Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms
- Molecular Biology Division, Biomedical Center, Ludwig-Maximilians-University, Germany
- Bioinformatics Unit, Biomedical Center, Ludwig-Maximilians-University, Germany
- Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Germany
- Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, United Kingdom
Figures
Figure 1 with 1 supplement
DOMINO isoform-specific affinity enrichment reveals distinct DOM-A and DOM-B complexes.
(A) Schematic representation of the DOM-A (dom-RA) and DOM-B (dom-RE) isoforms. The two proteins are derived through alternative splicing and differ in their C-termini. (B) Western blot showing the …
Figure 1—figure supplement 1
DOMINO isoform-specific affinity enrichment reveals distinct DOM-A and DOM-B complexes.
(A) Agarose gel analysis of CRISPR clones.The insertion of a 3XFLAG tag results in a PCR product 72 bp longer than in the untagged cells (-). The presence of two bands indicates heterozygosity. NEB …
Figure 2 with 1 supplement
Isoform-specific depletion of DOM-A and DOM-B causes distinct transcriptional effects.
(A) Western blot showing the expression of DOM-A, DOM-B and TIP60 in nuclear extracts of Kc167 cells treated with dsRNA against GST (CTRL), H2A.V, both DOM isoforms (AB), DOM-A (A), DOM-B (B) and …
Figure 2—figure supplement 1
Isoform-specific depletion of DOM-A and DOM-B causes distinct transcriptional effects.
(A) Plot showing relative log2-expression of tip60 mRNA as measured by RNAseq upon knock-down of protein indicated on the x-axis and by color-coding. The values of 3 biological replicates are shown …
Figure 3 with 1 supplement
DOM-B is responsible for H2A.V incorporation into chromatin in an ATP-dependent manner.
(A) Western blot showing the expression of H2A.V in nuclear extracts derived from Kc167 cells treated with dsRNA against GST (CTRL), H2A.V, DOM-A (A), DOM-B (B) and TIP60. Histone H4 (H4): loading …
Figure 3—figure supplement 1
DOM-B is responsible for H2A.V incorporation into chromatin in an ATP-dependent manner.
(A) Replicate western blot showing the expression of H2A.V in nuclear fractions derived from cells treated with dsRNA against GST/GFP, H2A.V, DOM-A, DOM-B and TIP60. Numbers indicate the dsRNA …
Figure 4 with 1 supplement
The DOM-A.C acetylates H4K12.
(A) Heatmap shows scaled acetylation levels for various histone H4 residues (measured by mass-spectrometry) in Kc167 cells treated with dsRNA against GST or GFP (CTRL), H2A.V, DOM-A (A), DOM-B (B) …
Figure 4—figure supplement 1
The DOM-A.C acetylates H4K12.
(A) Schematic comparison of S. cerevisiae EAF1 and D. melanogaster DOM-A. Conserved domain structure is highlighted, (B) Barplot showing the average fraction of acetylated peptide (over …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (D. melanogaster) | Kc167 | DGRC | FLYB;FBtc0000001 | |
| Cell line (D. melanogaster) | S2 (Clone L2-4) | Villa et al., 2016 | Gift from P Heun lab | |
| Cell line (D. virilis) | 79f7Dv3 | Albig et al., 2019 | Gift from BV Adrianov | |
| Antibody | DOM-A (17F4) (rat monoclonal) | Börner and Becker, 2016 and this publication | 1:5 (WB) | |
| Antibody | DOM-A (SA-8977) (rabbit polyclonal) | This publication | 1:1000 (WB) | |
| Antibody | DOM-B (SA-8979) (rabbit polyclonal) | This publication | 1:1000 (WB) | |
| Antibody | TIP60 (11B10) (rat monoclonal) | This publication | 1:20 (WB) | |
| Antibody | H2A.V (Rb-H2Av) (rabbit polyclonal) | Börner and Becker, 2016 | 1:1000 (WB) 1:2500 (IF) 25 μl/IP (ChIP) | |
| Antibody | H4 (rabbit polyclonal) | abcam | ab10158 | 1:5000 (WB) |
| Antibody | H4K12ac (rabbit polyclonal) | Merck-Millipore | 07–595 | 1:2500 (IF) 2 μl/IP (ChIP) |
| Antibody | FLAG-m2 (mouse monoclonal) | Sigma-Aldrich | F3165 | 1:1000 (WB) |
| Antibody | GFP (mouse monoclonal) | Roche | 11814460001 | 1:500 (IF) |
| Antibody | Lamin (mouse monoclonal) | Gift from H Saumweber | 1:1000 (WB) | |
| Recombinant DNA reagent | pENTR3C (plasmid) | Thermo Fischer Scientific | A10464 | |
| Recombinant DNA reagent | pHWG (plasmid) | DGRC | Kind gift from P Korber |
Additional files
-
Source code 1
R script for analysis of AP-MS data.
- https://cdn.elifesciences.org/articles/56325/elife-56325-code1-v1.r
-
Source code 2
Java script for quantification of immunofluorescence pictures.
- https://cdn.elifesciences.org/articles/56325/elife-56325-code2-v1.ijm
-
Source code 3
R script for quantification of acetylated peptides.
- https://cdn.elifesciences.org/articles/56325/elife-56325-code3-v1.r
-
Source code 4
R script for analysis of acetylated peptides.
- https://cdn.elifesciences.org/articles/56325/elife-56325-code4-v1.r
-
Supplementary file 1
Excel spreadsheet containing imputed LFQ values obtained from the MaxLFQ algorithm, limma output and DOM-A or DOM-B specific interactors.
- https://cdn.elifesciences.org/articles/56325/elife-56325-supp1-v1.xlsx
-
Supplementary file 2
Excel spreadsheet containing result tables from DEseq2 analysis.
- https://cdn.elifesciences.org/articles/56325/elife-56325-supp2-v1.xlsx
-
Supplementary file 3
Comparison of the known subunits of SWR1- and NuA4-type complexes between D. melanogaster, S. cerevisiae and H. sapiens.
Subunit composition of the yeast SWR1 and NuA4 were obtained from the manually-curated SGD database (https://www.yeastgenome.org) (CPX-2122 and CPX3155). For the human complexes, we refer to the EP400 complex subunits described in Dalvai et al., 2015 and to the SRCAP subunits described in Feng et al., 2018.
- https://cdn.elifesciences.org/articles/56325/elife-56325-supp3-v1.docx
-
Supplementary file 4
gRNAs, repair templates and primers used in this study.
- https://cdn.elifesciences.org/articles/56325/elife-56325-supp4-v1.xlsx
-
Supplementary file 5
Excel spreadsheet containing raw output from Skyline analysis and results from quantification of acetylated peptides.
- https://cdn.elifesciences.org/articles/56325/elife-56325-supp5-v1.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/56325/elife-56325-transrepform-v1.docx
