Project description:Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and exclude a significant role for termination by a torpedomechanism. They further reveal the existence of an asymmetric Boundary Complex formed by CTCF, Cohesin and three phased nucleosomes lying adjacent to the rDNA Enhancer and an arrested RNA Polymerase I complex. We find that this complex is the only site of active histone modification in the whole 45kbp rDNA repeat. Strikingly, the Enhancer Boundary Complex not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define the poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for the chromatin remodelling complexes.
Project description:Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using conditional gene deletion in mouse, we now show that UBF is indeed essential for transcription of the rRNA genes. Unexpectedly, arrest of rRNA synthesis does not affect RPIII transcription of 5S or tRNA genes, nor RPII expression of the hundreds of mRNAs implicated in ribosome biogenesis, but does upreguglate snRNAs and snoRNAs. Thus, rRNA gene activity does not coordinate global gene expression required for ribosome biogenesis. Conditional UBF flox/flox MEFs and immortalized MEFs and isogenic controls all carrying homozygous ER-Cre alleles were treated with 4-HT and mRNA analyzedat 0h and at 24h intervals posttreatment over 4 days.
Project description:Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using conditional gene deletion in mouse, we now show that UBF is indeed essential for transcription of the rRNA genes. Unexpectedly, arrest of rRNA synthesis does not affect RPIII transcription of 5S or tRNA genes, nor RPII expression of the hundreds of mRNAs implicated in ribosome biogenesis, but does upreguglate snRNAs and snoRNAs. Thus, rRNA gene activity does not coordinate global gene expression required for ribosome biogenesis.
Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells.
Project description:Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
Project description:Ribosomal DNA (rDNA) arrays are highly repetitive regions of the genome which encode essential genes required to produce ribosomes. DNA double-stranded breaks (DSBs) generated within rDNA genes elicit a unique cellular response involving robust transcriptional silencing and nucleolar reorganization into ‘cap’ structures at the nucleolar periphery. This process is coordinated by the nucleolar scaffolding protein TCOF1, which functions to recruit the DNA repair proteins NBS1 and TOPBP1 that activate the ATM and ATR kinases, resulting in ribosomal RNA (rRNA) transcriptional silencing and nucleolar segregation. However, the DNA damage and repair response at rDNA arrays remains incompletely understood. Here, we investigate the cellular response to rDNA DSBs using proteomics and genetic CRISPR-Cas9 screening. We show that the protein UFMylation pathway and the HUSH complex are important for cell viability and survival in response to rDNA DSBs, and that the E3 UFM1-ligase UFL1 and its heterodimer DDRGK1 are associated with TCOF1 at nucleolar caps. Loss of UFL1 leads to impaired ATM activation, reduced rRNA transcriptional silencing, and an overall reduction in nucleolar segregation. We identified ATM, UNC45A and SMC6 as UFMylated proteins, in which UFMylation may facilitate ATM activation and segregation of damaged rDNA to the nucleolar periphery. Altogether, our findings provide the first evidence for a role for UFMylation in rDNA DSB repair.
Project description:The nucleolus is a subnuclear compartment with a key role in ribosome biogenesis and cellular stress responses. These mechanisms are governed by a complex interaction of proteins, including the NOC1 network. This study reveals a novel relationship between NOC1 and MYC transcription factor, known for its key role in controlling ribosomal biogenesis and cell growth and proliferation. Here, we demonstrate that NOC1 functions as a direct target of MYC, as it is transcriptionally induced through a functional MYC-binding E-box sequence in the NOC1 promoter region. Furthermore, interactome analysis reveals that NOC1-complex includes the nucleolar proteins NOC2 and NOC3 and other nucleolar components such as Nucleostemin1 Ns1 and mainly components for the transport of ribosomal subunits and maturation and with components for rRNA processing. In response to MYC, NOC1 expression and localization within the nucleolus significantly increase, suggesting a direct functional link between MYC activity and NOC1 function. Notably, NOC1 over-expression leads to the formation of large nuclear granules and enlarged nucleoli, which co-localize with nucleolar fibrillarin and Ns1. Additionally, we demonstrate that NOC1 expression is necessary for Ns1 nucleolar localization, suggesting a role for NOC1 in maintaining nucleolar structure. Finally, the co-expression of NOC1 and MYC enhances the formation of abnormal structures formed by NOC1 within the nucleolus, outlining another aspect of NOC1 and MYC cooperation in nucleolar dynamics. This study reveals NOC1's interactions with proteins involved in RNA processing, modification, and splicing, such as Ythdc1, Flacc, and spenito. These proteins play key roles in mediating N6-methyladenosine (m6A) methylation of mRNAs. NOC1's potential involvement in coordinating RNA splicing and nuclear mRNA export highlights its significance in regulating gene expression. In summary, our study uncovers novel roles for NOC1 in nucleolar homeostasis and establishes its direct connection with MYC in the network governing nucleolar structure and function. These findings also highlight NOC1's interaction with proteins relevant for RNA processing, modification, and splicing, suggesting a broader role in addition to its control of nucleolar homeostasis, providing new insight into NOC1 function that can be further investigated into its function in regulating cellular growth and proliferation.
Project description:In recent years, several small molecule cytotoxic drugs have been identified as potential inhibitors of ribosome biogenesis (Drygin et al., 2011; Peltonen et al., 2014a; Peltonen et al., 2014b). CX-5461 is one such drug that has also demonstrated anticancer potential for a wide range of malignancies (Bywater et al., 2012; Cornelison et al., 2017; Devlin et al., 2015; Drygin et al., 2011; Hald et al., 2019; Hein et al., 2017; Ismael et al., 2019; Lawrence et al., 2018; Lee et al., 2017; Negi and Brown, 2015; Taylor et al., 2019; Xu et al., 2017; Yan et al., 2017) (Haddach et al., 2012), and is presently under phase I trials for the treatment of both hematological cancers and solid tumours (Group, 2016; Khot et al., 2019). CX-5461 was initially characterized as an inhibitor of RNA Polymerase I (RPI/PolR1/PolI) that is responsible for the synthesis of the major ribosomal RNAs and the initial step in ribosome biogenesis (Drygin et al., 2011). Since RPI and its corresponding core transcription factors are dedicated to this task alone, they present ideal molecular targets by which to modulate ribosome biogenesis. However, the specificity of CX-5461 has been questioned and it has been suggested that this drug may also act by stabilizing DNA G-quadruplexes or by “poisoning” topoisomerase II (Topo II). Thus, the primary target of this drug and its mode of action are still in doubt. Here we used Deconvolution-ChIP-Seq in NIH3T3 and HEK293T cells treated for different times with CX-5461. The data show that the primary target of CX5461 is the initiation of ribosomal RNA gene (rDNA) transcription. CX-5461 blocks transcription initiation in vitro and in vivo by arresting RNA polymerase I (RPI/Pol1) within the preinitiation complex. In contrast to previous suggestions, CX-5461 does not effect recruitment of the TBP-TAF complex SL1 to the rDNA promoter, the recruitment of the initiation competent RPI-Rrn3 complex or ongoing transcription elongation, arguing against a role for G-quadruplex stabilization or topoisomerase II poisoning. Inhibition of transcription by CX-5461 is not reversible, the RPI-Rrn3 complex remains arrested in the preinitiation complex even after drug removal. This leads to nucleolar stress, extensive DNA damage and cell senescence. Our data show that the cytotoxicity of CX-5461 is the downstream result of the highly specific inhibition of rDNA transcription. The observation that this inhibition is irreversible will be important for the future design of chemotherapeutic strategies and the avoidance of drug resistance.