Minichromosome maintenance 2) to initiate DNA replication. Cdc7 is a target for cancer research because there is a Cdc7 ortholog in humans that is necessary for DNA replication and cell survival. Our goal is to characterise a putative Cdc7 homolog in Drosophila melanogaster (dCdc7). We have previously shown that expression of the known Drosophila Dbf4 ortholog, Chiffon, and dCdc7 can rescue yeast cells deficient in active Cdc7. Our hypothesis is that the dCdc7 is activated by Chiffon to phosphorylate MCM2. To test this hypothesis, we will determine if Chiffon interacts with dCdc7 and if Chiffon triggers kinase activity of dCdc7. To do this we purified dCdc7, a putative kinase dead substitution mutant of dCdc7, Chiffon, and Chiffon truncations that are predicted to contain the interacting domains. To produce these proteins we performed a combination of subcloning, SLIC(sequence and ligation independent cloning), and MultiBac Baculovirus techniques. SLIC is a recently developed method of cloning that uses single strand sequence complements instead of ligation to combine vector and insert. This allows us to clone any insert without regard to enzyme restriction sites within the insert’s DNA sequence. MultiBac is a system that allows us to infect insect cells with a Baculovirus to coexpress multiple proteins. We purified our proteins through affinity chromatography using various resins that bind to tags on the proteins. These proteins will then be used for protein binding pulldowns and kinase activity assays to determine if Chiffon and dCdc7 have a physical interaction, and if dCdc7 is a kinase. The results from the protein binding pulldown and kinase assay are pending. The results from these experiments will show us whether or not this protein is the Cdc7 ortholog in Drosophila

In Vivo Tissue-Specific Chromatin Profiling in Drosophila Melanogaster Using GFP-Tagged Nuclei

The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila

A Screen to Identify SAGA-activated Genes that are required for Proper Photoreceptor Axon Targeting in Drosophila melanogaster

The inherited human genetic disease spinocerebellar ataxia type 7 (SCA7) is characterized by progressive neurodegeneration and visual impairment that ultimately leads to blindness. SCA7 results from a mutation in the human ATXN7 gene that causes an expansion of polyglutamine tracts in this gene’s corresponding protein. Human ATXN7 protein serves as a component of the deubiquitylase (DUB) module of the large, multi-subunit complex Spt-Ada-Gcn acetyltransferase, or SAGA. SAGA is a transcriptional coactivator and histone modifier that functions to deubiquitylate histone H2B and allow for transcription of SAGA-mediated genes to occur. In Drosophila, mutations in SAGA DUB’s Nonstop and sgf11 components compromise its deubiquitylase activity and result in mistargeting of photoreceptor axons R1-R6 within the developing eye-brain. Here, this work describes a screen to identify SAGA misregulated genes that are required for proper photoreceptor axon targeting in the developing visual system. Candidate genes were previously identified as targets of SAGA DUB by RNA-seq, and a previously optimized X-Gal staining protocol is used to visualize photoreceptor axon targeting in the Drosophila eye-brain upon candidate gene knockdown by RNAi. The results show that the transcriptional targets of SAGA are required cell-autonomously in glial cells for proper photoreceptor axon targeting. These data also suggest that genes involved in cell motility and photoreceptor axon targeting are pertinent players in proper visual and neurological development. By knowing the identity of these genes, it is possible to more clearly understand the pathogenesis of diseases such as spinocerebellar ataxia type 7

Determining the Binding Between SAGA Subunits and Spliceosomal Components

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. Here, we examine the specific binding partners between SAGA and splicing components utilizing the yeast two-hybrid system in spt7Δ Saccharomyces cerevisiae as a validation for the preliminary yeast two-hybrid performed, producing recombinant proteins through sequence and ligation-independent cloning (SLIC) and Baculovirus transfections to obtain purified proteins, and co-immunoprecipitation (co-IP) to detect specific protein-protein interactions from recombinant proteins. Yeast two-hybrid results reveal that Spt7 is necessary for the transcription of reporter genes used in this assay. Therefore, this assay cannot validate previous results or detect false positives. Currently, recombinant proteins are being produced to perform co-IPs to test direct protein interactions. The results from these experiments will demonstrate the type of binding between SAGA subunits and splicing factors and provide direct evidence of a link between all three of the elements of gene regulation

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Regulation of DNA Synthesis: The Identification of New Drosophila melanogaster Cdc7 Regulatory Subunits

Cell division cycle 7 (Cdc7) is an enzyme required for the initiation of DNA replication. Cdc7 cannot act alone requiring the binding of its regulatory subunit Dbf4 to perform its enzymatic function. Previous studies show that Dbf4 and Cdc7 are well conserved across eukaryotic organisms. Humans and Xenopus have multiple Cdc7 regulatory subunits, and recent studies suggest that Drosophila melanogaster might as well. Human Dbf4 was discovered because of its similarity to yeast Dbf4. It is possible that finding additional Cdc7 regulatory subunits in D. melanogaster could reveal related proteins in humans. As cancer is a disease caused by improper cell cycling, furthering our understanding of Cdc7 and the cell cycle regulation could lead to advances in cancer treatment. This study seeks to identify possible Cdc7 regulatory subunits by screening for D. melanogaster proteins that directly interact with Cdc7. The first goal was to use a Yeast 2-Hybrid assay to repeat results that indicated an interaction between Cdc7 and Drosophila Dbf4, known as Chiffon. This allowed for testing media and the effectiveness of the assay. While not preformed yet, screening will be completed using a Yeast 2-Hybrid assay to determine interactions between Cdc7 and proteins from a D. melanogaster cDNA library. Further testing will remove false positives. Any remaining plasmids be sequenced and identified by the sequence comparison software, BLAST. Our study will test for D. melanogaster proteins that interact with Cdc7, but once these proteins are found further experimentation will be required to confirm interaction and function with Cdc7