Promoter nucleosome dynamics regulated by signalling through the CTD code
- University of Namur, Belgium
- Universidad de Salamanca, Spain
- Universitat Pompeu Fabra, Spain
Figures
Figure 1 with 1 supplement
The RNA polymerase II S2P affects nucleosome dynamic at the promoter of a subset of genes.
(A) Left panel: the percentage of dynamic nucleosomes aligned relative to the transcription start site (TSS) in the fission yeast genome. The red line indicates the average percentage of dynamic …
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Figure 1—source data 1
List of the synthetic lethal genetic interactions uncovered with the lsk1Δ and CTD S2A mutant strains.
- https://doi.org/10.7554/eLife.09008.004
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Figure 1—source data 2
General features of the dynamic nucleosomes and the associated genes.
List of the genes showing dynamic changes in the −1 nucleosome (TSS) with false discovery rate < 0.5% and list of the 10% genes showing the strongest promoter NDR size decrease in the S2A mutant (see Figure 1A).
- https://doi.org/10.7554/eLife.09008.005
Figure 1—figure supplement 1
Genome-wide synthetic lethal interaction mappings of the lsk1Δ and rpb1 S2A mutants link Rpb1 CTD S2P to chromatin biology.
(A) The Bioneer deletion library was screened in quadruplicate for synthetic lethal interactions with either the lsk1 deletion or the rpb1 CTD S2A strains. 98 interactions were detected for lsk1, …
Figure 2 with 1 supplement
The CTD S2P regulates histone occupancy and acetylation at the promoter of the ste11 gene.
(A) Left panel: the occupancy of H3 measured by ChIP using the indicated amplicons in the 5′ proximal region of the ste11 and adh1 genes in the indicated strains. Right panel: similar to the left …
Figure 2—figure supplement 1
Nucleosome scanning of the ste11 5′ region reveals increased occupancy in the CTD S2A mutant.
′(A) Nucleosome scanning analysis of the wt and rpb1 CTD S2A strains. Nucleosomal DNA enrichment at the indicated positions of the ste11 locus was determined by ChIP experiment on MNase-digested …
Figure 3 with 2 supplements
The increase of CTD S2P nearby the promoter region is necessary to reverse the CTD S5P-dependent deacetylation of nucleosomes.
(A) Nucleosome scanning analysis of the S2A and H3K36R strains. Nucleosomal DNA enrichment at the indicated positions of the ste11 locus was determined by ChIP experiment on MNase-digested …
Figure 3—figure supplement 1
HDAC-dependent control of ste11 expression by CTD S2P independently of Gcn5.
(A) The occupancy of Gcn5-TAP was measured by ChIP at indicated loci in a wt strain and a lsk1Δ mutant. Each column represents the mean percentage immunoprecipitation value ± SEM (n = 3) normalized …
Figure 3—figure supplement 2
Untagged control experiments for the chromatin immunoprecipitations.
(A) Similar to Figure 3C except that an untagged wild-type strain was used. (B) Similar to Figure 5C except that an untagged wild-type strain was used. (C) Similar to Figure 5D except that an …
Figure 4
The S2P antagonizes Set1 binding to the CTD.
(A) RQ of the ste11 mRNA determined by quantitative RT-PCR using the ΔΔct method in the wt, set1Δ, and H3K4R strains. Each column represents the averaged value ± SEM (n = 2). a.u.: arbitrary units. …
Figure 5 with 1 supplement
The phosphorylation of Lsk1 by Sty1 is required for CTD S2 phosphorylation in the 5′ proximal region of the ste11 gene.
(A) The Sty1-TAP protein was precipitated and used on beads for in vitro kinase assays using wt and mutated (4SA or 12SA) forms of the GST-Lsk1 protein as indicated. The MAP kinase was activated by …
Figure 5—figure supplement 1
The phosphorylation of the CTD S2 kinase Lsk1 on 12 sites in vivo is not required for its activity.
(A) Lsk1-TAP was purified from 1 g of soluble extract and the final eluted fraction was separated on SDS–PAGE, silver stained, and analyzed by mass spectrometry. (B) Protein sequence alignment of …
Figure 6
A chimeric HMG-Lsk1-HA protein induces ste11 expression independently of nitrogen starvation.
(A) A schematic of the chimeric HMG-Lsk1-HA protein. The wild-type Ste11 and Lsk1 proteins are depicted and the size of various regions indicated in amino acids. (B) Western blot analysis (anti-HA) …
Figure 7 with 1 supplement
The induction of inv1 upon glucose deprivation is regulated by S2P-dependent control of promoter nucleosome acetylation.
(A) Northern blot analysis of inv1 expression in the indicated strains after an hour shift from high glucose (2%) to low glucose (0.1%). Note that the mRNA is not detected in high glucose (not …
Figure 7—figure supplement 1
The RNA polymerase II CTD S2P reaches its maximal level early during the induction of the ste11 and inv1 genes.
The level of S2P (based on a ChIP using the 3E10 antibody) was determined and normalized on the total level of polymerase (based on a ChIP using the 8WG16 antibody). The ratio of the normalized S2P …
Figure 8 with 1 supplement
Model of the control of promoter nucleosome dynamics by CTD S2 phosphorylation.
When gene expression is low in non-induced conditions, the phosphorylation of the CTD on S5 recruits the Set1 methylase and the SET3C or the RPD3L HDAC complexes. The Set3 and Png2 subunits of these …
Figure 8—figure supplement 1
The role of CTD S2P in promoter histone acetylation is conserved in budding yeast.
(A) The wt and ctk1Δ strains were starved for nitrogen at the indicated time points (hours). The occupancy of acetylated H3K14 at the IME1 TSS was determined by ChIP using anti-H3-K14-ac normalized …
Additional files
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Supplementary file 1
Oligonucleotides used in this study.
- https://doi.org/10.7554/eLife.09008.020
