Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.
ABSTRACT: The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.
Project description:RNA interference (RNAi) is implicated in maintaining tandem DNA arrays as constitutive heterochromatin. We used chromatin immunoprecipitation with antibodies to RNA polymerase II (RNAPol-ChIP) to test for transcription of the following repeat arrays in human cells: subtelomeric D4Z4, pericentromeric satellite 2, and centromeric satellite alpha. D4Z4 has a promoter-like sequence upstream of an ORF in its 3.3-kb repeat unit. A short D4Z4 array at 4q35 is linked to facioscapulohumeral muscular dystrophy (FSHD). By RNAPol-ChIP and RT-PCR, little or no transcription of D4Z4 was detected in FSHD and normal myoblasts; lymphoblasts from an FSHD patient, a control, and a patient with D4Z4 hypomethylation due to mutation of DNMT3B (ICF syndrome); and normal or cancer tissues. However, RNAPol-ChIP assays indicated transcription of D4Z4 in a chromosome 4-containing human-mouse somatic cell hybrid. ChIP and RT-PCR showed satellite DNA transcription in some cancers and lymphoblastoid cell lines, although only at a low level. Given the evidence for the involvement of RNAi in satellite DNA heterochromatinization, it is surprising that, at most, a very small fraction of satellite DNA was associated with RNA Pol II. In addition, our results do not support the previously hypothesized disease-linked differential transcription of D4Z4 sequences in short, FSHD-linked arrays.
Project description:RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing.
Project description:RPCC (RNA pol II ChIP-on-chip) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: ChIP-chip There are 4 different strains: rap1-sil (without the silencing domain), RAP1 (both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). The IP was done using an Ab against the RNApol II CTD (8WG16 Covance).There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.
Project description:Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4? is synthetically sick when paired with rpa12? and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4?, a ccr4? rpa12? rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.
Project description:Ribosome biogenesis is critical for proliferating cells and requires the coordinated activities of three eukaryotic RNA polymerases. We recently showed that the small ubiquitin-like modifier (SUMO) system controls the global level of RNA polymerase II (Pol II)-controlled transcription in mammalian cells by regulating cyclin-dependent kinase 9 activity. Here, we present evidence that the SUMO system also plays a critical role in the control of Pol I transcription. Using an siRNA-based knockdown approach, we found that multiple SUMO E3 ligases of the PIAS (protein inhibitor of activated STAT) family are involved in SUMO-mediated repression of ribosomal DNA (rDNA) gene transcription. We demonstrate that endogenous SUMO represses rDNA transcription primarily by repressing upstream-binding factor and proto-oncogene c-Myc expression and that ectopic overexpression of SUMO-associated enzymes additionally represses rDNA transcription via c-Myc SUMOylation and its subsequent degradation. The results of our study reveal a critical role of SUMOylation in the control of rDNA transcription, uncover the underlying mechanisms involved, and indicate that the SUMO system coordinates Pol I- and Pol II-mediated transcription in mammalian cells.
Project description:SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5' end of the gene. In snf6? cells there was a ?2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5' end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.
Project description:Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes.
Project description:Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49? mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.
Project description:Analysis of genome-wide differences of transcription using Genomic run-on (GRO), RNApol II ChIP-on-Chip, cDNA analysis and ChIP-on-Chip. This SuperSeries is composed of the following subset Series: GSE14060 RNA pol II ChIP on chip (RPCC) GSE14077 RPCC analysis of rap1-sil, tpk1 & tpk2 GSE14080 GRO analysis of rap1-sil, tpk1 & tpk2 GSE14082 Analysis of Spt16 depletion GSE1002 YPD to YPGal timecourse Refer to individual Series