ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

Abstract

eThe retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.T he retinoblastoma tumor suppressor gene (RB) is prevalently mutated at the initiation of retinoblastoma, osteosarcoma, and small-cell lung cancer. However, in the majority of human cancers, retinoblastoma protein (pRB) inactivation is frequently detected during tumor progression and is implicated in various facets of malignant behaviors (1). This suggests that pRB may possess more functions beyond its well-known roles in the control of the cell cycle and differentiation (2, 3). Previous studies demonstrated the direct interaction of pRB with a number of epigenetic modifiers carrying the LXCXE motif, including DNA methyltransferase 1 (DNMT1) (4, 5). pRB has been suggested to play a certain role in nuclear reprogramming in embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, mouse embryonic fibroblasts (MEFs), as well as tumor cells (6-10). Furthermore, RB loss was recently shown to contribute to the progression of human retinoblastoma via epigenetic modifications (11). However, the precise mechanism and target genes of the pRB epigenetic function are still largely unknown.pRB inactivation induces DNA double-strand breaks (DSBs) via multiple pathways (12-17). The MRN (Mre11, Rad50, and NBS1) sensor complex detects DSBs, rapidly activates ataxia telangiectasia mutated (ATM), and recruits ATM to the site of DSBs. ATM then activates a number of transducers, such as MDC1, 53BP1, and BRCA1, and effector kinases, such as Chk2. These proteins subsequently activate molecules required for DSB repair, including the histone variant H2AX (18)(19)(20). Our previous study demonstrated that some components of the DNA damage response (DDR) are activated upon Rb loss in vivo and in vitro (15). We therefore speculated that ATM might mediate pRB functions to regulate the DDR pathway.C cell (calcitonin-producing cell) tumors developed in Rb ϩ/Ϫ mice are precancerous and display many features of the DDR and cellular s...

The Rb binding domain of HPV31 E7 is required to maintain high levels of DNA repair factors in infected cells

“…Interestingly, p300 has also been shown to bind directly to the LXCXE domain of HPV16 E7 (Bernat et al, 2003), providing a potential direct link between the Rb binding site of E7 and the regulation of NBS1 stability. Furthermore, previous studies have linked Rb inactivation to ATM activation and DSB induction (Pickering and Kowalik, 2006; Shamma et al, 2013, 2009), as well as the control of Tip60-dependent acetylation of ATM (Shamma et al, 2013), which is required for ATM autophosphorylation and activation. Importantly, recent studies have shown that Tip60 is required for productive replication of HPV31 (Hong et al, 2015b), presumably through facilitating ATM activation.…”

“…High-risk E7 expression has been shown to lead to DSB induction and genomic instability in a manner thought to be dependent on Rb inactivation and deregulation of E2F transcription factors (Bester et al, 2011; Duensing and Munger, 2002; Pickering and Kowalik, 2006). Inactivation of Rb outside the context of HPV infection is sufficient to induce DSBs, formation of γH2AX and MRE11 positive foci, and increase levels of ATM, Chk2, and Chk1 (Frame et al, 2006; Pickering and Kowalik, 2006; Shamma et al, 2013, 2009). Previous studies demonstrated that expression of high-risk HPV31 E7 alone is sufficient to induce ATM signaling (Moody and Laimins, 2009), raising the possibility that E7 contributes to the differentiation-dependent phase of the life cycle through modulation of the DDR.…”

Discussion of Pathogen and Potential Therapeutic Strategy for Triple Negative Breast Cancer Based on a Rare Case Study

“…On the other hand, TP53 polymorphism has shown modulator function in DNA methylation of carcinoma [7]. ATM is another gene mediating DNA methylation [8]. ATM inactivating mutant had been shown its resistant to those DNA damaging drugs probably through the immune system, which affected efficacy of chemotherapy to leukemia [9].…”

Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts

“…However, determining the functions of RB in these contexts is challenging because of complicated cellular response to the loss of function of RB . For example, the germline loss of Rb results in increased intracellular signaling, DNA damage responses, and cellular senescence ; this generated technical difficulties in addressing RB functions especially in controlling undifferentiated state.…”