Independent manipulation of histone H3 modifications in individual nucleosomes reveals the contributions of sister histones to transcription
- Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
- Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- Shanghai Tech University, China
Figures
Figure 1 with 1 supplement
The complementary mutants of histone H3A110D and H3L130H are assembled into nucleosomes in vivo.
(A) Schematic illustration of the production of histone H3 mutants that are able to complement and form heterodimers. (B and D) Schematic strategy for screening histone H3 mutants. Genomic HHF1, HHF2…
Figure 1—figure supplement 1
The pairwise histone H3 mutants other than H3A110D and H3L130H cannot grow when the WT histone H3 gene is counter-selected using 5-FOA.
A dotting assay was performed as in Figure 1E. The wild-type strain (LHT001) that survived on the 5-FOA plate is highlighted in red.
Figure 2 with 2 supplements
Characterization of the H3D/H3H strain.
(A) Mononucleosomes prepared from both the cells expressing Myc-tagged H3 (ZL38) and the cells expressing untagged H3 (LHT001) were mixed (Myc-H3 +H3) and immunoprecipitated with the anti-Myc …
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Figure 2—source data 1
Characterization of the H3D/H3H strain.
- https://doi.org/10.7554/eLife.30178.008
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Figure 2—source data 2
Analysis of RNA-Seq data.
- https://doi.org/10.7554/eLife.30178.009
Figure 2—figure supplement 1
Mononucleosome preparation from H3D/H3H cells.
(A) Mononucleosomes were prepared from H3D/H3H cells, and purified by fast protein liquid chromatography. Molecular standards are labeled above the elution profile. (B) Fractions around the peak …
Figure 2—figure supplement 2
Sample-to-sample reproducibility for the RNA-Seq assay of WT (LHT001) and H3D/H3H strains.
(A) The scatter plot shows the FPKM distribution for the two WT replicates. The Pearson's product-moment correlation for WT_rep1 and WT_rep2 is 0.9907. The red line is the fitted curve, which has a …
Figure 3
Examination of an asymmetric deletion in the N-terminus of histone H3.
(A) Schematic illustration of asymmetrical N-terminal-deleted histone H3 mutants of H3DΔ4–15/H3H, H3D/H3HΔ4–15 and H3DΔ4–15/H3HΔ4–15 derived from the H3D/H3H strain. The genotype of each mutant is …
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Figure 3—source data 1
Examination of an asymmetric deletion in the N-terminus of histone H3.
- https://doi.org/10.7554/eLife.30178.011
Figure 4
Asymmetrically methylated K4 on sister H3s independently upregulates GAL1 transcription efficiency.
(A) Schematic illustration of asymmetrical mutation of histone H3K4R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is marked by ® on the histone H3 tails. The color labeling of each …
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Figure 4—source data 1
Asymmetrically methylated K4 on sister H3s independently upregulates GAL1 transcription efficiency.
- https://doi.org/10.7554/eLife.30178.013
Figure 5
The H3K36me3 marks on sister histones regulate transcription independently.
(A) Schematic illustration of asymmetrical mutation of histone H3K36R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is marked by ® on the histone H3. The color labeling of each …
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Figure 5—source data 1
The H3K36me3 marks on sister histones regulate transcription independently.
- https://doi.org/10.7554/eLife.30178.015
Figure 6
The H3K79me marks on both sister histones contribute to the regulation of sub-telomeric chromatin structure.
(A) Schematic illustration of the asymmetrical mutation of histone H3K79R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is marked by on the histone H3. The color labeling of each …
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Figure 6—source data 1
The H3K79me marks on both sister histones contribute to the regulation of sub-telomeric chromatin structure.
- https://doi.org/10.7554/eLife.30178.017
Figure 7
The performance of histone mutants challenged by multiple DNA damage reagents.
Dotting assay was performed in the indicated mutants as in Figure 2D. Plates were photographed after incubation at 30°C on YPD medium or YPD medium containing DNA damage reagents, that is, 5 mg/ml …
Figure 8 with 1 supplement
Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on sister H3s in response to glucose starvation.
(A) Histogram showing the gene skewness score (GSS) of asymmetrical K4R mutants in set MID calculated by a GSS model (see the Materials and methods for details). Bidirectional arrows with gradient …
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Figure 8—source data 1
Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on sister H3s in response to glucose starvation.
- https://doi.org/10.7554/eLife.30178.021
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Figure 8—source data 2
Genes regulated by H3K4R mutation under glucose starvation.
- https://doi.org/10.7554/eLife.30178.022
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Figure 8—source data 3
GSS of K4R mutants.
- https://doi.org/10.7554/eLife.30178.023
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Figure 8—source data 4
Gene type classification.
- https://doi.org/10.7554/eLife.30178.024
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Figure 8—source data 5
KEGG pathway analysis of RNA-Seq data.
- https://doi.org/10.7554/eLife.30178.025
Figure 8—figure supplement 1
Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on sister H3s in response to glucose starvation.
(A) Heatmap showing the log2 fold-change of 2500 genes that exhibit significant differences (p<0.05, t-test) between H3D/H3H and H3DK4R/H3HK4R cells. The set MID was the ~1500 gene collection of …
Author response image 1
H3D/H3H strain grow as well as WT cell when challenged by rapamycin.
WT (LHT001) and H3D/H3H cells were grown on YP plate with 100nM rapamycin and photographed at Day1-3 as indicated on the top. Cells grown on YPD plate was set as a control which was displayed in Figu…
Author response image 2
Linear range of antibodies.
Nucleosomes were immunoprecipitated and loaded in gradient as indicated. Western blot was performed using the indicated antibodies and quantified by ImageJ software. The relationship (black line) …
Author response image 3
Immunoprecipitation analysis using anti-Myc antibody.
Additional files
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Supplementary file 1
S. cerevisiae strains used in this study.
- https://doi.org/10.7554/eLife.30178.026
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Supplementary file 2
Antibodies used in this study.
- https://doi.org/10.7554/eLife.30178.027
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Supplementary file 3
Oligonucleotides used in this study.
- https://doi.org/10.7554/eLife.30178.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.30178.029
