DOI: 10.5061/dryad.1rn8pk0qt
title: Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms

description: Histone acetylation and deposition of H2A.Z variant are integral aspects
 of active transcription. In Drosophila, the single DOMINO chromatin
 regulator complex is thought to combine both activities via 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. Drosophila generates the same
 diversity by alternative splicing of a single gene.
description: <b>Complementation assays and
 immunofluorescence</b> 1-2 million Kc167 cells in
 2 ml complete Schneider’s Drosophila Medium were seeded in each well of a
 6-well plate. After 4 h, cells were transfected with 500 ng of
 complementation plasmid (described before) + 25 ng of pCoBlast (Thermo
 Fischer, Cat. No K5150-01) using Effectene Transfection Reagent (QIAgen,
 Cat. No 301425). 48 h after transfection, 2 ml of the cells were
 collected, transferred into T-25 flasks and diluted with 4 ml of complete
 Schneider’s Drosophila Medium + Blasticidin at a final concentration of 50
 ng/ul. 7-8 days after selection the cells were collected and treated with
 dsRNA as described before. For immunofluorescence,
 0.2-0.4 million cells in 200 µL of complete Schneider’s Drosophila Medium
 were seeded onto a round 12 mm coverslips (Paul Marienfeld GmbH &
 Co., Cat No. 0117520) placed separately inside wells of 12-well plates.
 Cell were allowed to attach for 2-4 h and the coverslips were gently
 rinsed with 500 µL of PBS. Cells were fixed in 500 µL  of ice-cold PBS +
 2% formaldehyde for 7.5 min. After removal of fixative, cells were
 permeabilized by adding 500 µL of ice-cold PBS + 0.25% Triton-X-100 + 1%
 formaldehyde and incubating for 7.5 min. Coverslips were washed two times
 with 1 ml of PBS and blocked with PBS + 3% BSA for 1h at room temperature.
 Coverslip were transferred onto a piece of parafilm, placed into a wet
 chamber and 40 µL of primary antibody solution was gently added onto the
 coverslip. After overnight incubation at 4°C, coverslips were transferred
 back to 12-well plates and washed twice with 1 ml of PBS. Coverslip were
 transferred again onto a piece of parafilm, placed into a wet chamber and
 40 µL of secondary antibody was gently added onto the coverslip. After 1 h
 incubation at room temperature, coverslips were transferred back to
 12-well plates and washed twice with 1 ml of PBS. Cells were incubated
 with 1ml of 0.2 µg/ml DAPI (Sigma-Aldrich, Cat. No 10236276001) for 5 min
 at room temperature. Coverslips were washed with PBS and with deionized
 water, mounted on slides with 8 µL of Vectashield (Vector Laboratories,
 Cat. No H-1000) and sealed with nail polish. Images were taken on a Leica
 SP5 confocal microscope. Images were processed and analyzed using
 Fiji. Script available as source code file.