Chromatin organization and expression

A report on the 29th Lorne Genome Conference on the Organization and Expression of the Genome, Lorne, Australia, 17-21 February 2008

Epstein-Barr virus LMP1 blocks p16INK4a–RB pathway by promoting nuclear export of E2F4/5

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway

The potential of targeting ribosome biogenesis in high-grade serous ovarian cancer

Overall survival for patients with ovarian cancer (OC) has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC). HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR) and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC)-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.This work was supported by the National Health and Medical Research Council (NHMRC) of Australia, project grants (#1043884, 251608, 566702, 166908, 251688, 509087, 400116, 400120, 566876) and a NHMRC Program Grant (#1053792). Cancer Council Victoria research grant (#1065118). Ross D. Hannan and Richard B. Pearson were funded by NHMRC Fellowships

c-MYC coordinately regulates ribosomal gene chromatin remodeling and Pol I availability during granulocyte differentiation

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

UBF levels determine the number of active ribosomal RNA genes in mammals

In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation

Widespread FRA1-Dependent Control of Mesenchymal Transdifferentiation Programs in Colorectal Cancer Cells

Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.This work was supported by project grants 1026228 and 1044168 (to A.S.D.) and Senior Research Fellowships (to R.D.H., R.B.P. and J.M.M.) from the National Health and Medical Research Council of Australia

rDNA Chromatin Activity Status as a Biomarker of Sensitivity to the RNA Polymerase I Transcription Inhibitor CX-5461

Hyperactivation of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes (rDNA) is a key determinant of growth and proliferation and a consistent feature of cancer cells. We have demonstrated that inhibition of rDNA transcription by the Pol I transcription inhibitor CX-5461 selectively kills tumor cells in vivo. Moreover, the first-in human trial of CX-5461 has demonstrated CX-5461 is well-tolerated in patients and has single-agent anti-tumor activity in hematologic malignancies. However, the mechanisms underlying tumor cell sensitivity to CX-5461 remain unclear. Understanding these mechanisms is crucial for the development of predictive biomarkers of response that can be utilized for stratifying patients who may benefit from CX-5461. The rDNA repeats exist in four different and dynamic chromatin states: inactive rDNA can be either methylated silent or unmethylated pseudo-silent; while active rDNA repeats are described as either transcriptionally competent but non-transcribed or actively transcribed, depending on the level of rDNA promoter methylation, loading of the essential rDNA chromatin remodeler UBF and histone marks status. In addition, the number of rDNA repeats per human cell can reach hundreds of copies. Here, we tested the hypothesis that the number and/or chromatin status of the rDNA repeats, is a critical determinant of tumor cell sensitivity to Pol I therapy. We systematically examined a panel of ovarian cancer (OVCA) cell lines to identify rDNA chromatin associated biomarkers that might predict sensitivity to CX-5461. We demonstrated that an increased proportion of active to inactive rDNA repeats, independent of rDNA copy number, determines OVCA cell line sensitivity to CX-5461. Further, using zinc finger nuclease genome editing we identified that reducing rDNA copy number leads to an increase in the proportion of active rDNA repeats and confers sensitivity to CX-5461 but also induces genome-wide instability and sensitivity to DNA damage. We propose that the proportion of active to inactive rDNA repeats may serve as a biomarker to identify cancer patients who will benefit from CX-5461 therapy in future clinical trials. The data also reinforces the notion that rDNA instability is a threat to genomic integrity and cellular homeostasis.This work was supported by the National Health and Medical Research Council (NHMRC) of Australia project grants (#1100654 and #1162052). RH and RP were supported by NHMRC fellowships. AG was supported by the New Zealand Marsden Fund (14-MAU-053)

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serious ovarian cancer

Background: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Methods: Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. Results: We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Conclusions: Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.The China Scholarship Council University of Melbourne Ph.D. Scholarship supported S.Y. A National Health and Medical Research Council (NHMRC) Grant and NHMRC Senior Research Fellowship to R.B.P. supported this work. The Victorian Centre for Functional Genomics (K.J.S.) is funded by the Australian Cancer Research Foundation (ACRF), the Australian Phenomics Network (APN) through funding from the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS) programme, the Peter MacCallum Cancer Centre Foundation and the University of Melbourne Research Collaborative Infrastructure Programm