Abstract The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.
// Jian Kang 1 , Eric P. Kusnadi 1 , Allison J. Ogden 1 , Rodney J. Hicks 2,3 , Lukas Bammert 4 , Ulrike Kutay 4 , Sandy Hung 5 , Elaine Sanij 1 , Ross D. Hannan 1,2,6,7,8,9 , Katherine M. Hannan 6,9 and Richard B. Pearson 1,2,6,7 1 Oncogenic Signaling and Growth Control Program, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia 2 Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia 3 Molecular Imaging and Targeted Therapeutics Laboratory, Cancer Therapeutics Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia 4 Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland 5 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia 6 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia 7 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 8 School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia 9 Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberaa, ACT, Australia Correspondence to: Richard B. Pearson, email: // Katherine M. Hannan, email: // Keywords : amino acids, rDNA transcription, S6K1, MYC, Gerotarget Received : June 08, 2016 Accepted : June 15, 2016 Published : June 30, 2016 Abstract Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer.
Loss of c-MYC is required for downregulation of ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) during granulocyte differentiation. Here, we demonstrate a robust reduction of Pol I loading onto rDNA that along with a depletion of the MYC target gene upstream binding factor (UBF) and a switch from epigenetically active to silent rDNA accompanies this MYC reduction. We hypothesized that MYC may coordinate these mechanisms via direct regulation of multiple components of the Pol I transcription apparatus. Using gene expression arrays we identified a ‘regulon’ of Pol I factors that are both downregulated during differentiation and reinduced in differentiated granulocytes upon activation of the MYC-ER transgene. This regulon includes the novel c-MYC target genes RRN3 and POLR1B. Although enforced MYC expression during granulocyte differentiation was sufficient to increase the number of active rRNA genes, its activation in terminally differentiated cells did not alter the active to inactive gene ratio despite increased rDNA transcription. Thus, c-MYC dynamically controls rDNA transcription during granulocytic differentiation through the orchestrated transcriptional regulation of core Pol I factors and epigenetic modulation of number of active rRNA genes.
The upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. We recently reported in Sanij et al. (2014) [1] that the two isoforms of UBF (UBF1/2) are enriched at Pol II-transcribed genes throughout the mouse and human genomes. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of UBF1/2, Pol I, Pol II, H3K9me3, H3K4me4, H3K9ac and H4 hyperacetylation, we reported a correlation of UBF1/2 binding with enrichments in Pol II and markers of active chromatin. In addition, we examined a functional role for UBF1/2 in mediating Pol II transcription by performing expression array analysis in control and UBF1/2 depleted NIH3T3 cells. Our data demonstrate that UBF1/2 bind highly active Pol II-transcribed genes and mediate their expression without recruiting Pol I. Furthermore, we reported ChIP-sequencing analysis of UBF1/2 in immortalized human epithelial cells and their isogenically matched transformed counterparts. Here we report the experimental design and the description of the ChIP-sequencing and microarray expression datasets uploaded to NCBI Sequence Research Archive (SRA) and Gene Expression Omnibus (GEO).
<title>Abstract</title> The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PI) has revolutionized the treatment of MM. However, resistance to PI is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461 has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here we show that CX-5461 has potent antimyeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell cycle arrest and apoptotic cell death. Surprisingly, the addition of PI does not enhance the therapeutic benefit of CX-5461. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk*MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM.
Acquired drug resistance leads to poor clinical outcome in high grade serous ovarian cancer (HGSOC). We have demonstrated the efficacy of the novel drug CX-5461 in HGSOC is mediated through destabilization of DNA replication forks. The data highlights the potential of CX-5461 in overcoming a general mechanism of chemotherapeutic resistance.
