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Published OnlineFirst July 12, 2018; DOI: 10.1158/1541-7786.MCR-18-0415

Review Molecular Cancer Research Mitotic Gene Bookmarking: An Epigenetic Program to Maintain Normal and Cancer Phenotypes Sayyed K. Zaidi1, Jeffrey A. Nickerson2, Anthony N. Imbalzano3, Jane B. Lian1, Janet L. Stein1, and Gary S. Stein1

Abstract

Reconfiguration of nuclear structure and function during chromosomes is essential to sustain lineage commitment; (ii) mitosis presents a significant challenge to resume the next cell Select chromatin modifiers and posttranslational histone cycle in the progeny cells without compromising structural modifications/variants retain competency of mitotic chroma- and functional identity of the cells. Equally important is the tin for gene reactivation as cells exit mitosis; and (iii) Func- requirement for cancer cells to retain the transformed pheno- tional components of RNAP I and II transcription complexes type, that is, unrestricted proliferative potential, suppression (e.g., UBF and TBP, respectively) are retained on genes of cell phenotype, and activation of oncogenic pathways. poised for reactivation immediately following mitosis. Impor- Mitotic gene bookmarking retention of key regulatory proteins tantly, recent findings have identified oncogenes that are that include sequence-specific transcription factors, chroma- associated with target genes on mitotic chromosomes in cancer tin-modifying factors, and components of RNA Pol (RNAP) cells. The current review proposes that mitotic gene book- I and II regulatory machineries at gene loci on mitotic chro- marking is an extensively utilized epigenetic mechanism for mosomes plays key roles in coordinate control of cell pheno- stringent control of proliferation and identity in normal type, growth, and proliferation postmitotically. There is grow- cells and hypothesizes that bookmarking plays a pivotal role ing recognition that three distinct protein types, mechanisti- in maintenance of tumor phenotypes, that is, unrestricted cally, play obligatory roles in mitotic gene bookmarking: (i) proliferation and compromised control of differentiation. Retention of phenotypic transcription factors on mitotic Mol Cancer Res; 16(11); 1617–24. 2018 AACR.

Introduction by our group, and others, have shown that mitotic gene bookmarking is mediated by key regulatory proteins that include Functional compartmentalization of regulatory proteins and components of RNAP I and II machineries, chromatin-modifying nucleic acids in the interphase nuclear microenvironments is factors and sequence-specific transcription factors and coregula- essential for physiologic control of gene expression (1–6). This tory proteins as well as variants and selective modifications of organization is disrupted in cancer, leading to deregulated nucleosomal histones (15–37). The functional outcome of mitot- transcriptional programming during the onset and progression ic gene bookmarking is the sustained normal cell phenotype of tumorigenesis (6–11). Mitosis is an essential cellular process across successive cell divisions. Evidence is accruing for role(s) that requires structural and functional remodeling of regulatory of mitotic gene bookmarking in regulating stem cell plasticity machinery in the nucleus (12–14). Disruption of this physiologic and in the onset, progression and maintenance of the tumor process poses a serious challenge to cell phenotype and identity phenotype. In this review, we present evolution of the concept and every cell cycle. Over the past two decades, mitotic gene book- discuss recent findings that indicate mitotic gene bookmarking is a marking—retention of regulatory proteins and selective histone specific and broadly relevant epigenetic program for coordinate variants and modifications at gene loci that are poised for imme- control of cell growth and identity through regulation of RNAP I diate reactivation postmitotically—has emerged as a key epige- and Pol II-mediated gene transcription and is obligatory to netic mechanism that plays a pivotal role in maintaining cell maintain normal and cancer phenotypes. phenotype and identity through successive cell divisions. Studies Mitotic Gene Bookmarking: Evolution of the 1Department of Biochemistry and University of Vermont Cancer Centre, Concept and Evidence for Specificity University of Vermont, Burlington Vermont. 2Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts. A conceptual framework for mitotic gene bookmarking was 3Graduate Program in Cell Biology and Department of Biochemistry initiated with the identification of a limited number of nuclease and Molecular Pharmacology, University of Massachusetts Medical School, accessible sites on the condensed mitotic chromatin that persist Worcester, Massachusetts. through the cell cycle. In the 1990s, Levens and colleagues showed Corresponding Author: Gary S. Stein, University of Vermont College of Medicine, that the chromatin is conformationally distorted at transcription 89 Beaumont Avenue, Burlington, VT 05405. Phone: 802-656-6613; Fax: 802-656- start sites (TSS) in genes poised for reactivation following mitosis 6613; E-mail: [email protected] and proposed that a subset of factors remains bound to mitotic doi: 10.1158/1541-7786.MCR-18-0415 chromosomes, providing a molecular bookmark to restore 2018 American Association for Cancer Research. chromatin conformation and gene expression postmitotically

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(20, 38). Consistent with this model, Wu and colleagues showed Mitotic retention of key components of RNA polymerase that the hsp70i gene promoter contains nuclease accessible sites (RNAP) machineries support competency for a basal level of that persist through mitosis. However, Wu and colleague also transcription throughout mitosis found that several sequence-specific transcription factors were Mitosis is accompanied by striking biochemical changes, displaced from the condensed mitotic chromatin (39). In 2003, including a decline in nuclear RNA transcription (12, 14, 42). our group identified the osteogenic master regulator RUNX2 as Mitotic repression of transcription was first noted over 60 years the first sequence-specific and phenotypic bookmark that remains ago in studies analyzing incorporation of radiolabeled RNA associated with target genes on mitotic chromosomes (40). precursors during the cell cycle (47). Initial studies examining Subsequent studies identified mitotic retention of several mechanisms that repress transcription in mitotic cells reported tissue-restricted transcription factors, indicating that mitotic that most of the RNAP II elongation complexes were physically bookmarking is a key epigenetic mechanism for regulation of excluded from mitotic chromosomes, although some genes genes that coordinately control cell growth and lineage mainte- retained active RNAP II complexes (48). The authors suggested nance following mitosis (15, 18, 22, 25, 41). that limitations of pulse-labeling approaches may prevent detec- It was long thought that highly condensed mitotic chromo- tion of low levels of mitotic transcriptional activity. Recent somes interfere with accessibility of gene regulatory factors, a approaches designed to detect subtle transcriptional changes have concept that extended to accessibility of antibodies to detect identified waves of transcriptional activity throughout mitosis. endogenous proteins on mitotic chromosomes by immunofluo- Using cell-permeable 5-ethynyluridine to pulse-label nascent rescence microscopy (42, 43). However, advances in genome- transcripts, Zaret and colleagues showed that mitotic cells main- wide biochemical and cell biological approaches have supported tain a steady-state level of transcriptional activity, and that the unbiased examination of transcriptional and epigenetic states genes involved in fundamental cellular functions, for example, during mitosis. Recent studies by several groups demonstrate that cell growth and proliferation, are among the first to be transcribed, mitotic gene bookmarking and downstream transcriptional while lineage-specific genes are expressed to establish cell identity events are a rule, and not the exception. The Tjian group has as cells exit mitosis (46). These findings are consistent with reports reported that the observation of sequence-specific regulatory from our group, and others, that components of RNAP I machin- protein displacement from mitotic chromosomes is an artifact ery that include upstream binding factor 1 (UBF1) remain asso- of formaldehyde fixation (44). Using live cell microscopy, the ciated with ribosomal RNA genes during mitosis (49, 50). A recent authors show that the kinetics of formaldehyde fixation prevents report from the Tjian group further shows that TBP, a key com- detection of sequence-specific transcription factors that are ponent of the RNAP II machinery, is also stably retained on retained on mitotic chromosomes, further strengthening the mitotic chromosomes and facilitates recruitment of RNAP II to role of mitotic gene bookmarking as a physiologically relevant genes for transcription as cells progress through and exit mitosis epigenetic mechanism. Studies from the Blobel group have estab- (51). These studies support a mechanism for transcriptional lished that nuclease accessibility of mitotic chromatin is a wide- memory through successive cell divisions that is defined by the spread phenomenon (45). Consistent with these observations, a retention of essential components of basal transcriptional recent report by Zaret and colleagues has shown that gene tran- machineries to support low levels of transcription throughout scription occurs in waves throughout mitosis; genes required for mitosis. essential cellular processes, for example, proliferation and growth are expressed from mid to late mitosis, while genes associated Modifications and variants of nucleosomal histones establish a with cell identity and phenotype are reactivated immediately after transcription-permissive mitotic chromatin state mitosis (46). These findings, together with studies over the last Low level, yet persistent, transcriptional activity during mitosis two decades, indicate that mitotic gene bookmarking is a specific, suggests that mitotic chromosomes are, at least partially, acces- selective, and wide-spread epigenetic program to sustain cell sible to the necessary transcriptional regulatory complexes. How- identity and retain options for plasticity. ever, the highly condensed nature of mitotic chromosomes has prevented a systematic, gene-level analysis of chromosome acces- Mitotic Gene Bookmarking as a Broadly sibility during mitosis. Advances in approaches to examine three- Relevant Epigenetic Program for Cell dimensional genome organization have produced high-resolution mapping of chromosome architecture in the interphase Identity nucleus and during mitosis (12, 52, 53). Chromosome confor- Advances in genome-wide molecular, biochemical and cell mation capture assays have identified a linearly organized and biological approaches, as well as improved techniques for enrich- longitudinally compressed array of consecutive chromatin loops ment of pure mitotic cell populations without using chemical that is shared by all chromosomes and is consistent across cell inhibitors, have allowed unbiased examination of transcriptional types (54). Despite a high degree of compaction, regions of activity during mitosis under physiologic conditions. (See Table 1 mitotic chromosomes remain in an open conformation and for current approaches being used to study mitotic gene book- accessible to regulatory proteins. These regions often contain marking.) Accruing evidence indicates that mitotic gene book- mitotically bookmarked genes and share several properties that marking is a broadly relevant and fundamental epigenetic pro- include (i) nuclease accessibility, (ii) enrichment of specific gram—with multiple converging and overlapping mechanisms— histone variants/modifications, and (iii) association of the mitot- by which pluripotent cells exercise options for differentiation ic compaction protein cohesin with actively transcribed genes. into different lineages and committed cells maintain identity Several studies in the mid-1970s revealed that condensed through successive cell divisions (Fig. 1). We discuss key mechan- mitotic chromosomes are amenable to nuclease digestion (55). isms underlying mitotic gene bookmarking as an epigenetic Subsequent studies using ligation-mediated polymerase chain program that are based on established and emerging evidence. reaction identified specific genes that are nuclease accessible,

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Table 1. Current approaches to study mitotic gene bookmarking by transcription factors Approach Application Limitations Tissue culture Fluorescence activated cell sorting Purified population of mitotic cells using Requires large number of cells depending upon antibodies against mitosis specific histone downstream application (e.g., a transcription factor modifications (e.g., H3S10 and H3S28) ChIP-seq requires larger cell number than an RNA-seq experiments). Can be cost-prohibitive. Cell synchronization Enrichment of mitotic population using cell-cycle Potential artifacts caused by treatment with chemicals. inhibitors (e.g., nocodazole) Imprecise enrichment of mitotic population (e.g., depending on the inhibitor used, cells may be

synchronized at the boundary of G2–M phases, thus resulting in a heterogenous population of late G2–early M phase cells). Cell biological Fixed cell immunofluorescence Visualization of protein of interest (POI) Antibody specificity. Antibody accessibility to condensed microscopy localization to mitotic chromosomes mitotic chromosomes. Fixation artifacts. Live cell microscopy Dynamics of protein localization during mitosis Most approaches require fusion of POI with fluorescence proteins with possible issues associated with overexpression and/or interference of fluorescence proteins with physiologic activity of POI. High-throughput Imaging (e.g., Covisualization of multiple genes, transcripts and Developing reagents that work together is challenging. high-throughput imaging proteins Specialized instrumentation and training is required for positioning mapping) execution and interpretation of experiments. Biochemical Transcription factor (TF) Gene-specific (by qPCR) or genome-wide (by Antibody specificity is a key variable and must be chromatin immunoprecipitation sequencing) protein-chromatin interactions determined empirically using supporting approaches (ChIP) using antibodies against POI (e.g., IP and Western blot). Antibody accessibility to condensed mitotic chromosomes. Histone posttranslational Often done in combination with TF-ChIP to Although antibodies against most histone PTMs are well- modification (PTM) ChIP identify epigenetic characteristics of genes characterized, multiple histone PTMs coexist on bookmarked by POI histone amino terminal tails. A limitation is the chromatin inaccessibility of an antibody against one histone PTM when another PTM is present, thus potentially missing a subset of genes that otherwise contain the PTM under experimental conditions. Functional Global run-on sequencing Whole transcriptome analysis of nascently Requires complex experimental design to ensure specific transcribed RNA in mitotically enriched cells in and selective detection of transcripts nascently which the POI expression has been modified. transcribed during and immediately following mitosis in the presence or the absence of POI. Inducible POI expression/ Regulated expression of protein of interest at and Functional link between mitotic gene bookmarking and downregulation using lentivirus. during mitosis to determine functional activity of POI requires precise expression (or Degron systems for inducible relevance of mitotic gene bookmarking downregulation) of POI at mitosis, which can be degradation of POI at mitosis. challenging because of the short length of mitosis.

permanganate sensitive [i.e., contain single-stranded (ss) DNA] Similarly, nucleosomes immediately downstream of the tran- and exhibit regulatory protein occupancy during mitosis. For scription start sites (TSS) contain H2A.Z and shift upstream to example, the Myc gene is reactivated by ssDNA-binding proteins occupy TSSs during mitosis, thus reducing nucleosome-depleted [e.g., far upstream element-binding protein (FBP)] immediately regulatory regions (57). This change appears to be specificto after cells divide. Consistent with a role for mitotic gene book- active genes that are silenced during mitosis and rapidly reacti- marking in reactivating genes postmitotically, the Myc gene is vated postmitotically. These authors report that, among other nuclease accessible and permanganate sensitive. Importantly, FBP histone modifications, the activating histone mark—trimethyla- remains associated with the gene during mitosis. Recent findings tion of lysine 4 at histone H3 (H3K4me3)—is enriched at these at higher resolution and genome-wide levels indicate larger chro- promoters during mitosis, whereas other epigenetic markers of matin domains of moderate nuclease accessibility remain open active chromatin are relinquished. More recent studies have during mitosis, and the more dynamic changes in accessibility are identified H3K27ac, a histone mark that is enriched in the locally dynamic. Furthermore, DNA hypomethylation marks a interphase enhancer regions, is also associated with mitotically subset of promoters that retain accessibility during mitosis (45). bookmarked genes (26). These findings are consistent with a These findings are consistent with a unique and selective behavior distinct global epigenomic landscape of mitotic chromosomes, of bookmarked genes during mitosis, for example, nuclease that is, association of the activating H3K9ac, H3K27ac, and sensitivity and accessibility to single-strand DNA-binding pro- H3K4me3 modifications with the gene-rich regions and CpG teins for rapid reactivation postmitotically. Genome-wide studies islands, and enrichment of the repressive H3K27me3 mark in are required to further assess whether nuclease accessibility is a distinct bands that show very little overlap with gene-rich regions common feature of all mitotically bookmarked genes. (58). Together, these observations point to a specialized chroma- Emerging evidence indicates that the nucleosomes of book- tin landscape of mitotically bookmarked genes that is defined by marked genes are enriched in histone variants, particularly in selective incorporation of histone variants and retention of spe- H2A.Z and H3.3. For example, nucleosomes at the myogenic gene cific posttranslational histone modifications and supports rapid MyoD are enriched in histone H3.3, and this enrichment supports recruitment of transcriptional regulatory machinery for gene persistent expression of the gene through 24 cell divisions (56). reactivation immediately following cell division.

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Although selective nuclease accessibility, histone modifica- proteins that include transcriptional activators and repressors. tions and histone variants are associated with mitotically (See more below.) bookmarked genes, highly condensed nature of mitotic chromosomes, and global reorganization of regulatory machinery Regulatory Contributions of Mitotic Gene during cell division suggests that additional mechanisms must Bookmarking to Biological Control and exist to maintain structural integrity of bookmarked genes during mitosis. Genome-wide chromatin immunoprecipita- Cancer tion-sequencing studies have revealed that transcription factors Coordination of RNAP I and II transcription are bound to genes in a highly clustered manner (59, 60). At The role for mitotic gene bookmarking in coordinating cell the chromatin architectural level, most clusters are formed growth, proliferation and identity is illustrated by RUNX proteins, around cohesin, a protein required for chromosome conden- master regulators of osteogenesis, hematopoiesis, neurogenesis, sation during mitosis. Mechanistically, cohesin plays two key and gastrointestinal development (69). During mitosis, all RUNX roles: (i) during the S-phase of the cell cycle, it holds the proteins (RUNX1, 2, and 3) associate with RNA Pol I-transcribed replicating strands together at the TF cluster sites; and (ii) ribosomal RNA genes and RNA Pol II-transcribed genes that are during mitosis, it remains bound to the clusters when the involved in control of the cell cycle, phenotype, and differenti- transcription factors have been displaced from target genes ation (49, 67, 68). RUNX transcription factors are equally parti- (61). Functionally, loss of cohesin decreases both DNA acces- tioned in progeny cells at the completion of cell division (40). The sibility and binding of TFs to clusters (59). These results association of RUNX factors with ribosomal and cell-cycle– provide a mechanistic explanation for nuclease accessibility regulatory genes (e.g., the cell-cycle inhibitor p21) during mitosis of mitotic chromosomes and identify additional chromatin bookmarks these genes for regulation during the early G1 phase of properties of mitotically bookmarked genes. Furthermore, the cell cycle. In addition, RUNX bookmarking of differentiation- these observations suggest that cohesin binding promotes related genes that include Smads, downstream effectors of the reestablishment of TF clusters and reactivation of target genes transforming growth factor b/bone morphogenetic protein sig- after DNA replication as well as after mitosis, thus adding naling pathway, by RUNX proteins during mitosis provides a a structural component to the mechanism for maintaining mechanistic basis for lineage-restricted transcriptional memory in cellular memory through cell divisions (59, 60). progeny cells (70). Occupancy and regulation of RNA Pol I- and Consistent with accessible mitotic chromatin that is enriched in RNA Pol II-transcribed genes by RUNX proteins during interphase specific histone variants and posttranslational modifications, key and mitosis enables coordination of cell proliferation, growth, chromatin remodeling proteins are retained on mitotic chromo- and differentiation by acting both at genetic and epigenetic levels. somes. These include histone acetyl transferases (e.g., p300), DNA demethylases (e.g., DNMT1), ATP-dependent chromatin remo- Maintenance of cell plasticity and lineage identity deling proteins (e.g., ISWI1), methyltransferases (e.g., MLL), Mesenchymal stem cells (MSC) have the capacity to differen- and readers of histone modifications (e.g., BRD4; refs. 17, 32, tiate into multiple lineages that include osteoblasts, adipocytes, 40, 62–65). Although it remains to be established whether every and myoblasts. Differentiation of MSCs into myoblasts requires bookmarked gene retains all or some of the chromatin-modifying the basic helix–loop–helix myogenic regulatory factors (including proteins, it is important to point out that many of these coregu- MyoD, Myf5, and MRF4) that bind to E-boxes in target gene lators interact with mitotically retained phenotypic proteins (e.g., promoters and play crucial roles in skeletal-muscle development. RUNX) that occupy target genes in a sequence-specific manner Together with Mef2 proteins and E-box factors, these transcription and provide scaffolds for organization and assembly of regulatory factors are responsible for coordinating muscle-specific gene complexes to coordinately control gene expression. expression by negatively regulating proliferation and promoting differentiation. During the proliferative stage of mesenchymal Phenotypic transcription factors provide specific and selective stem cells, MyoD is localized to mitotic chromosomes and gene regulatory activity required for cell identity associates with ribosomal RNA genes and nucleolar-organizing As discussed above, components of RNAP machineries are regions (NOR; ref. 41). The association of MyoD with the inter- retained on chromosomes during mitosis, and together with the phase nucleolus in early stages of myogenesis and its replacement mitotic retention of selective chromatin modifiers, create a reg- by myogenin in later stages results in the downregulation ulatory environment that is permissive to steady-state transcrip- of ribosomal RNA genes, concomitant with initiation of the tion during mitosis. Another level of specificity is provided by skeletal-muscle differentiation program. Consistent with these sequence-specific transcription factors that are often master reg- observations, adipocyte differentiation of MSCs recruits CCAAT/ ulators of their respective lineages. To date, more than 20 enhancer-binding proteins a and d (C/EBPa and d) to the C/EBP sequence-specific and pioneering transcription factors—proteins regulatory element in the C/EBPb gene promoter, upregulating with the ability to reprogram one cell type into another—have the C/EBPb protein, which functions as a transcriptional activator been reported to be retained on mitotic chromosomes (66). These of late-adipocyte genes. Studies from our lab demonstrate C/EBP include the osteogenic and hematopoietic RUNX transcription transcription factors occupy ribosomal RNA genes during mitosis factors (40, 67, 68), the key erythroid regulator GATA1 (22, 45), (41). As preadipocytes complete cell division, C/EBP proteins the muscle-restricted MyoD (41), and the liver-related FOXA1 downregulate ribosomal RNA genes, consistent with their role in transcription factor (18). Among the properties, these regulatory initiating adipocyte differentiation when ribosomal gene expres- proteins share are (i) specific intranuclear localization in the sion is decreased. The association of muscle and adipocyte- interphase nucleus; (ii) sequence-specific and dynamic interac- specific transcription factors with mitotic chromosomes in their tions with the chromatin; (iii) lineage-restricted physiologic respective lineages and the subsequent downregulation of ribo- gene regulation; and (iv) interaction with multiple coregulatory somal RNA genes in the interphase suggest that these phenotypic

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Figure 1. Mitotic gene bookmarking is a broadly relevant epigenetic program for cell identity. Studies over the past two decades have identified mitotic gene bookmarking as a broadly relevant epigenetic mechanism to coordinate cell proliferation, growth, and identity in progeny cells. Several regulatory proteins and transcription factors involved in key cellular processes have been identified to bookmark target genes for postmitotic reactivation/regulation. A, A simplified representation of the differentiation potential of pluripotent stem cells. Only those lineages are shown for which a "master" phenotypic regulator has been identified to bookmark target genes in mitosis (labeled in red). In addition, recent findings demonstrate that key pluripotent transcription factors that include SOX2 and KLF4 mitotically bookmark genes in pluripotent stem cells. B, Select signaling pathways, each involved in and essential for cell proliferation, growth, and differentiation, are depicted. Dashed arrows represent multiple steps that are not shown for simplicity. For each pathway, downstream effectors that mitotically bookmark genes are shown in red (e.g., FOXL1, RBPJ, REX, and HSF2). C, Accruing evidence has established key properties of chromatin of mitotically bookmark genes. Shown here are chromatin architectural proteins, cohesin (blue ring) and CTCF (red triangles), histone variants H3.3 and H2A.Z, as well as chromatin regulators that mediate histone acetylation (e.g., p300), deposit methyl moieties on nucleosomal histones (e.g., MLL complex), methylate DNA (e.g., DNMT1), and facilitate nucleosome remodeling (e.g., ISWI). regulatory proteins mediate lineage commitment and mainte- H3K4me3 marked. These findings are further corroborated by nance through bookmarking of target gene loci. recent studies showing that in addition to bivalent chromatin Pluripotent stem cells divide rapidly and exhibit an abbre- marks—the activating H3K4me3 and the repressive viated G1 phase of the cell cycle (71). The pluripotent stem cell H3K27me3—the enhancer-associated H3K27ac mark as well phenotype exhibits the capacity for unrestricted, but highly as pluripotent transcription factors SOX2 and KLF4 are retained regulated proliferation, and plasticity to differentiate into any during mitosis in pluripotent stem cells (26). A significant cell lineage, providing an optimal model to investigate mitotic recent finding is the partial recapitulation of chromatin biva- bookmarking. Mitotically purified populations of undifferen- lency in early-stage cancer cells. This "oncofetal epigenetic tiated stem cells retain the activating H3K4me3 mark on control" indicates that the bivalent chromatin plays a role in selective genes necessary for lineage commitment, thus poising acquisition of tumor phenotype (73, 74). It remains to be them for expression (72). In the absence of extracellular established whether some, all, or none of the genes that differentiation cues, these genes reacquire the repressive reacquire the activating H3K4me3 and the repressive H3K27me3 until the next cell division. In response to an H3K27me3 histone marks in early-stage cancer cells also relin- extracellular differentiation signal, pluripotent cells exercise quish the repressive H3K27me3 mark during mitosis to sustain the option to commit to a defined lineage and the genes remain plasticity of cancer cells. Together, these and other studies have

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identified mitotic gene bookmarking as a central epigenetic proliferation, growth, and differentiation. Importantly, mitotic mechanism for lineage identity in committed cells and cell gene bookmarking by oncogenes in cancer cells may be necessary plasticity in pluripotent stem cells. to maintain the tumor phenotype. Open-ended questions related to mitotic gene bookmarking that can provide mechanistic and Sustained tumor phenotype clinically relevant insights into compromised epigenetic control The tumor phenotype is characterized by deregulated differen- in the onset and progression of cancer include (i) Are mitotically tiation program and unrestricted cell proliferation that, unlike in bookmarked genes organized in shared nuclear microenviron- pluripotent stem cells, is not physiologically controlled. Recent ments in G1 cells to facilitate coordinate control? And/or is mitotic studies suggest that mitotic gene bookmarking has an important gene bookmarking a mechanism to assemble coordinately regu- role in the onset, progression, and perpetuation of disease. A key lated genes in shared nuclear regulatory microenvironments? (ii) example is provided by the leukemic fusion protein AML1-ETO What is the role of coregulatory proteins of transcription factor that blocks myeloid differentiation and enhances proliferative bookmarks in gene reactivation as cells exit mitosis? (iii) To what potential (75). The leukemic AML1-ETO mitotically bookmarks extent are genes bookmarked that are not expressed immediately rRNA genes, as well as genes controlling cell proliferation and after mitosis but in subsequent cell-cycle stages (e.g., histone myeloid cell differentiation (67). Functionally, AML1-ETO upre- genes that are specifically upregulated in S-phase.)? (iv) What is gulates rRNA and cell proliferation-related genes but downregu- the core regulatory network that is required to maintain cell lates gene-mediating myeloid cell differentiation, promoting the identity? (v) Is mitotic bookmarking operative in cells that divide transformed phenotype. Another example of cancer-related asymmetrically? and (vi) What is the contribution of mitotic mitotic gene bookmarking is the mixed lineage leukemia protein bookmarking in tumorigenesis? It has been traditionally (MLL). MLL is a chromatin remodeling factor that is associated difficult to target transcription factors due to unfavorable with leukemia and regulates transcription by recruiting chroma- pharmacokinetics and substantial off-target effects. From trans- tin-modifying machinery to target genes. MLL mitotic retention lational and clinical perspectives, mitotic bookmarking has favors rapid reactivation of target genes required for the onset and the potential to provide preferential and selective therapeutic progression of MLL postmitotically (17). In-depth and genome- intervention. wide studies are required to establish whether mitotic bookmarking of cancer-related genes is a shared activity of sequence-specific Disclosure of Potential Conflicts of Interest oncogenic proteins. No potential conflicts of interest were disclosed.

Concluding Remarks Acknowledgments The studies were supported by P01 CA 082834 from The National Cancer It is increasingly evident that mitotic bookmarking is a central Institute and by the Charlotte Perelman Fund for Cancer Research. G.S. Stein is epigenetic mechanism to maintain cellular identity through cell the Arthur J. Perelman Professor in Cancer Research. divisions (Fig. 1). Emerging evidence indicates that phenotypic transcription factors mitotically bookmark a subset of target genes Received April 25, 2018; revised May 24, 2018; accepted June 22, 2018; and this bookmarking contributes to coordinate control of cell published ﬁrst July 12, 2018.

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1624 Mol Cancer Res; 16(11) November 2018 Molecular Cancer Research

Mitotic Gene Bookmarking: An Epigenetic Program to Maintain Normal and Cancer Phenotypes

Sayyed K. Zaidi, Jeffrey A. Nickerson, Anthony N. Imbalzano, et al.

Mol Cancer Res 2018;16:1617-1624. Published OnlineFirst July 12, 2018.

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