Epigenetics and Malaria Susceptibility/Protection: A Missing Piece of the Puzzle

A better understanding of stable changes in regulation of gene expression that result from epigenetic events is of great relevance in the development of strategies to prevent and treat infectious diseases. Histone modification and DNA methylation are key epigenetic mechanisms that can be regarded as marks, which ensure an accurate transmission of the chromatin states and gene expression profiles over generations of cells. There is an increasing list of these modifications, and the complexity of their action is just beginning to be understood. It is clear that the epigenetic landscape plays a fundamental role in most biological processes that involve the manipulation and expression of DNA. Although the molecular mechanism of gene regulation is relatively well understood, the hierarchical order of events and dependencies that lead to protection against infection remain largely unknown. In this review, we propose that host epigenetics is an essential, though relatively under studied, factor in the protection or susceptibility to malaria

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

Investigating the p53-independent responses to inhibition of RNA Polymerase I transcription by CX-5461

© 2017 Dr. Jaclyn QuinIncreased rates of DNA-dependent RNA Polymerase I (Pol I) transcription of the 47S pre-ribosomal RNA (rRNA) genes are observed in almost all cancer types. Cancer cells may require high rates of Pol I transcription and ribosome biogenesis to achieve their unrestrained growth and proliferative capacity, thus presenting a therapeutic window for selectively targeting cancer cells with inhibitors of Pol I transcription. Our laboratory helped develop and validate a first-in-class small molecule selective inhibitor of Pol I transcription, CX-5461 (Senhwa Biosciences). Here, we have investigated the response of cells at defined stages of malignant transformation to inhibition of Pol I transcription, utilising a panel of isogenically matched BJ fibroblast cell lines. We compared the response of non-transformed and transformed cells of the same genetic background, and demonstrated that CX-5461 can selectively induce cell death in cancer cell lines in vitro. We investigated the phenotypic response of a nontransformed BJ fibroblast cell line minimally immortalized with hTERT (BJ-T) to CX- 5461, and demonstrated that they display a proliferation defect. The proliferation defect is associated with the activation of p53 and a p53-dependent G1 cell cycle checkpoint, as well as p53-independent S-phase and G2 cell cycle checkpoints and senescence. Escape from cell cycle arrest in transformed BJ fibroblast cell lines is associated with increased rates of cell death in response to CX-5461. To identify pathways mediating the p53-independent responses to inhibition of Pol I transcription, we have performed RNA-sequencing analysis in CX-5461 treated BJ-T cells in which p53 was silenced (BJ-T p53shRNA). The analysis identified ATM (Ataxia-telangiectasia mutated) / ATR (ATM and RAD3-related) signaling and transcriptional programs associated with senescence to be modulated following treatment with CX-5461. Further, we have demonstrated that inhibition of Pol I transcription by CX-5461 rapidly and potently activates the ATM/ATR kinase signaling pathways in the absence of global DNA damage. Combined ATM/ATR inhibition and CX-5461 treatment results in bypass of CX-5461 mediated S-phase and G2 arrest, and induced cell death in the BJ-T p53shRNA cell line. We investigated the mechanisms by which inhibition of Pol I transcription by CX-5461 activates the ATM/ATR signaling pathways. We demonstrated that inhibition of Pol I transcription initiation by CX-5461 results in ‘exposed’ rRNA genes (rDNA) that are in an open chromatin conformation but devoid of Pol I. Inhibition of Pol I transcription by CX-5461 also results in reorganization of nucleolar structure and translocation of proteins to and from the nucleoli. We observed increased levels of NBS1 (Nijmegen Breakage Syndrome 1) activation by ATM specifically within the nucleoli during S/G2. We propose CX-5461 treatment induces an unusual chromatin structure at the rDNA that is sufficient to activate ATM/ATR in the nucleoli. Finally, we have shown that DNA damage repair is attenuated following treatment with CX-5461. Together, our studies identify activation of ATM/ATR signaling as a key p53-independent pathway of response to inhibition of Pol I transcription, that can be targeted to improve the efficacy of CX-5461 in cancer therapy

Targeting the nucleolus for cancer intervention

The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells. The dependence of certain tumor cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumor cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun to scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with "suppression of nucleolar stress" representing an emerging "hallmark" of cancer. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease

Targeting the nucleolus for cancer intervention

The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells. The dependence of certain tumor cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumor cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun to scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with "suppression of nucleolar stress" representing an emerging "hallmark" of cancer. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease

Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne).Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy

Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy