Its function in flies is unknown. Division of labor between chromatin modifying enzymes is key to ensure efficient Linderane regulation of nuclear processes. GUID:?072FC39C-D5A8-4342-8CFA-2288C9525604 Source code 4: R script for analysis of acetylated peptides. elife-56325-code4.r (32K) GUID:?239B5D15-83C9-4077-9AB6-CACD2EF4201E Supplementary file 1: Excel spreadsheet containing imputed LFQ values obtained from the MaxLFQ algorithm, output and DOM-A or DOM-B specific interactors. elife-56325-supp1.xlsx (1.3M) GUID:?1C83096A-6DB4-4127-B285-4E75FF7890A1 Supplementary file 2: Excel spreadsheet containing result tables from DEseq2 analysis. elife-56325-supp2.xlsx (5.2M) GUID:?782B5365-52F2-4E8B-B7BE-0DC7AF8D9543 Supplementary file 3: Comparison of the known subunits of SWR1- and NuA4-type complexes between and scaling and input normalization for ChIP-seq are available on GitHub (https://github.com/tschauer/Domino_ChIPseq_2020;?Schauer, 2020a; copy archived at https://github.com/elifesciences-publications/Domino_ChIPseq_2020). Scripts for RNA-seq analysis are available on GitHub (https://github.com/tschauer/Domino_RNAseq_2020;?Schauer, 2020b; copy archived at https://github.com/elifesciences-publications/Domino_RNAseq_2020). Immunofluorescence images used for quantification of the complementation assays are available on Dryad (https://doi.org/10.5061/dryad.1rn8pk0qt). Sequencing data have been deposited in GEO under accession code “type”:”entrez-geo”,”attrs”:”text”:”GSE145738″,”term_id”:”145738″GSE145738. Targeted proteomics data are available at ProteomeXchange under accession number PXD017729. Immunofluoresce images are available at Dryad under accession number https://doi.org/10.5061/dryad.1rn8pk0qt. The following datasets were generated: Scacchetti A, Schauer TR, Apostolou Z, Sparr AC, Krause S, Heun P, Wierer M, Becker PB. 2020. Drosophila SWR1 and NuA4 complexes originate from DOMINO splice isoforms. NCBI Gene Expression Omnibus. GSE145738 Scacchetti A, Schauer TR, Apostolou Z, Sparr AC, Krause S, Heun P, Wierer M, Becker PB. 2020. Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoform. ProteomeXchange. PXD017729 Scacchetti A, Schauer TR, Apostolou Z, Sparr AC, Krause S, Heun P, Wierer M, Becker PB. 2020. Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoform. Dryad Digital Repository. [CrossRef] Abstract Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. generates the same diversity by alternative splicing of a single gene. (transcripts are alternatively spliced to generate two major isoforms, DOM-A and DOM-B (Ruhf et al., 2001). We and others previously found that the two splice variants play non-redundant, essential roles during development with interesting phenotypic differences (B?rner and Becker, 2016; Liu et al., 2019). In this work, we systematically Linderane characterized the molecular context and function of each DOM splice variant in cell lines and assessed Linderane their contribution to the activity of the HNRNPA1L2 DOM.C in the context of transcription. We discovered the existence of two separate, isoform-specific complexes with characteristic composition. Both are involved in transcription regulation, but through different mechanisms. On the one hand, we found that the DOM-B.C is the main ATP-dependent remodeler for H2A.V, responsible for its deposition across the genome and specifically at active promoters. On the other hand, we discovered that DOM-A.C is not involved in bulk H2A.V incorporation, despite the presence of an ATPase domain and many shared subunits with DOM-B.C. Rather, we realized that DOM-A.C might be the missing acetyltransferase NuA4.C of gene in embryonic cell lines using CRISPR/Cas9. The sites were chosen such that either DOM-A (DOM-RA) or DOM-B (DOM-RE) would be tagged at their C-termini. Of note, the editing of DOM-A C-terminus results in the additional tagging of a longer, DOM-A-like isoform (DOM-RG, which compared to DOM-RA has an insertion of 35 residues at its N-terminus starting from residue 401), but leaves a second DOM-A-like isoform untagged (DOM-RD, 16 residues shorter than DOM-RA at the very C-terminus). We obtained three different clonal cell lines for each isoform (3 homozygous clones for DOM-A, 2 homozygous and 1 heterozygous clone for DOM-B) (Figure 1figure supplement 1A,B). The gene editing resulted in the expression of 3XFLAG-tagged proteins of the correct size and with similar expression levels across clones (Figure.