Recruitment of Saccharomyces cerevisiae Cmr1/Ydl156w to Coding Regions Promotes Transcription Genome Wide

Abstract

Cmr1 (changed mutation rate 1) is a largely uncharacterized nuclear protein that has recently emerged in several global genetic interaction and protein localization studies. It clusters with proteins involved in DNA damage and replication stress response, suggesting a role in maintaining genome integrity. Under conditions of proteasome inhibition or replication stress, this protein localizes to distinct sub-nuclear foci termed as intranuclear quality control (INQ) compartments, which sequester proteins for their subsequent degradation. Interestingly, it also interacts with histones, chromatin remodelers and modifiers, as well as with proteins involved in transcription including subunits of RNA Pol I and Pol III, but not with those of Pol II. It is not known whether Cmr1 plays a role in regulating transcription of Pol II target genes. Here, we show that Cmr1 is recruited to the coding regions of transcribed genes of S. cerevisiae. Cmr1 occupancy correlates with the Pol II occupancy genome-wide, indicating that it is recruited to coding sequences in a transcription-dependent manner. Cmr1-enriched genes include Gcn4 targets and ribosomal protein genes. Furthermore, our results show that Cmr1 recruitment to coding sequences is stimulated by Pol II CTD kinase, Kin28, and the histone deacetylases, Rpd3 and Hos2. Finally, our genome-wide analyses implicate Cmr1 in regulating Pol II occupancy at transcribed coding sequences. However, it is dispensable for maintaining co-transcriptional histone occupancy and histone modification (acetylation and methylation). Collectively, our results show that Cmr1 facilitates transcription by directly engaging with transcribed coding regions.

Fig 1. Cmr1 is recruited to the transcribed coding sequences of Gcn4 and Gal4 target genes.

Cmr1-Myc tagged strains were treated with SM (0.6 μM) to induce Gcn4-target genes and processed for chromatin immunoprecipitation (ChIP) using anti-Myc antibodies. A) Cmr1 ChIP occupancies at the ARG1 TATA, 5’ and 3’ ORFs are shown under Gcn4 non-inducing (-SM) and inducing conditions (+ SM). B) Cmr1 occupancy was measured at various time-points (0 min to 60 min) during the course of ARG1 induction (+SM). Cmr1occupancy in a gcn4Δ at 60 minutes, post-induction is also shown. C) Cmr1 occupancy at the HIS4 5’ and 3’ ORFs under non-inducing and inducing conditions. D-E) Cmr1 occupancies at the 5’ and 3’ ORFs of ADH1 (D) and of PMA1 (E) are shown. Since both ADH1 and PMA1 are constitutively expressed, untagged WT strain was used as a control. F) Cells were grown in raffinose (Raf) and GAL1 transcription was induced by adding 2% galactose (Gal) and repressed by adding 4% glucose (Glu). Cmr1 occupancy was determined at the GAL1 TATA, 5’ and 3’ ORFs.

Fig 2. Transcription-dependent recruitment of Cmr1 to coding regions.

A) Scatter plot showing correlation between Rpb3 and Cmr1 occupancies (averaged over ORF) in WT cells. The data points representing Gcn4 targets are shown by red circles, and ARG1, HIS4, ADH1 and PMA1 are marked (green circles) in the plot. B) Quartiles were generated based on average Rpb3 ORF occupancies (n = 1387, each quartile) and Cmr1 occupancies in each quartile are presented as a box-plot. Genes in quartile Q1 showed the greatest Rpb3 occupancy, and Q4 showed the least. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software, and outliers are represented by dots. The notches represent 95% confidence intervals for each median. C) Cmr1 occupancy is shown at the metagene comprised of the top 10% and the bottom 10% of genes showing the greatest and the lowest Rpb3 occupancies, respectively. D) Average Cmr1 occupancy profile for the 94 Gcn4 targets harboring Rpb3 occupancy >1.0 log₂ ratio (ChIP/input) is shown in WT and gcn4Δ cells. E) Cmr1 average occupancy profile at ribosomal protein (RP) genes (n = 135) is shown for the WT and gcn4Δ cells.

Fig 3. Pol II CTD kinase Kin28 stimulates Cmr1 recruitment to ARG1 coding regions.

A-C) Cmr1-Myc tagged strains (WT, gcn4Δ, kin28as and kin28as/bur2Δ) were treated with an ATP-analog NA-PP1 to inactivate Kin28 kinase activity, and Cmr1-Myc and Rpb3 (Pol II subunit) occupancies were determined by ChIP. Occupancies of Cmr1 (A), and Rpb3 (B) at the ARG1 TATA, 5’ and 3’ ORFs are shown for the indicated strains. C-D) Cmr1-Myc tagged strains (WT, gcn4Δ, bur1as and bur1as/ctk1Δ) were treated with ATP-analog 3MB-PP1 to inactivate Bur1 kinase activity, and Cmr1 and Rpb3 occupancies were measured by ChIP. Occupancies of Cmr1 (C), and Cmr1/Rpb3 ratios (D) at the 5’ and 3’ ORFs of ARG1 are shown for the indicated strains.

Fig 4. HDACs Rpd3 and Hos2 promote Cmr1 binding in the ARG1 coding regions.

A) Cmr1 occupancy in the WT and histone methyltransferase mutants (set1Δ and set2Δ) at ARG1 5’ and 3’ ORFs is shown. B) Occupancy of Cmr1 at the induced ARG1 gene in WT, gcn4Δ and histone acetyltransferase mutant (gcn5Δ/esa1ts) is shown. C-D) Cmr1 ChIP occupancies in the induced WT and in histone deacetylase (HDACs) mutants (hos2Δ, rpd3Δ, and hos2Δ/rpd3Δ) at the ORFs of ARG1 (C) and HIS4 (D) are shown.

Fig 5. Cmr1 does not regulate histone occupancy or cotranscriptional histone modifications.

WT and cmr1Δ strains were treated with SM to induce ARG1 transcription and occupancies of histone H3 and histone modifications were determined by ChIP. A) H3 ChIP occupancies at ARG1 and HIS4 in SM-induced WT and cmr1Δ cells, and in the non-induced WT cells are shown. B) Occupancies of histone H3 modifications (acetylation, Ac; methylation, me; 2, di; 3, tri) normalized to H3 occupancy at ARG1 in SM-induced WT and cmr1Δ strains are shown. The levels of these modifications for an un-induced WT strain are also shown. C) Trimethylated (me3) H3K4 and K3K36 occupancies normalized to H3 levels at HIS4 ORFs in the WT and cmr1Δ cells. D) Whole cell extracts were prepared from WT and cmr1Δ cells and H3 acetylation levels were detected by western blot. H3 and Gcd6 (a translational factor) were used as loading controls.

Fig 6. Cmr1 promotes transcription of ARG1 and HIS4.

A) Pol II occupancy profile for the 500 genes (among those genes harboring average Rpb3 occupancies >0.5 log₂ ratio) showing the greatest Rpb3 binding defect in cmr1Δ cells. B) Venn diagram showing enrichment of Gcn4 and RP (ribosomal protein) genes among the top 500 Cmr1-affected genes. C) Rpb3 enrichments at the Gcn4 (n = 77) and RP genes (n = 43) among the top 500 Cmr1-affected genes (left) and gene average profile (right) in WT and cmr1Δ are shown. Notches represent 95% confidence intervals for each median. D) Rpb3 occupancy at the indicated regions of ARG1 (left) and HIS4 (right) was determined at various time-points during induction by ChIP in the WT and cmr1Δ strain.