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

Functional compartmentalization of regulatory proteins and nucleic acids in the interphase nuclear microenvironments is essential for physiologic control of gene expression (1–6). This organization is disrupted in cancer, leading to deregulated transcriptional programming during the onset and progression of tumorigenesis (6–11). Mitosis is an essential cellular process that requires structural and functional remodeling of regulatory machinery in the nucleus (12–14). Disruption of this physiologic process poses a serious challenge to cell phenotype and identity every cell cycle. Over the past two decades, mitotic gene bookmarking—retention of regulatory proteins and selective histone variants and modifications at gene loci that are poised for immediate reactivation postmitotically—has emerged as a key epigenetic mechanism that plays a pivotal role in maintaining cell phenotype and identity through successive cell divisions. Studies by our group, and others, have shown that mitotic gene bookmarking is mediated by key regulatory proteins that include components of RNAP I and II machineries, chromatin-modifying factors and sequence-specific transcription factors and coregulatory proteins as well as variants and selective modifications of nucleosomal histones (15–37). The functional outcome of mitotic gene bookmarking is the sustained normal cell phenotype across successive cell divisions. Evidence is accruing for role(s) of mitotic gene bookmarking in regulating stem cell plasticity and in the onset, progression and maintenance of the tumor phenotype. In this review, we present evolution of the concept and discuss recent findings that indicate mitotic gene bookmarking is a specific and broadly relevant epigenetic program for coordinate control of cell growth and identity through regulation of RNAP I and Pol II-mediated gene transcription and is obligatory to maintain normal and cancer phenotypes.

A conceptual framework for mitotic gene bookmarking was initiated with the identification of a limited number of nuclease accessible sites on the condensed mitotic chromatin that persist through the cell cycle. In the 1990s, Levens and colleagues showed that the chromatin is conformationally distorted at transcription start sites (TSS) in genes poised for reactivation following mitosis and proposed that a subset of factors remains bound to mitotic chromosomes, providing a molecular bookmark to restore chromatin conformation and gene expression postmitotically (20, 38). Consistent with this model, Wu and colleagues showed that the hsp70i gene promoter contains nuclease accessible sites that persist through mitosis. However, Wu and colleague also found that several sequence-specific transcription factors were displaced from the condensed mitotic chromatin (39). In 2003, our group identified the osteogenic master regulator RUNX2 as the first sequence-specific and phenotypic bookmark that remains associated with target genes on mitotic chromosomes (40). Subsequent studies identified mitotic retention of several tissue-restricted transcription factors, indicating that mitotic bookmarking is a key epigenetic mechanism for regulation of genes that coordinately control cell growth and lineage maintenance following mitosis (15, 18, 22, 25, 41).

It was long thought that highly condensed mitotic chromosomes interfere with accessibility of gene regulatory factors, a concept that extended to accessibility of antibodies to detect endogenous proteins on mitotic chromosomes by immunofluorescence microscopy (42, 43). However, advances in genome-wide biochemical and cell biological approaches have supported unbiased examination of transcriptional and epigenetic states during mitosis. Recent studies by several groups demonstrate that mitotic gene bookmarking and downstream transcriptional events are a rule, and not the exception. The Tjian group has reported that the observation of sequence-specific regulatory protein displacement from mitotic chromosomes is an artifact of formaldehyde fixation (44). Using live cell microscopy, the authors show that the kinetics of formaldehyde fixation prevents detection of sequence-specific transcription factors that are retained on mitotic chromosomes, further strengthening the role of mitotic gene bookmarking as a physiologically relevant epigenetic mechanism. Studies from the Blobel group have established that nuclease accessibility of mitotic chromatin is a wide-spread phenomenon (45). Consistent with these observations, a recent report by Zaret and colleagues has shown that gene transcription occurs in waves throughout mitosis; genes required for essential cellular processes, for example, proliferation and growth are expressed from mid to late mitosis, while genes associated with cell identity and phenotype are reactivated immediately after mitosis (46). These findings, together with studies over the last two decades, indicate that mitotic gene bookmarking is a specific, selective, and wide-spread epigenetic program to sustain cell identity and retain options for plasticity.

Advances in genome-wide molecular, biochemical and cell biological approaches, as well as improved techniques for enrichment of pure mitotic cell populations without using chemical inhibitors, have allowed unbiased examination of transcriptional activity during mitosis under physiologic conditions. (See Table 1 for current approaches being used to study mitotic gene bookmarking.) Accruing evidence indicates that mitotic gene bookmarking is a broadly relevant and fundamental epigenetic program—with multiple converging and overlapping mechanisms—by which pluripotent cells exercise options for differentiation into different lineages and committed cells maintain identity through successive cell divisions (Fig. 1). We discuss key mechanisms underlying mitotic gene bookmarking as an epigenetic program that are based on established and emerging evidence.

Table 1.

Current approaches to study mitotic gene bookmarking by transcription factors

ApproachApplicationLimitations
Tissue culture Fluorescence activated cell sorting Purified population of mitotic cells using antibodies against mitosis specific histone modifications (e.g., H3S10 and H3S28) Requires large number of cells depending upon downstream application (e.g., a transcription factor ChIP-seq requires larger cell number than an RNA-seq experiments). Can be cost-prohibitive. 
 Cell synchronization Enrichment of mitotic population using cell-cycle inhibitors (e.g., nocodazole) Potential artifacts caused by treatment with chemicals. 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 microscopy Visualization of protein of interest (POI) localization to mitotic chromosomes Antibody specificity. Antibody accessibility to condensed 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., high-throughput imaging positioning mapping) Covisualization of multiple genes, transcripts and proteins Developing reagents that work together is challenging. Specialized instrumentation and training is required for execution and interpretation of experiments. 
Biochemical Transcription factor (TF) chromatin immunoprecipitation (ChIP) Gene-specific (by qPCR) or genome-wide (by sequencing) protein-chromatin interactions using antibodies against POI Antibody specificity is a key variable and must be determined empirically using supporting approaches (e.g., IP and Western blot). Antibody accessibility to condensed mitotic chromosomes. 
 Histone posttranslational modification (PTM) ChIP Often done in combination with TF-ChIP to identify epigenetic characteristics of genes bookmarked by POI Although antibodies against most histone PTMs are well-characterized, multiple histone PTMs coexist on 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 transcribed RNA in mitotically enriched cells in which the POI expression has been modified. Requires complex experimental design to ensure specific and selective detection of transcripts nascently transcribed during and immediately following mitosis in the presence or the absence of POI. 
 Inducible POI expression/downregulation using lentivirus. Degron systems for inducible degradation of POI at mitosis. Regulated expression of protein of interest at and during mitosis to determine functional relevance of mitotic gene bookmarking Functional link between mitotic gene bookmarking and activity of POI requires precise expression (or downregulation) of POI at mitosis, which can be challenging because of the short length of mitosis. 
ApproachApplicationLimitations
Tissue culture Fluorescence activated cell sorting Purified population of mitotic cells using antibodies against mitosis specific histone modifications (e.g., H3S10 and H3S28) Requires large number of cells depending upon downstream application (e.g., a transcription factor ChIP-seq requires larger cell number than an RNA-seq experiments). Can be cost-prohibitive. 
 Cell synchronization Enrichment of mitotic population using cell-cycle inhibitors (e.g., nocodazole) Potential artifacts caused by treatment with chemicals. 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 microscopy Visualization of protein of interest (POI) localization to mitotic chromosomes Antibody specificity. Antibody accessibility to condensed 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., high-throughput imaging positioning mapping) Covisualization of multiple genes, transcripts and proteins Developing reagents that work together is challenging. Specialized instrumentation and training is required for execution and interpretation of experiments. 
Biochemical Transcription factor (TF) chromatin immunoprecipitation (ChIP) Gene-specific (by qPCR) or genome-wide (by sequencing) protein-chromatin interactions using antibodies against POI Antibody specificity is a key variable and must be determined empirically using supporting approaches (e.g., IP and Western blot). Antibody accessibility to condensed mitotic chromosomes. 
 Histone posttranslational modification (PTM) ChIP Often done in combination with TF-ChIP to identify epigenetic characteristics of genes bookmarked by POI Although antibodies against most histone PTMs are well-characterized, multiple histone PTMs coexist on 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 transcribed RNA in mitotically enriched cells in which the POI expression has been modified. Requires complex experimental design to ensure specific and selective detection of transcripts nascently transcribed during and immediately following mitosis in the presence or the absence of POI. 
 Inducible POI expression/downregulation using lentivirus. Degron systems for inducible degradation of POI at mitosis. Regulated expression of protein of interest at and during mitosis to determine functional relevance of mitotic gene bookmarking Functional link between mitotic gene bookmarking and activity of POI requires precise expression (or downregulation) of POI at mitosis, which can be challenging because of the short length of mitosis. 
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).

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).

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Mitotic retention of key components of RNA polymerase (RNAP) machineries support competency for a basal level of transcription throughout mitosis

Mitosis is accompanied by striking biochemical changes, including a decline in nuclear RNA transcription (12, 14, 42). Mitotic repression of transcription was first noted over 60 years ago in studies analyzing incorporation of radiolabeled RNA precursors during the cell cycle (47). Initial studies examining mechanisms that repress transcription in mitotic cells reported that most of the RNAP II elongation complexes were physically excluded from mitotic chromosomes, although some genes retained active RNAP II complexes (48). The authors suggested that limitations of pulse-labeling approaches may prevent detection of low levels of mitotic transcriptional activity. Recent approaches designed to detect subtle transcriptional changes have identified waves of transcriptional activity throughout mitosis. Using cell-permeable 5-ethynyluridine to pulse-label nascent transcripts, Zaret and colleagues showed that mitotic cells maintain a steady-state level of transcriptional activity, and that the genes involved in fundamental cellular functions, for example, cell growth and proliferation, are among the first to be transcribed, while lineage-specific genes are expressed to establish cell identity as cells exit mitosis (46). These findings are consistent with reports from our group, and others, that components of RNAP I machinery that include upstream binding factor 1 (UBF1) remain associated with ribosomal RNA genes during mitosis (49, 50). A recent report from the Tjian group further shows that TBP, a key component of the RNAP II machinery, is also stably retained on mitotic chromosomes and facilitates recruitment of RNAP II to genes for transcription as cells progress through and exit mitosis (51). These studies support a mechanism for transcriptional memory through successive cell divisions that is defined by the retention of essential components of basal transcriptional machineries to support low levels of transcription throughout mitosis.

Modifications and variants of nucleosomal histones establish a transcription-permissive mitotic chromatin state

Low level, yet persistent, transcriptional activity during mitosis suggests that mitotic chromosomes are, at least partially, accessible to the necessary transcriptional regulatory complexes. However, the highly condensed nature of mitotic chromosomes has prevented a systematic, gene-level analysis of chromosome accessibility during mitosis. Advances in approaches to examine three-dimensional genome organization have produced high-resolution mapping of chromosome architecture in the interphase nucleus and during mitosis (12, 52, 53). Chromosome conformation capture assays have identified a linearly organized and longitudinally compressed array of consecutive chromatin loops that is shared by all chromosomes and is consistent across cell types (54). Despite a high degree of compaction, regions of mitotic chromosomes remain in an open conformation and accessible to regulatory proteins. These regions often contain mitotically bookmarked genes and share several properties that include (i) nuclease accessibility, (ii) enrichment of specific histone variants/modifications, and (iii) association of the mitotic compaction protein cohesin with actively transcribed genes.

Several studies in the mid-1970s revealed that condensed mitotic chromosomes are amenable to nuclease digestion (55). Subsequent studies using ligation-mediated polymerase chain reaction identified specific genes that are nuclease accessible, permanganate sensitive [i.e., contain single-stranded (ss) DNA] and exhibit regulatory protein occupancy during mitosis. For example, the Myc gene is reactivated by ssDNA-binding proteins [e.g., far upstream element-binding protein (FBP)] immediately after cells divide. Consistent with a role for mitotic gene bookmarking in reactivating genes postmitotically, the Myc gene is nuclease accessible and permanganate sensitive. Importantly, FBP remains associated with the gene during mitosis. Recent findings at higher resolution and genome-wide levels indicate larger chromatin domains of moderate nuclease accessibility remain open during mitosis, and the more dynamic changes in accessibility are locally dynamic. Furthermore, DNA hypomethylation marks a subset of promoters that retain accessibility during mitosis (45). These findings are consistent with a unique and selective behavior of bookmarked genes during mitosis, for example, nuclease sensitivity and accessibility to single-strand DNA-binding proteins for rapid reactivation postmitotically. Genome-wide studies are required to further assess whether nuclease accessibility is a common feature of all mitotically bookmarked genes.

Emerging evidence indicates that the nucleosomes of bookmarked genes are enriched in histone variants, particularly in H2A.Z and H3.3. For example, nucleosomes at the myogenic gene MyoD are enriched in histone H3.3, and this enrichment supports persistent expression of the gene through 24 cell divisions (56). Similarly, nucleosomes immediately downstream of the transcription start sites (TSS) contain H2A.Z and shift upstream to occupy TSSs during mitosis, thus reducing nucleosome-depleted regulatory regions (57). This change appears to be specific to active genes that are silenced during mitosis and rapidly reactivated postmitotically. These authors report that, among other histone modifications, the activating histone mark—trimethylation of lysine 4 at histone H3 (H3K4me3)—is enriched at these promoters during mitosis, whereas other epigenetic markers of active chromatin are relinquished. More recent studies have identified H3K27ac, a histone mark that is enriched in the interphase enhancer regions, is also associated with mitotically bookmarked genes (26). These findings are consistent with a distinct global epigenomic landscape of mitotic chromosomes, that is, association of the activating H3K9ac, H3K27ac, and H3K4me3 modifications with the gene-rich regions and CpG islands, and enrichment of the repressive H3K27me3 mark in distinct bands that show very little overlap with gene-rich regions (58). Together, these observations point to a specialized chromatin landscape of mitotically bookmarked genes that is defined by selective incorporation of histone variants and retention of specific posttranslational histone modifications and supports rapid recruitment of transcriptional regulatory machinery for gene reactivation immediately following cell division.

Although selective nuclease accessibility, histone modifications and histone variants are associated with mitotically bookmarked genes, highly condensed nature of mitotic chromosomes, and global reorganization of regulatory machinery during cell division suggests that additional mechanisms must exist to maintain structural integrity of bookmarked genes during mitosis. Genome-wide chromatin immunoprecipitation-sequencing studies have revealed that transcription factors are bound to genes in a highly clustered manner (59, 60). At the chromatin architectural level, most clusters are formed around cohesin, a protein required for chromosome condensation during mitosis. Mechanistically, cohesin plays two key roles: (i) during the S-phase of the cell cycle, it holds the replicating strands together at the TF cluster sites; and (ii) during mitosis, it remains bound to the clusters when the transcription factors have been displaced from target genes (61). Functionally, loss of cohesin decreases both DNA accessibility and binding of TFs to clusters (59). These results provide a mechanistic explanation for nuclease accessibility of mitotic chromosomes and identify additional chromatin properties of mitotically bookmarked genes. Furthermore, these observations suggest that cohesin binding promotes reestablishment of TF clusters and reactivation of target genes after DNA replication as well as after mitosis, thus adding a structural component to the mechanism for maintaining cellular memory through cell divisions (59, 60).

Consistent with accessible mitotic chromatin that is enriched in specific histone variants and posttranslational modifications, key chromatin remodeling proteins are retained on mitotic chromosomes. These include histone acetyl transferases (e.g., p300), DNA demethylases (e.g., DNMT1), ATP-dependent chromatin remodeling proteins (e.g., ISWI1), methyltransferases (e.g., MLL), and readers of histone modifications (e.g., BRD4; refs. 17, 32, 40, 62–65). Although it remains to be established whether every bookmarked gene retains all or some of the chromatin-modifying proteins, it is important to point out that many of these coregulators interact with mitotically retained phenotypic proteins (e.g., RUNX) that occupy target genes in a sequence-specific manner and provide scaffolds for organization and assembly of regulatory complexes to coordinately control gene expression.

Phenotypic transcription factors provide specific and selective gene regulatory activity required for cell identity

As discussed above, components of RNAP machineries are retained on chromosomes during mitosis, and together with the mitotic retention of selective chromatin modifiers, create a regulatory environment that is permissive to steady-state transcription during mitosis. Another level of specificity is provided by sequence-specific transcription factors that are often master regulators of their respective lineages. To date, more than 20 sequence-specific and pioneering transcription factors—proteins with the ability to reprogram one cell type into another—have been reported to be retained on mitotic chromosomes (66). These include the osteogenic and hematopoietic RUNX transcription factors (40, 67, 68), the key erythroid regulator GATA1 (22, 45), the muscle-restricted MyoD (41), and the liver-related FOXA1 transcription factor (18). Among the properties, these regulatory proteins share are (i) specific intranuclear localization in the interphase nucleus; (ii) sequence-specific and dynamic interactions with the chromatin; (iii) lineage-restricted physiologic gene regulation; and (iv) interaction with multiple coregulatory proteins that include transcriptional activators and repressors. (See more below.)

Coordination of RNAP I and II transcription

The role for mitotic gene bookmarking in coordinating cell growth, proliferation and identity is illustrated by RUNX proteins, master regulators of osteogenesis, hematopoiesis, neurogenesis, and gastrointestinal development (69). During mitosis, all RUNX proteins (RUNX1, 2, and 3) associate with RNA Pol I-transcribed ribosomal RNA genes and RNA Pol II-transcribed genes that are involved in control of the cell cycle, phenotype, and differentiation (49, 67, 68). RUNX transcription factors are equally partitioned in progeny cells at the completion of cell division (40). The association of RUNX factors with ribosomal and cell-cycle–regulatory genes (e.g., the cell-cycle inhibitor p21) during mitosis bookmarks these genes for regulation during the early G1 phase of the cell cycle. In addition, RUNX bookmarking of differentiation-related genes that include Smads, downstream effectors of the transforming growth factor β/bone morphogenetic protein signaling pathway, by RUNX proteins during mitosis provides a mechanistic basis for lineage-restricted transcriptional memory in progeny cells (70). Occupancy and regulation of RNA Pol I- and RNA Pol II-transcribed genes by RUNX proteins during interphase and mitosis enables coordination of cell proliferation, growth, and differentiation by acting both at genetic and epigenetic levels.

Maintenance of cell plasticity and lineage identity

Mesenchymal stem cells (MSC) have the capacity to differentiate into multiple lineages that include osteoblasts, adipocytes, and myoblasts. Differentiation of MSCs into myoblasts requires the basic helix–loop–helix myogenic regulatory factors (including MyoD, Myf5, and MRF4) that bind to E-boxes in target gene promoters and play crucial roles in skeletal-muscle development. Together with Mef2 proteins and E-box factors, these transcription factors are responsible for coordinating muscle-specific gene expression by negatively regulating proliferation and promoting differentiation. During the proliferative stage of mesenchymal stem cells, MyoD is localized to mitotic chromosomes and associates with ribosomal RNA genes and nucleolar-organizing regions (NOR; ref. 41). The association of MyoD with the interphase nucleolus in early stages of myogenesis and its replacement by myogenin in later stages results in the downregulation of ribosomal RNA genes, concomitant with initiation of the skeletal-muscle differentiation program. Consistent with these observations, adipocyte differentiation of MSCs recruits CCAAT/enhancer-binding proteins α and δ (C/EBPα and δ) to the C/EBP regulatory element in the C/EBPβ gene promoter, upregulating the C/EBPβ protein, which functions as a transcriptional activator of late-adipocyte genes. Studies from our lab demonstrate C/EBP transcription factors occupy ribosomal RNA genes during mitosis (41). As preadipocytes complete cell division, C/EBP proteins downregulate ribosomal RNA genes, consistent with their role in initiating adipocyte differentiation when ribosomal gene expression is decreased. The association of muscle and adipocyte-specific transcription factors with mitotic chromosomes in their respective lineages and the subsequent downregulation of ribosomal RNA genes in the interphase suggest that these phenotypic regulatory proteins mediate lineage commitment and maintenance through bookmarking of target gene loci.

Pluripotent stem cells divide rapidly and exhibit an abbreviated G1 phase of the cell cycle (71). The pluripotent stem cell phenotype exhibits the capacity for unrestricted, but highly regulated proliferation, and plasticity to differentiate into any cell lineage, providing an optimal model to investigate mitotic bookmarking. Mitotically purified populations of undifferentiated stem cells retain the activating H3K4me3 mark on selective genes necessary for lineage commitment, thus poising them for expression (72). In the absence of extracellular differentiation cues, these genes reacquire the repressive H3K27me3 until the next cell division. In response to an extracellular differentiation signal, pluripotent cells exercise the option to commit to a defined lineage and the genes remain H3K4me3 marked. These findings are further corroborated by recent studies showing that in addition to bivalent chromatin marks—the activating H3K4me3 and the repressive H3K27me3—the enhancer-associated H3K27ac mark as well as pluripotent transcription factors SOX2 and KLF4 are retained during mitosis in pluripotent stem cells (26). A significant recent finding is the partial recapitulation of chromatin bivalency in early-stage cancer cells. This “oncofetal epigenetic control” indicates that the bivalent chromatin plays a role in acquisition of tumor phenotype (73, 74). It remains to be established whether some, all, or none of the genes that reacquire the activating H3K4me3 and the repressive H3K27me3 histone marks in early-stage cancer cells also relinquish the repressive H3K27me3 mark during mitosis to sustain plasticity of cancer cells. Together, these and other studies have identified mitotic gene bookmarking as a central epigenetic mechanism for lineage identity in committed cells and cell plasticity in pluripotent stem cells.

Sustained tumor phenotype

The tumor phenotype is characterized by deregulated differentiation program and unrestricted cell proliferation that, unlike in pluripotent stem cells, is not physiologically controlled. Recent studies suggest that mitotic gene bookmarking has an important role in the onset, progression, and perpetuation of disease. A key example is provided by the leukemic fusion protein AML1-ETO that blocks myeloid differentiation and enhances proliferative potential (75). The leukemic AML1-ETO mitotically bookmarks rRNA genes, as well as genes controlling cell proliferation and myeloid cell differentiation (67). Functionally, AML1-ETO upregulates rRNA and cell proliferation-related genes but downregulates gene-mediating myeloid cell differentiation, promoting the transformed phenotype. Another example of cancer-related mitotic gene bookmarking is the mixed lineage leukemia protein (MLL). MLL is a chromatin remodeling factor that is associated with leukemia and regulates transcription by recruiting chromatin-modifying machinery to target genes. MLL mitotic retention favors rapid reactivation of target genes required for the onset and progression of MLL postmitotically (17). In-depth and genome-wide studies are required to establish whether mitotic bookmarking of cancer-related genes is a shared activity of sequence-specific oncogenic proteins.

It is increasingly evident that mitotic bookmarking is a central epigenetic mechanism to maintain cellular identity through cell divisions (Fig. 1). Emerging evidence indicates that phenotypic transcription factors mitotically bookmark a subset of target genes and this bookmarking contributes to coordinate control of cell proliferation, growth, and differentiation. Importantly, mitotic gene bookmarking by oncogenes in cancer cells may be necessary to maintain the tumor phenotype. Open-ended questions related to mitotic gene bookmarking that can provide mechanistic and clinically relevant insights into compromised epigenetic control in the onset and progression of cancer include (i) Are mitotically bookmarked genes organized in shared nuclear microenvironments in G1 cells to facilitate coordinate control? And/or is mitotic gene bookmarking a mechanism to assemble coordinately regulated genes in shared nuclear regulatory microenvironments? (ii) What is the role of coregulatory proteins of transcription factor bookmarks in gene reactivation as cells exit mitosis? (iii) To what extent are genes bookmarked that are not expressed immediately after mitosis but in subsequent cell-cycle stages (e.g., histone genes that are specifically upregulated in S-phase.)? (iv) What is the core regulatory network that is required to maintain cell identity? (v) Is mitotic bookmarking operative in cells that divide asymmetrically? and (vi) What is the contribution of mitotic bookmarking in tumorigenesis? It has been traditionally difficult to target transcription factors due to unfavorable pharmacokinetics and substantial off-target effects. From translational and clinical perspectives, mitotic bookmarking has the potential to provide preferential and selective therapeutic intervention.

No potential conflicts of interest were disclosed.

The studies were supported by P01 CA 082834 from The National Cancer Institute and by the Charlotte Perelman Fund for Cancer Research. G.S. Stein is the Arthur J. Perelman Professor in Cancer Research.

1.
Dundr
M
. 
Nuclear bodies: multifunctional companions of the genome
.
Curr Opin Cell Biol
2012
;
24
:
415
22
.
2.
Schneider
R
,
Grosschedl
R
. 
Dynamics and interplay of nuclear architecture, genome organization, and gene expression
.
Genes Dev
2007
;
21
:
3027
43
.
3.
Sleeman
JE
,
Trinkle-Mulcahy
L
. 
Nuclear bodies: new insights into assembly/dynamics and disease relevance
.
Curr Opin Cell Biol
2014
;
28
:
76
83
.
4.
Zaidi
SK
,
Medina
RF
,
Pockwinse
SM
,
Bakshi
R
,
Kota
KP
,
Ali
SA
, et al
Subnuclear localization and intranuclear trafficking of transcription factors
.
Methods Mol Biol
2010
;
647
:
77
93
.
5.
Zaidi
SK
,
Young
DW
,
Choi
JY
,
Pratap
J
,
Javed
A
,
Montecino
M
, et al
The dynamic organization of gene-regulatory machinery in nuclear microenvironments
.
EMBO Rep
2005
;
6
:
128
33
.
6.
Zaidi
SK
,
Young
DW
,
Javed
A
,
Pratap
J
,
Montecino
M
,
van Wijnen
A
, et al
Nuclear microenvironments in biological control and cancer
.
Nat Rev Cancer
2007
;
7
:
454
63
.
7.
Dey
P
. 
Cancer nucleus: morphology and beyond
.
Diagn Cytopathol
2010
;
38
:
382
90
.
8.
Drobic
B
,
Dunn
KL
,
Espino
PS
,
Davie
JR
. 
Abnormalities of chromatin in tumor cells
.
EXS
2006
;
96
:
25
47
.
9.
Lever
E
,
Sheer
D
. 
The role of nuclear organization in cancer
.
J Pathol
2010
;
220
:
114
25
.
10.
Misteli
T
. 
Beyond the sequence: cellular organization of genome function
.
Cell
2007
;
128
:
787
800
.
11.
Tai
PW
,
Zaidi
SK
,
Wu
H
,
Grandy
RA
,
Montecino
M
,
van Wijnen
AJ
, et al
The dynamic architectural and epigenetic nuclear landscape: developing the genomic almanac of biology and disease
.
J Cell Physiol
2014
;
229
:
711
27
.
12.
Naumova
N
,
Imakaev
M
,
Fudenberg
G
,
Zhan
Y
,
Lajoie
BR
,
Mirny
LA
, et al
Organization of the mitotic chromosome
.
Science
2013
;
342
:
948
53
.
13.
Ohta
S
,
Wood
L
,
Bukowski-Wills
JC
,
Rappsilber
J
,
Earnshaw
WC
. 
Building mitotic chromosomes
.
Curr Opin Cell Biol
2011
;
23
:
114
21
.
14.
Scholey
JM
,
Brust-Mascher
I
,
Mogilner
A
. 
Cell division
.
Nature
2003
;
422
:
746
52
.
15.
Arampatzi
P
,
Gialitakis
M
,
Makatounakis
T
,
Papamatheakis
J
. 
Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements
.
Nucleic Acids Res
2013
;
41
:
2202
15
.
16.
Arora
M
,
Packard
CZ
,
Banerjee
T
,
Parvin
JD
. 
RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins
.
Nucleic Acids Res
2016
;
44
:
2136
44
.
17.
Blobel
GA
,
Kadauke
S
,
Wang
E
,
Lau
AW
,
Zuber
J
,
Chou
MM
, et al
A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit
.
Mol Cell
2009
;
36
:
970
83
.
18.
Caravaca
JM
,
Donahue
G
,
Becker
JS
,
He
X
,
Vinson
C
,
Zaret
KS
. 
Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes
.
Genes Dev
2013
;
27
:
251
60
.
19.
Festuccia
N
,
Dubois
A
,
Vandormael-Pournin
S
,
Gallego Tejeda
E
,
Mouren
A
,
Bessonnard
S
, et al
Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network
.
Nat Cell Biol
2016
;
18
:
1139
48
.
20.
John
S
,
Workman
JL
. 
Bookmarking genes for activation in condensed mitotic chromosomes
.
Bioessays
1998
;
20
:
275
9
.
21.
Kadauke
S
,
Blobel
GA
. 
Mitotic bookmarking by transcription factors
.
Epigenetics Chromatin
2013
;
6
:
6
.
22.
Kadauke
S
,
Udugama
MI
,
Pawlicki
JM
,
Achtman
JC
,
Jain
DP
,
Cheng
Y
, et al
Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1
.
Cell
2012
;
150
:
725
37
.
23.
Kelly
TK
,
Jones
PA
. 
Role of nucleosomes in mitotic bookmarking
.
Cell Cycle
2011
;
10
:
370
1
.
24.
Lake
RJ
,
Tsai
PF
,
Choi
I
,
Won
KJ
,
Fan
HY
. 
RBPJ, the major transcriptional effector of Notch signaling, remains associated with chromatin throughout mitosis, suggesting a role in mitotic bookmarking
.
PLoS Genet
2014
;
10
:
e1004204
.
25.
Lerner
J
,
Bagattin
A
,
Verdeguer
F
,
Makinistoglu
MP
,
Garbay
S
,
Felix
T
, et al
Human mutations affect the epigenetic/bookmarking function of HNF1B
.
Nucleic Acids Res
2016
;
44
:
8097
111
.
26.
Liu
Y
,
Pelham-Webb
B
,
Di Giammartino
DC
,
Li
J
,
Kim
D
,
Kita
K
, et al
Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells
.
Cell Rep
2017
;
19
:
1283
93
.
27.
Lodhi
N
,
Ji
Y
,
Tulin
A
. 
Mitotic bookmarking: maintaining post-mitotic reprogramming of transcription reactivation
.
Curr Mol Biol Rep
2016
;
2
:
10
6
.
28.
Lodhi
N
,
Kossenkov
AV
,
Tulin
AV
. 
Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark
.
Nucleic Acids Res
2014
;
42
:
7028
38
.
29.
Sarge
KD
,
Park-Sarge
OK
. 
Gene bookmarking: keeping the pages open
.
Trends Biochem Sci
2005
;
30
:
605
10
.
30.
Sarge
KD
,
Park-Sarge
OK
. 
Mitotic bookmarking of formerly active genes: keeping epigenetic memories from fading
.
Cell Cycle
2009
;
8
:
818
23
.
31.
Verdeguer
F
,
Le Corre
S
,
Fischer
E
,
Callens
C
,
Garbay
S
,
Doyen
A
, et al
A mitotic transcriptional switch in polycystic kidney disease
.
Nat Med
2010
;
16
:
106
10
.
32.
Wong
MM
,
Byun
JS
,
Sacta
M
,
Jin
Q
,
Baek
S
,
Gardner
K
. 
Promoter-bound p300 complexes facilitate post-mitotic transmission of transcriptional memory
.
PLoS One
2014
;
9
:
e99989
.
33.
Xing
H
,
Wilkerson
DC
,
Mayhew
CN
,
Lubert
EJ
,
Skaggs
HS
,
Goodson
ML
, et al
Mechanism of hsp70i gene bookmarking
.
Science
2005
;
307
:
421
3
.
34.
Zaidi
SK
,
Grandy
RA
,
Lopez-Camacho
C
,
Montecino
M
,
van Wijnen
AJ
,
Lian
JB
, et al
Bookmarking target genes in mitosis: a shared epigenetic trait of phenotypic transcription factors and oncogenes?
Cancer Res
2014
;
74
:
420
5
.
35.
Zaidi
SK
,
Young
DW
,
Montecino
MA
,
Lian
JB
,
van Wijnen
AJ
,
Stein
JL
, et al
Mitotic bookmarking of genes: a novel dimension to epigenetic control
.
Nat Rev Genet
2010
;
11
:
583
9
.
36.
Zaret
KS
. 
Genome reactivation after the silence in mitosis: recapitulating mechanisms of development?
Dev Cell
2014
;
29
:
132
4
.
37.
Zhao
R
,
Nakamura
T
,
Fu
Y
,
Lazar
Z
,
Spector
DL
. 
Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation
.
Nat Cell Biol
2011
;
13
:
1295
304
.
38.
Michelotti
EF
,
Sanford
S
,
Levens
D
. 
Marking of active genes on mitotic chromosomes
.
Nature
1997
;
388
:
895
9
.
39.
Martinez-Balbas
MA
,
Dey
A
,
Rabindran
SK
,
Ozato
K
,
Wu
C
. 
Displacement of sequence-specific transcription factors from mitotic chromatin
.
Cell
1995
;
83
:
29
38
.
40.
Zaidi
SK
,
Young
DW
,
Pockwinse
SM
,
Javed
A
,
Lian
JB
,
Stein
JL
, et al
Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells
.
Proc Natl Acad Sci U S A
2003
;
100
:
14852
7
.
41.
Ali
SA
,
Zaidi
SK
,
Dacwag
CS
,
Salma
N
,
Young
DW
,
Shakoori
AR
, et al
Phenotypic transcription factors epigenetically mediate cell growth control
.
Proc Natl Acad Sci U S A
2008
;
105
:
6632
7
.
42.
Gottesfeld
JM
,
Forbes
DJ
. 
Mitotic repression of the transcriptional machinery
.
Trends Biochem Sci
1997
;
22
:
197
202
.
43.
Hartl
P
,
Gottesfeld
J
,
Forbes
DJ
. 
Mitotic repression of transcription in vitro
.
J Cell Biol
1993
;
120
:
613
24
.
44.
Teves
SS
,
An
L
,
Hansen
AS
,
Xie
L
,
Darzacq
X
,
Tjian
R
. 
A dynamic mode of mitotic bookmarking by transcription factors
.
Elife
2016
;
5
:
e22280
.
45.
Hsiung
CC
,
Morrissey
CS
,
Udugama
M
,
Frank
CL
,
Keller
CA
,
Baek
S
, et al
Genome accessibility is widely preserved and locally modulated during mitosis
.
Genome Res
2015
;
25
:
213
25
.
46.
Palozola
KC
,
Donahue
G
,
Liu
H
,
Grant
GR
,
Becker
JS
,
Cote
A
, et al
Mitotic transcription and waves of gene reactivation during mitotic exit
.
Science
2017
;
358
:
119
22
.
47.
Prescott
DM
,
Bender
MA
. 
Synthesis of RNA and protein during mitosis in mammalian tissue culture cells
.
Exp Cell Res
1962
;
26
:
260
8
.
48.
Parsons
GG
,
Spencer
CA
. 
Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes
.
Mol Cell Biol
1997
;
17
:
5791
802
.
49.
Young
DW
,
Hassan
MQ
,
Pratap
J
,
Galindo
M
,
Zaidi
SK
,
Lee
SH
, et al
Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2
.
Nature
2007
;
445
:
442
6
.
50.
Roussel
P
,
Andre
C
,
Comai
L
,
HernandezVerdun
D
. 
The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs
.
J Cell Biol
1996
;
133
:
235
46
.
51.
Teves
SS
,
An
L
,
Bhargava-Shah
A
,
Xie
L
,
Darzacq
X
,
Tjian
R
. 
A stable mode of bookmarking by TBP recruits RNA Polymerase II to mitotic chromosomes
.
eLife
2018
;
7
:
e35621
.
52.
Dekker
J
,
Rippe
K
,
Dekker
M
,
Kleckner
N
. 
Capturing chromosome conformation
.
Science
2002
;
295
:
1306
11
.
53.
Gibcus
JH
,
Dekker
J
. 
The hierarchy of the 3D genome
.
Mol Cell
2013
;
49
:
773
82
.
54.
Gibcus
JH
,
Samejima
K
,
Goloborodko
A
,
Samejima
I
,
Naumova
N
,
Nuebler
J
, et al
A pathway for mitotic chromosome formation
.
Science
2018
;
359:eaa06135
.
55.
Bostock
CJ
,
Christie
S
,
Hatch
FT
. 
Accessibility of DNA in condensed chromatin to nuclease digestion
.
Nature
1976
;
262
:
516
9
.
56.
Ng
RK
,
Gurdon
JB
. 
Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription
.
Nat Cell Biol
2008
;
10
:
102
9
.
57.
Kelly
TK
,
Miranda
TB
,
Liang
G
,
Berman
BP
,
Lin
JC
,
Tanay
A
, et al
H2A.Z maintenance during mitosis reveals nucleosome shifting on mitotically silenced genes
.
Mol Cell
2010
;
39
:
901
11
.
58.
Terrenoire
E
,
McRonald
F
,
Halsall
JA
,
Page
P
,
Illingworth
RS
,
Taylor
AM
, et al
Immunostaining of modified histones defines high-level features of the human metaphase epigenome
.
Genome Biol
2010
;
11
:
R110
.
59.
Yan
J
,
Enge
M
,
Whitington
T
,
Dave
K
,
Liu
J
,
Sur
I
, et al
Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites
.
Cell
2013
;
154
:
801
13
.
60.
Zuin
J
,
Dixon
JR
,
van der Reijden
MI
,
Ye
Z
,
Kolovos
P
,
Brouwer
RW
, et al
Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells
.
Proc Natl Acad Sci U S A
2014
;
111
:
996
1001
.
61.
Bernardi
G
. 
Genome organization and chromosome architecture
.
Cold Spring Harb Symp Quant Biol
2015
;
80
:
83
91
.
62.
Easwaran
HP
,
Schermelleh
L
,
Leonhardt
H
,
Cardoso
MC
. 
Replication-independent chromatin loading of Dnmt1 during G2 and M phases
.
EMBO Rep
2004
;
5
:
1181
6
.
63.
Yokoyama
H
,
Rybina
S
,
Santarella-Mellwig
R
,
Mattaj
IW
,
Karsenti
E
. 
ISWI is a RanGTP-dependent MAP required for chromosome segregation
.
J Cell Biol
2009
;
187
:
813
29
.
64.
Dey
A
,
Chitsaz
F
,
Abbasi
A
,
Misteli
T
,
Ozato
K
. 
The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis
.
Proc Natl Acad Sci U S A
2003
;
100
:
8758
63
.
65.
Dey
A
,
Nishiyama
A
,
Karpova
T
,
McNally
J
,
Ozato
K
. 
Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription
.
Mol Biol Cell
2009
;
20
:
4899
909
.
66.
Festuccia
N
,
Gonzalez
I
,
Owens
N
,
Navarro
P
. 
Mitotic bookmarking in development and stem cells
.
Development
2017
;
144
:
3633
45
.
67.
Bakshi
R
,
Zaidi
SK
,
Pande
S
,
Hassan
MQ
,
Young
DW
,
Montecino
M
, et al
The leukemogenic t(8;21) fusion protein AML1-ETO controls rRNA genes and associates with nucleolar-organizing regions at mitotic chromosomes
.
J Cell Sci
2008
;
121
(
Pt 23
):
3981
90
.
68.
Pande
S
,
Ali
SA
,
Dowdy
C
,
Zaidi
SK
,
Ito
K
,
Ito
Y
, et al
Subnuclear targeting of the Runx3 tumor suppressor and its epigenetic association with mitotic chromosomes
.
J Cell Physiol
2009
;
218
:
473
9
.
69.
Otto
F
,
Lubbert
M
,
Stock
M
. 
Upstream and downstream targets of RUNX proteins
.
J Cell Biochem
2003
;
89
:
9
18
.
70.
Young
DW
,
Hassan
MQ
,
Yang
XQ
,
Galindo
M
,
Javed
A
,
Zaidi
SK
, et al
Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2
.
Proc Natl Acad Sci U S A
2007
;
104
:
3189
94
.
71.
Kapinas
K
,
Grandy
R
,
Ghule
P
,
Medina
R
,
Becker
K
,
Pardee
A
, et al
The abbreviated pluripotent cell cycle
.
J Cell Physiol
2013
;
228
:
9
20
.
72.
Grandy
RA
,
Whitfield
TW
,
Wu
H
,
Fitzgerald
MP
,
VanOudenhove
JJ
,
Zaidi
SK
, et al
Genome-wide studies reveal that H3K4me3 modification in bivalent genes is dynamically regulated during the pluripotent cell cycle and stabilized upon differentiation
.
Mol Cell Biol
2015
;
36
:
615
27
.
73.
Messier
TL
,
Boyd
JR
,
Gordon
JA
,
Stein
JL
,
Lian
JB
,
Stein
GS
. 
Oncofetal epigenetic bivalency in breast cancer cells: H3K4 and H3K27 Tri-methylation as a biomarker for phenotypic plasticity
.
J Cell Physiol
2016
;
231
:
2474
81
.
74.
Zaidi
SK
,
Frietze
SE
,
Gordon
JA
,
Heath
JL
,
Messier
T
,
Hong
D
, et al
Bivalent epigenetic control of oncofetal gene expression in cancer
.
Mol Cell Biol
2017
;
37
:
e00352
17
.
75.
Peterson
LF
,
Zhang
DE
. 
The 8;21 translocation in leukemogenesis
.
Oncogene
2004
;
23
:
4255
62
.