cancers Review Retinoblastoma Tumor Suppressor Protein Roles in Epigenetic Regulation Frederick Guzman 1, Yasamin Fazeli 2, Meagan Khuu 2, Kelsey Salcido 2, Sarah Singh 2 and Claudia A. Benavente 1,2,3,4,* 1 Department of Pharmaceutical Sciences, University of California, 101 Theory #100, Irvine, CA 92617, USA; [email protected] 2 School of Biological Sciences, University of California, 5120 Natural Sciences II, Irvine, CA 92697, USA; [email protected] (Y.F.); [email protected] (M.K.); [email protected] (K.S.); [email protected] (S.S.) 3 Department of Developmental and Cell Biology, University of California, 2011 Biological Sciences III, Irvine, CA 92697, USA 4 Chao Family Comprehensive Cancer Center, University of California, 839 Medical Sciences Rd, Irvine, CA 92697, USA * Correspondence: [email protected] Received: 1 September 2020; Accepted: 27 September 2020; Published: 29 September 2020 Simple Summary: Loss of function of the retinoblastoma gene (RB1) is the rate-limiting step in the initiation of both the hereditary and sporadic forms of retinoblastoma tumor. Furthermore, loss of function of the retinoblastoma tumor suppressor protein (pRB) is frequently found in most human cancers. In retinoblastoma, tumor progression is driven by epigenetic changes following pRB loss. This review focuses on the diverse functions of pRB in epigenetic regulation. Abstract: Mutations that result in the loss of function of pRB were first identified in retinoblastoma and since then have been associated with the propagation of various forms of cancer. pRB is best known for its key role as a transcriptional regulator during cell cycle exit. Beyond the ability of pRB to regulate transcription of cell cycle progression genes, pRB can remodel chromatin to exert several of its other biological roles. In this review, we discuss the diverse functions of pRB in epigenetic regulation including nucleosome mobilization, histone modifications, DNA methylation and non-coding RNAs. Keywords: retinoblastoma; RB1; E2F; epigenetic; DNA methylation; histone modification; nucleosomes; miRNA; ncRNA; chromatin 1. Introduction Mutations leading to the loss of the retinoblastoma transcriptional corepressor 1 gene (RB1) or deregulation of its gene product, the retinoblastoma tumor suppressor protein (pRB), are associated with several forms of cancer including retinoblastoma, osteosarcoma, adenocarcinomas, small cell lung cancer, breast cancer, prostate cancer, and others [1]. pRB controls transcription and is a negative regulator of cell proliferation through repression of the E2F family of transcription factors (E2Fs). Transcriptional repression in this model occurs through the binding of pRB’s “pocket” domain to E2Fs’ C-terminal transactivation domain [2]. In addition to its interactions with E2Fs, pRB can further regulate transcription through the binding to chromatin remodelers. Current models of pRB functions are increasingly complex and yet to be completely elucidated. The pRB-E2F association is known to play a role in transcriptional regulation of E2F targets in at least three different mechanism: (1) direct pRB repression on E2F transcription (Figure1A), (2) pRB recruitment of transcriptional corepressors to E2F targets (Figure1B), and (3) association of the pRB-E2F complex with transcriptional coactivators, Cancers 2020, 12, 2807; doi:10.3390/cancers12102807 www.mdpi.com/journal/cancers Cancers 2020, 12, 2807 2 of 14 Cancers 2020, 12, x FOR PEER REVIEW 2 of 15 whichalso noteworthy result in increased that beyond expression its E2F of E2Ftranscriptional targets (Figure regulation,1C) [ 3]. ItpRB is also may noteworthy regulate transcription that beyond itsthrough E2F transcriptional its interaction regulation, with other pRB transcription may regulate factors transcription and has through transcription its interaction-independent with otherroles transcriptionthroughout the factors cell (Figure and has 1D) transcription-independent [4]. roles throughout the cell (Figure1D) [4]. Figure 1. Transcriptional regulation by pRB. (A–C) The pRB can regulate E2F targets in at least threeFigure di 1.fferent Transcriptional mechanisms: regulation (A) direct by pRB. pRB ( repressionA–C) The pRB on E2F can transcription;regulate E2F targets (B) pRB in at recruitment least three ofdifferent transcriptional mechanism corepressors,s: (A) direct like pRB HDACs repression and histone on E2F methyltransferases transcription; (B (e.g.,) pRB SUV39H1) recruitment to E2F of targets;transcriptional and (C) associationcorepressors, of thelike pRB-E2F HDACs complex and histone with transcriptionalmethyltransferases coactivators, (e.g., SUV39H1) which results to E2F in increasedtargets; and expression (C) association of E2F of targets. the pRB (D-)E2F pRB complex can regulate with transcriptiontranscriptional through coactivators, its interaction which result withs otherin increased transcription expression factors of (e.g.,E2F targets. CBFA1). (D) pRB can regulate transcription through its interaction with other transcription factors (e.g., CBFA1). Within the cell cycle, CYCLIN D: CDK4/6 mono-phosphorylates pRB at active sites and unstructuredWithin the regions cell ofcycle, the proteinCYCLIN during D: CDK4/6 early G1 mono phase,-phosphorylate CYCLIN E: CDK2s pRB hyper-phosphorylates at active sites and pRBunstructured in late G1, regions and CYCLIN of the protein B: CDK1 during maintain early pRBG1 phase, in a hyper-phosphorylated CYCLIN E: CDK2 hyper state-phosphorylate during S, G2,s andpRB M in phases late G1, [5 ].and pRB CYCLIN mono-phosphorylation B: CDK1 maintain inactivates pRB in a unphosphorylatedhyper-phosphorylated pRB G0state functions. during S, pRB G2, hyper-phosphorylationand M phases [5]. pRB inhibitsmono-phosphorylation its ability to bind inactivates to E2Fs, allowing unphosphorylated E2Fs to drive pRB transcription G0 functions. of genes pRB necessaryhyper-phosphorylation for G1/S phase inhibits cell cycle its progression.ability to bind While to E2Fs, hyper-phosphorylation allowing E2Fs to dri largelyve transcription disrupts pRB of bindinggenes necessary to other for proteins, G1/S phase some cell protein cycle interactionsprogression. persist While followinghyper-phosphorylation pRB hyper-phosphorylation. largely disrupts ThepRB protein–protein binding to other interactions proteins, that co-existsome protein with pRB interactions in a hyper-phosphorylated persist following form pRB suggest hyper that- pRBphosphorylation. hyper-phosphorylation The protein does–protein not inhibit interactions all pRB t functionshat co-exist [6]. with While pRB pRB in a binding hyper- tophosphorylated E2Fs occur at form suggest that pRB hyper-phosphorylation does not inhibit all pRB functions [6]. While pRB binding to E2Fs occur at the pocket domain, pRB interactions with epigenetically-relevant proteins CancersCancers 20202020,,12 12,, 2807x FOR PEER REVIEW 33 ofof 1415 involve the LxCxE-binding domain [7]. The availability of the LxCxE binding site, even when pRB is thebound pocket to E2F, domain, presents pRB expanded interactions functions with epigenetically-relevant of pRB in the recruitment proteins of involvechromatin the remodelers LxCxE-binding and domainother protein [7]. The complexes. availability Indeed, of the pRB LxCxE has been binding repor site,ted to even interact when with pRB over is bound 300 proteins to E2F, and presents many expandedprotein interactions functions ofare, pRB or are in theinvolved recruitment with, chromatin of chromatin modifier remodelers proteins and [8] other. pRB protein therefore complexes. extends Indeed,relevance pRB into has protein been reported complexes to interactbeyond withthose over within 300 the proteins canonical and manypRB/E2F protein pathway. interactions Studies are, in orretinoblastoma are involved with,indicate chromatin that these modifier tumors proteins develop [ 8quickly]. pRB thereforeas a result extends of epigenetic relevance deregulation into protein of complexeskey cancer beyondpathways those as a withindirect result the canonical of pRB loss pRB [9]/E2F. Studies pathway. in the Studies last decade in retinoblastoma have found significant indicate thatevidence these regarding tumors develop the relationship quickly as between a result ofpRB epigenetic and chromatin deregulation remodeling of key cancerproteins, pathways pushing as the a directboundaries result of of knowledge pRB loss [9 ].regarding Studies inpRB the functions last decade. This have review found provides significant an in evidence-depth examination regarding the of relationshipthe literature between involving pRB pRB and in chromatin epigenetic remodeling regulation, proteins, including pushing nucleosome the boundaries mobilization, of knowledge histone regardingacetylation, pRB histone functions. methylation, This review DNA provides methylation an in-depth, and non examination-coding RNAs of the. literature involving pRB in epigenetic regulation, including nucleosome mobilization, histone acetylation, histone methylation, DNA2. pRB methylation, Protein Structure and non-coding RNAs. 2. pRBHuman Protein pRB Structure consists of 928 amino acids, which can be divided into three domains: the N- terminal domain (RBN), the A/B region or “pocket” domain (RBP), and the C-terminal domain (RBC) Human pRB consists of 928 amino acids, which can be divided into three domains: the N-terminal (Figure 2) [10,11]. The RBP contains a Leu-X-Cys-X-Glu (LxCxE) binding cleft which mediates domain (RBN), the A/B region or “pocket” domain (RBP), and the C-terminal