ASH1-catalyzed H3K36 methylation drives gene repression and marks H3K27me2/3-competent chromatin
- University of Oregon, United States
- Charles University, Czech Republic
- St. George's University School of Medicine, Caribbean
- University of Fukui, Japan
Figures
Figure 1
H3K36 KMT activity in Neurospora crassa.
(A) Schematic of ASH1-orthologs in Neurospora crassa, Drosophila melanogaster, and Homo sapiens. (B) Multiple sequence alignment of the SET domain of ASH1 orthologs. Highlighted: F/Y Switch and …
Figure 2 with 1 supplement
ASH1 and SET-2 specificities segregate the genome into compartments of poorly and robustly transcribed genes.
(A) Representative IGV tracks of H3K36me2 ChIP-seq in WT, Δset-2, and ash1(Y888F) backgrounds. Gene location, DNA methylation (to highlight constitutive heterochromatin), and ‘input’ tracks are …
Figure 2—figure supplement 1
(A) Representative IGV tracks of H3K36me3 ChIP-seq in WT, ash1(Y888F), and Δset-2 backgrounds are shown for LGIII.
Gene location and DNA methylation are included for reference. All of LGIII is shown. (B) Average H3K36me2 signal was determined for each gene in the Δset-2 background and the distribution of signal …
Figure 3 with 1 supplement
ASH1 catalytic activity maintains repression of poorly transcribed genes.
(A) Gene expression changes are displayed as scatter plots of log2-fold changes vs. mean of normalized counts for ash1(Y888F) and Δset-2 strains compared to WT controls. Duplicate biological …
Figure 3—figure supplement 1
(A) Average genic H3K36me2 levels (X-axis) catalyzed by ASH1 (defined in Figure 2) are plotted against change in gene expression (Y-axis) in the ash1(Y888F) background.
https://doi.org/10.7554/eLife.41497.007
Figure 4
ASH1-catalyzed H3K36me2 delineates H3K27me2/3-competent chromatin.
(A) Representative IGV tracks for H3K27me2/3 in WT and H3K36me2 as catalyzed by ASH1. All of LGIII is shown. (B) Heatmap showing the distribution of average H3K27me2/3 signal intensity in WT (left) …
Figure 5 with 2 supplements
ASH1 activity differentially regulates H3K27me2/3 accumulation.
(A) Representative IGV tracks of H3K27me2/3 in WT and ash1(Y888F) are shown for LGVI. Gene locations are included for reference. (B) Heatmap highlighting frequency and intensity of H3K27me2/3 loss …
Figure 5—figure supplement 1
(A, B) H3K36me2 ChIPseq in a set-2; set-7 strain displayed on IGV with WT H3K27me2/3 and set-2 H3K36me2.
All of LGIV is included in (A) and a zoomed-in segment of LGIII in (B). (C) Immunoblotting H3K36me2 and H3 in WT, set-2, and set-2; set-7 strains.
Figure 5—figure supplement 2
Gene expression changes are summarized for genes marked by ASH1-catalyzed H3K36me2 (Top) and genes comarked by ASH1-catalyzed H3K36me2 and SET-7-catalyzed H3K27me2/3 (Bottom) in the ash1(Y888F) and Δset-7 strains.
Results for Δset-7 are derived from Klocko et al., 2016.
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Figure 5—figure supplement 2—Source data 1
Gene expression changes (log2 ash1(Y888F)/wt).
- https://doi.org/10.7554/eLife.41497.012
Figure 6
Model for H3K36me deposition in Neurospora crassa.
Neurospora genes can be divided into two groups depending upon their source of H3K36me. SET-2 marks the gene body of actively transcribed genes, and conversion to the trimethylated state is tied to …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (N. crassa) | WT | FGSC#6103 | N623 | mat A; his-3 |
| Strain, strain background (N. crassa) | WT | Honda and Selker, 2008 | N2930 | mat A; his-3; ∆mus-52::bar |
| Strain, strain background (N. crassa) | WT | FGSC#2489 | N3752 | mat A |
| Strain, strain background (N. crassa) | WT | FGSC#4200 | N3753 | mat a |
| Strain, strain background (N. crassa) | ash1-3xFLAG | this study | N4865 | mat A; his-3; ash1- 3xFLAG::hph |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N4877 | mat A; his-3; ash1(Y888F)− 3xFLAG::hph; ∆mus-52::bar |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N4878 | mat A; his-3; ash1(Y888F) −3xFLAG::hph |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N6268 | mat A; his-3; ash1(Y888F) −3xFLAG::hph |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N6269 | mat A; his-3; ash1(Y888F) −3xFLAG::hph |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N6875 | mat a; ash1(Y888F)−3x FLAG::nat |
| Strain, strain background (N. crassa) | ash1(Y888F) | this study | N6878 | mat a; ash1(Y888F) −3xFLAG::nat; ∆mus-52::bar |
| Strain, strain background (N. crassa) | ∆set-2 | FGSC#15504 | N5761 | mat a; ∆set-2::hph |
| Strain, strain background (N. crassa) | ∆set-2 | this study | N6335 | mat A; ∆set-2::hph |
| Strain, strain background (N. crassa) | set-2(∆SRI) | this study | N6956 | mat A; set-2(∆SRI) ::nat |
| Strain, strain background (N. crassa) | ∆set-7 | FGSC# 11182 | N4718 | mat a; ∆set-7::hph |
| Strain, strain background (N. crassa) | ∆set-7 | Jamieson et al., 2018 | N4730 | mat A; ∆set-7::bar |
| Strain, strain background (N. crassa) | ∆npf | FGSC# 13915 | N4721 | mat a; ∆npf::hph |
| Strain, strain background (N. crassa) | ash1(Y888F); ∆set-2::nat | this study | N6266 | mat A; his-3; ash1(Y888F) −3xFLAG::hph; ∆set-2::nat; ∆mus-52::bar |
| Strain, strain background (N. crassa) | ash1(Y888F); ∆set-2 | this study | N6267 | mat A; his-3; ash1(Y888F) −3xFLAG::hph; ∆set-2::nat |
| Strain, strain background (N. crassa) | ∆set-2; ∆set-7 | this study | N6333 | mat ?; ∆ set-2::hph; ∆set-7::bar |
| Strain, strain background (N. crassa) | ∆set-2; ∆set-7 | this study | N6334 | mat ?; ∆set-2::hph; ∆set-7::bar |
| Strain, strain background (N. crassa) | csr-1: (TTAGGG)17 | Jamieson et al., 2018 | N6383 | mat a; csr-1: (TTAGGG)17; ∆mus-52::bar |
| Antibody | H3K36me3 | Cell Signaling | Cat#4909S, Clone (D5A7)) | immunoblot (1:1000) |
| Antibody | H3K36me2 | Abcam | Cat#ab9049 | immunoblot (1:1000) |
| Antibody | Histone H3 | Abcam | Cat#ab1791 | immunoblot (1:2000) |
| Antibody | IRDye 680RD Goat-anti-Rabbit secondary antibody | Licor | Cat#926–68071 | immunoblot (1:5000) |
| Antibody | H3K27me3 | Millipore | Cat#07–449 | Chromatin immuno precipitation |
| Antibody | H3K36me3 | Abcam | Cat#ab9050 | Chromatin immuno precipitation |
| Antibody | H3K36me2 | Abcam | Cat#ab9049 | Chromatin immuno precipitation |
| Antibody | H3K27ac | ActiveMotif | Cat#39133 | Chromatin immuno precipitation |
| Antibody | H3K27me2/3 | ActiveMotif | Cat#39535 | Chromatin immuno precipitation |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.41497.014
