Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms
Scacchetti, Alessandro; Becker, Peter (2020), Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms, Dryad, Dataset, https://doi.org/10.5061/dryad.1rn8pk0qt
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.
Complementation assays and immunofluorescence
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.
Deutsche Forschungsgemeinschaft, Award: Project ID 213249687 - SFB1064-A1