Epigenetic-mediated regulation of gene expression for biological control and cancer: fidelity of mechanisms governing the cell cycle

Abstract

The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on 1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, 2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and 3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.

1. An epigenetic perspective of cell cycle control

The cell cycle is controlled by complex and interdependent processes that require epigenetically-mediated remodeling of the genome and the structural and functional regulatory machinery that governs fidelity of DNA replication and cell division. Crosstalk between mechanisms that coordinate proliferation and cell growth is obligatory, with unique requirements that are phenotype dependent. Many normal and tumor cells exhibit unrestricted proliferation. However, stringent control of the cell cycle is compromised in tumor cells, often reaching the balance between proliferation, cell survival, and responsiveness to physiological cues, as well as acquiring resistance to therapy that targets cell cycle check points. We will provide an overview of the decisive regulatory parameters for cell cycle control that are mechanistically and clinically informative. Emphasis is on genome reconfiguration during the cell cycle that is required for epigenetically-mediated cell cycle and mitotic progression with competency to retain regulatory machinery during mitosis for transmission from parental to progeny cells.

2. Interplay between cell cycle progression, cellular transcriptional machinery, and epigenetics

Progression between distinct phases of the cell cycle depends on the correct timing of various events, which are coordinated by cellular transcriptional machinery and epigenetics controlling genome accessibility.

The cell cycle consists of interphase (G1, S, and G2 phases), mitotic phase (mitosis and cytokinesis, figure 1). During G1, cells nearly double their size, preparing for DNA replication. At the restriction point late in G1, the genes for deoxynucleotide biosynthesis are expressed, supporting competency for DNA synthesis. This preparation is carried out by assembling the pre-replicative complex at the replication origins. CDC6 is essential for the assembly of the pre-replicative complex (pre-RCs), which are located on the origins of DNA replication [1, 2]. At S-phase, histone gene expression is initiated. The pre-replication complexes become active, followed by the separation of the two DNA strands of the double helix, and by an elongation step catalyzed by DNA polymerase. Once DNA replication is completed, the chromosomes and Centrosomes are duplicated, and the amount of DNA is doubled in the cell. During G2 phase, the cells grow rapidly, and the checkpoint machinery ensures that the DNA is properly replicated and repaired when damage occurs. Centrosomes detach and undergo growth by the accumulation of pericentriolar material around the centrioles. This leads to centrosome maturation, which is a necessary step for assembly of the mitotic apparatus to enter the M-phase.

Figure 1. Chromosomes during interphase and mitotic M-phase.

a. The chromatin is present within chromosome territories during interphase. b. In prophase, the chromatin condenses into chromosomes and the nucleolus disappears. Centrosomes migrate to the opposite poles of the nucleus and initiate the formation of the mitotic spindle. c. During prometaphase-metaphase, the nuclear envelope breaks. Then, the chromosomes are oriented and aligned on the metaphase plate. d. In early anaphase, the sister chromatids separate, and the microtubules pull the chromatids apart toward the two opposite poles of the mitotic spindle. d• In late anaphase, the plasma membrane begins to invaginate to the equatorial plane. e. During telophase, the plasma membrane continues to invaginate, and the chromosomes decondense. f. During cytokinesis-abscission, the invagination of the plasma membrane appears through a contractile ring. The cleavage furrow progresses to create midbody between the progeny cells, which disappears during the abscission process. g. This results in the separation of the two progeny cells, which enter interphase and begin the process again.

Several studies investigated the structure, function, and regulation of the human histone genes, focusing especially on the cell cycle regulation of histone gene transcription and histone mRNA coupling with DNA replication. Genome-wide high-resolution chromosome conformation capture (HiC) technologies indicate that non-random higher-order organization of genes into structural domains supports regulatory activities involving non-contiguous sequences that influence transcription [3–6]. The human histone locus bodies (HLBs) are nonmembrane bound, phase-separated domains that provide microenvironments for dynamic regulation of histone genes. The human histone gene transcription factor HINFP mediates coordinate expression of multiple histone H4 genes at the G1/S transition in normal and cancer cells, as well as the abbreviated human embryonic stem cell cycle [1–5]. Importantly, HINFP loss-of-function causes increased nucleosome spacing, induces replicative stress, and leads to genomic instability [6]. The overall increase in nucleosome spacing could be due to the insufficiency of newly synthesized histone H4 during DNA replication. In in vivo mouse models, HINFP is essential for fidelity of histone H4 gene expression and required for early embryonic development [6, 7]. Furthermore, the role of HINFP in guarding normal chromatin organization is functionally conserved in flies [8]. NPAT is an essential prototypical HLB resident protein that regulates the expression of all replication-dependent histone gene classes (H4, H3, H2A, H2B, H1). HINFP is the critical anchor that tethers NPAT to histone H4 genes [9, 10]. In response to activation of the CDK2/cyclin E cell cycle signaling cascade at the onset of the S-phase, NPAT is phosphorylated to initiate histone gene transcription [11–13]. Furthermore, deregulation of histone gene expression upon sustained loss of HINFP or NPAT causes disruption of HLBs and has drastic consequences for cell division and cell survival in both normal and cancer cells [6, 14, 15]. These findings establish that regulatory interactions involving HINFP and NPAT are essential for chromatin conformations at HLBs during the cell cycle to regulate histone gene transcription. Thus, understanding molecular mechanisms that control histone synthesis at the onset of the S-phase would be a paradigm for transcriptional control that is obligatory for cell cycle progression.

GRO-seq, RNA-seq, and ChIP-seq analyzed the transcriptional and epigenetic dynamics during the cell cycle in breast cancer MCF-7 cells [16]. This study identified genes differentially transcribed at the G0/G1, G1/S, and M phases in MCF-7. There was no correlation between the transcription and steady-state mRNA abundance at the cell-cycle level. Active transcription occurred only during early mitosis. However, an overall increase in histone modification marks was reported at mitosis. Thousands of enhancer RNAs (eRNAs) and their associated transcription factors were identified, which were related to cell-cycle–regulated transcription but not to the steady-state expression.

Consequently, these results showed that the interplay between the transcriptional machinery and epigenetics is crucial for cell-cycle progression. Thus, the precise understanding of cell cycle control is of great interest for understanding such pathologies as cancers.

Epigenetic control during mitosis

Mitosis plays a fundamental role in transmitting stable inheritance of gene expression pattern from parental cells to their progeny [17]. During this phase, the nuclear envelop breaks down is disaggregated, triggered by the disassembly of nuclear pore complexes and lamina depolymerization [18, 19]. This process leads to chromatin becoming highly compacted into chromosomes [20]. During mitosis, many transcription factors dissociate from chromatin leading to significant downregulation of transcriptional activity [21]. However, there is little knowledge of how lineage-specific epigenetic information is faithfully maintained in progeny cells. Several studies suggest that the gene regulatory networks are controlled by epigenetic bookmarks that are maintained during mitosis [21–27] (figure 2). In human embryonic stem cells (hESCs), bivalent genes with both the activation mark (H3K4me3) and repressive mark (H3K27me3) are important for maintaining pluripotency [28, 29]. However, during mitosis, the bivalent genes containing H3K4me3 are the most upregulated genes upon differentiation of hESC, showing a new aspect of epigenetic regulation crucial for maintaining pluripotency [28–30].

Figure 2. Mitotic bookmarking establishes post-mitotic reactivation of gene expression.

During mitosis, histone marks and chromatin regulators bind the open regions on the chromatin, thus bookmarking specific loci for the memory program. Transcription factors can additionally associate with the chromatin targets. Consequently, these mechanisms result in a post-mitotic transcriptional activation after mitosis.

The entry of mitosis is controlled by CDK1/cyclin B kinase also known as MPF (M-phase Promoting Factors). CDK1/cyclin B is responsible for direct and indirect phosphorylation of substrates involved in mitosis. Histone H1 is a direct substrate of CDK1/cyclin B [31]. Other substrates, including CDK1 regulators like CDC25 and WEE1, and cytoskeletal proteins like Microtubule-Associated Proteins (MAPs), nuclear lamins, and vimentin, are required for the proper progression of mitosis [32–35]. Most recently, it was demonstrated that the S-phase initiator CDC6 plays a crucial role in controlling mitotic M-phase entry. CDC6 regulates CDK1 activity and determines the timing of mitosis in Xenopus cell-free extract [36–40]. Inhibition of CDC6 results in earlier M-phase entry in Xenopus cell-free extract and mouse zygotes. CDC6 modulates the activity of CDK1 via cyclin B and not cyclin A and acts through a bona fide CDK inhibitor XIC1 [36, 39]. Additionally, CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes [40]. This suggests that the time of mitosis is not only under the control of cyclins as it has been described in the last 20 years [41, 42], but additionally under the control of CDC6, which can counterbalance the cyclins to control CDK1 activation and subsequently to specify the timing of mitosis.

These findings suggest that the epigenetic control and correct timing of mitosis play a coordinating role between gene regulatory networks, cell cycle machinery, and the developmental program, ensuring accurate inheritance of the genome during cell division.

3. Mechanisms of chromosome behavior during mitosis

Cell division is the essential feature of all cancers and requires the orchestrated separation of the duplicated genome into two daughter cells during mitosis. Mitosis comprises four basic phases (figure 1): prophase, prometaphase-metaphase, anaphase, and telophase [43].

During prophase, the chromatin begins to condense into chromosomes and the nucleolus disappears. The chromosomes consist of two sister chromatids; each contains a DNA element called centromere that connects both chromatids [44]. The two sister chromatids are glued with cohesin. However, kinetochores are assembled at centromeres to ensure the interaction of chromosomes with spindle microtubules. In prophase, microtubules depolymerize, centrosomes migrate to opposite poles of the nucleus, and initiate the formation of the mitotic spindle.

During prometaphase-metaphase, the breakdown of the nuclear envelope (NEBD) occurs, and chromosome condensation begins. This is carried out by activation of the histone H1 kinase activity resulting in hyperphosphorylation of nuclear lamins, leading to NEBD, and shortening microtubules by phosphorylation of Microtubule-Associated Proteins (MAPs). Then, the chromosomes are captured by microtubules attached to kinetochores. At metaphase, the chromosomes are fully condensed and assembled uniformly on the equatorial plate of the spindle. Three types of microtubules nucleated at the centrosomes can be distinguished i) kinetochore microtubules, ii) polar microtubules pointing directly to the opposite spindle pole, iii) astral microtubules radiating out of the spindle. The balance between ejection and pulling forces exerted on chromosomes permits the alignment of chromosomes on the metaphase plate. At this stage, the ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C), which is involved in the degradation of cyclins, becomes active [45]. This activation is carried out by a series of phosphorylation on the APC/C complex induced by CDK1. Therefore, APC/C triggers degradation of the cyclins, allowing inactivation of CDK1 and subsequent separation of the sister-chromatid.

During anaphase, active segregation of sister chromatids takes place within the mitotic spindle. When APC/C becomes active, it catalyzes ubiquitination and degradation of securing, relieving the inhibition from separase. The latter is responsible for cohesion cleavage allowing separation of sister chromatids [46]. In parallel, the microtubules pull the chromatids apart toward the two opposite poles of the mitotic spindle. In late anaphase, the plasma membrane starts to invaginate to the equatorial plane, and the cell enters telophase.

In telophase, the nuclear envelope is reformed around the chromosomes entering the interphase state of de-condensation. The plasma membrane continues to invaginate, and the elongation of polar microtubules stops leading to the formation of the central spindle. In higher eukaryotes, this process is known as open mitosis, ensuring the inheritance of chromosomes to the newly formed cells [47]. However, because the nuclear envelope does not break down during mitosis in lower eucaryotes, the mode of mitosis is referred to as closed mitosis [48].

During cytokinesis, the cytoplasm, organelles, cell membrane, and the two formed nuclei split into two progeny cells. This is characterized by a cleavage furrow formed perpendicular to the mitotic spindle. The invagination of the plasma membrane appears through a contractile ring, where actin and myosin microfilaments play mechanical roles. The cleavage furrow progresses to create midbody between the progeny cells. The midbody disappears during the abscission process leading to the separation of the two daughter cells.

These results showed how accurately chromosomes are distributed and organized at each stage of mitosis. Chromosome organization must be highly dynamic during mitosis to ensure inheritance to the progeny cells.

Recent reports have provided an update about the current knowledge on mechanisms of chromosome regulation across mitosis [49–51]. They examined how the pairs of homologous chromosomes are continuously separated at mitosis. It has been shown that mammalian cells disrupted homologous chromosome pairing by keeping the two haploid chromosome sets separately and by positioning them on either edge of the nuclear division axis, defined by centrosomes. Loss of mitotic anti-pairing causes loss of heterozygosity (LOH) and is correlated with gene misregulation in cancer cells. This suggests that anti-pairing plays a significant role in the inhibition of genetic recombination or allelic misregulation across cell division. How mitotic recombination is prevented and how genomic stability is maintained during division are fundamental unanswered questions. For example, what are the regulatory mechanisms involved in haploid set chromosome sequestration? Is the cytoskeleton implicated in this process? In Drosophila, chromosome pairing can be actively inhibited, and several studies suggest that condensin II could play a role in regulating the anti-pairing activity [52–55]. Consequently, how does condensin II regulate mitotic anti-pairing in human cells? These are relevant questions that must be addressed.

4. Condensin-mediated remodeling of mitotic chromosomes

Nuclear DNA is highly condensed and wrapped around nucleosomes to fit inside the nucleus [56]. Each nucleosome is formed by a set of eight histone proteins — two copies each of the histone H2A, H2B, H3, H4, and 146-nucleotide pair DNA double helix. The latter is wrapped 1.65 times around the histone core to form a fiber structure of 10 nm width, described as ‘beads on a string’. Nucleosomes are stabilized by the histone H1 and compact the genomic DNA sevenfold. Additional compaction occurs during interphase to fully organize the DNA inside the nucleus. Upon mitotic entry, the chromatin highly condenses into mitotic chromosomes, positions at the metaphase plate, and avoids being disrupted during anaphase. The condensation of chromatin is controlled by condensins and post translational modifications [57]. The condensation events start before NEBD. The chromatin fibers fold and reach 700nm of diameter by the end of prophase approximately [58]. These events are in part due to the activation of CDK1/cyclin B and of condensins by phosphorylation [59]. Condensins depletion leads to a delay in mitotic compaction [60, 61].

Atomic force microscopy is an excellent technique to visualize condensins during mitosis [62]. Unlike other microscopes, it does not use photons nor electrons (like in electron microscopy) to produce images. Condensin is composed of two proteins of the family of SMC (Structural maintenance of chromosome) as well as three non-SMC subunits: a kleisin (NCAPH) and two HEAT domain proteins (NCAPG/D) [63]. There are two condensin types, condensin I and II, which differ by their non-SMC proteins. Condensin II binds the chromatin fiber throughout the cell cycle, while the condensin I complex binds the chromosomes only after NEBD. Both condensins I and II contribute to mitotic compaction [64]. However, it seems that condensin I acts on the fibers that condensin II did not compact, thus promoting lateral compaction of the fibers and subsequently sustaining stable condensed chromatin [65].

Cohesins interact with chromatin in G1 and attach the sister chromatids to each other during the S-phase. A fraction of condensins II associates with duplicated regions during S-phase then initiates sister chromatid resolution. In prophase, the majority of cohesins are released from the chromosomal arms, and condensins II associates with chromosomes inducing condensation. After NEBD, condensins I is directed to chromosomes and keep facilitating the condensation. In anaphase, the separase cleaves the kleisin subunit of cohesins, allowing the separation of sister chromatids.

Several studies have shown that binding of condensin I to prometaphase requires the mitotic kinase Aurora B [66]. In the presence of topoisomerases I, Condensins fold the chromatin fiber into super loops to form the mitotic chromosome [67]. Later in mitosis, the axial compaction of chromosomal arms requires condensins activities in combination with sister chromatid resolution mediated by topoisomerase II and release of cohesins [68].

The condensation can take place in condensing-depleted cells; however, it is associated with chromosomal structural defects [65]. In S. pombe yeast, mutants of SMC proteins are not able to condense their chromosomes but remain capable of carrying out the elongation of the mitotic spindle and cell division [69]. In animals, condensation is affected but not completely abolished by the loss of condensins [65]. Moreover, knock-out of SMC2 in chicken DT40 cells or knock-down by RNAi (RNA interference) in C. elegans affects the architecture of the mitotic chromosome but has a limited impact on the condensation itself [60, 61]. Together these results suggest that several factors are important for the chromosome compaction during mitosis.

5. Chromatin decondensation upon the mitotic exit

In mitosis, Haspin and Aurora B are dissociated from the chromosomes and recruited to the centromeres [70]. Therefore, the histone H3S10PO₄ and H3T3PO₄ modification levels gradually decrease during metaphase/anaphase transition. Despite the reduction in H3 phosphorylation, the chromosomes remain condensed until telophase. During telophase, the mitotic chromosomes decondense and regain their interphase nuclear structure.

The exit from mitosis requires inactivation of kinases and reversion of mitotic dephosphorylations of mitotic substrates by phosphatases PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [37, 38, 40]. Depletion of PP1 and PP2A delays the exit from mitosis [71]. PP1 is responsible for the dephosphorylation of Thr23, Ser10 and Ser28 of histone H3 [72]. These dephosphorylations allow histone H4K16 reacetylation and chromosome decondensation. However, PP1 is directed to chromosomes by its Repo-man subunit. Depletion of Repo-Man alters the reformation of the nuclear envelope but has no visible consequence on the chromatin decondensation [73, 74]. PP1 is recruited at chromosomes undergoing decondensation by Ki-67 (MKI67) [75], which could compensate for the loss of Repo-man. Because PP1 is recruited before the reformation of the nuclear envelope, this suggests a direct role of PP1 in mitotic decondensation; yet the mechanism is still unclear [76].

To study decondensation mechanisms upon mitosis exit, an interesting approach has been developed in vitro using Xenopus laevis cytoplasmic egg extract system which recapitulates mitotic decondensation [77]. These experiments showed that chromatin decondensation requires energy in the form of ATP and GTP, suggesting that decondensation is an active process and not just chromatin relaxation caused by the dissociation of condensation factors. This is consistent with the fact that decondensation is dependent on the presence of factors present in Xenopus eggs [77]. The dependency on ATP can be explained by the fact that the dissociation of Aurora B requires the ATPase protein p97/VCP [78]. Another ATPase complex has been involved in decondensation, the RuvB-like 1/2 complex [77]. In addition to its implications in many cellular processes, RuvB-like 1/2 is a component of several interphase ATP-dependent chromatin remodeling complexes [79], suggesting that it may act at the end of mitosis to regain an interphasic structure.

6. Involvement of TADs and CTCF in chromatin structure re-configuration after mitosis

Topologically Associating Domains (TADs) play a key role in regulating of gene expression along promoters, enhancers, and silencers respectively activate or repress transcription [80]. For example, by folding DNA in space, enhancers and promoters interact and modulate transcription. It is then obvious that an enhancer must be in the same TAD as its target genes. This allows for several genes within the same TAD to be co-regulated by the same enhancer.

The formation of TADs was demonstrated by validating the Loop extrusion model [80]. This mechanism involves cohesin and CCCTC-Binding Factor (CTCF) proteins which recognize motifs around TADs and drag chromatin through rings [81, 82]. Both cohesin and CTCF are enriched at TAD boundaries [83]; however, their depletion makes TADs less prominent [84].

During mitosis, TADs are absent; however, how TAD formation is controlled during the cell cycle remains unclear. Recent 5C analysis demonstrated that TADs and CTCF loops are present in interphase but absent on mitotic chromatin [85], as stated in previous studies [86]. Additionally, ATAC-seq and CUT&RUN analysis showed reduced accessibility and loss of CTCF binding in prometaphase.

More recently, it has been demonstrated that depletion of CTCF during the M- to G1-phase progression alters the re-formation of chromatin domain boundary and structural loops, leading to inappropriate interactions between cis-regulatory elements (CREs) [86]. Thus, CTCF plays a crucial role in the nuclear architecture and chromatin dynamics, thus resetting a functional G1 nucleus after mitosis.

7. Mitotic gene bookmarking: an epigenetic program for cell identity

After mitosis, cells remember their identity and faithfully reactivate their genetic programs [87]. This occurs through gene bookmarking, an epigenetic mechanism involved in transmitting of information during cell division [23, 24, 88]. It is defined as the retention of key regulatory proteins at gene loci on mitotic chromosomes (e.g., chromatin-modifying factors, transcription factors, and components of RNA Pol I and II) [24]. This epigenetic mechanism ensures that genes important for the cellular phenotype will be expressed immediately after mitosis.

RUNX1 is a well characterized bookmark for defining mammary epithelial cell identity [22]. Disruption of RUNX1 and its heterodimeric partner CBFB interaction and association with mitotic chromosomes leads to epithelial-to-mesenchymal transition (EMT) [22]. This shows the importance of bookmarking in stabilizing the mammary epithelial cell identity. Similarly, the HNF1B transcription factor is known to be involved in the conversion of fibroblast into hepatic stem cells and in renal tubular cells [89, 90]. When combined with three additional transcription factors involved in kidney functions, HNF1B induces conversation of mouse and human fibroblasts into induced renal tubular cells (iRECs) [90].

Nascent RNA sequencing technologies, such as precision run-on sequencing (PRO-seq) could be an excellent approach to study bookmarking mechanisms [91–95]. PRO-seq directly measures nascent RNA transcription by creating high-resolution maps of all transcriptionally engaged RNA polymerases, unlike the traditional RNA-seq or ChIP-seq analyses. PROseq can detect distinct steps of RNA transcription, including RNA polymerase recruitment, promoter-proximal pausing, and transcription elongation. It provides a direct measurement of enhancer activity and quantifies the transcription of enhancers and target genes simultaneously. Therefore, PROseq is extremely useful for studying bookmarking and enhancer-mediated regulation during mitosis.

7.1. Role of the histone post translational modifications (PTMs)

Histone post-translational modifications (PTMs) are responsible for the maintenance of the epigenetic memory, in this way, the daughter cells decide which genes need to be reactivated or repressed after division. During mitosis, histones undergo many post-translational modifications, including phosphorylation, methylation, acetylation, and ubiquitylation [96]. These modifications can modify the overall chemical charge of histone proteins, which normally carry an acidic, negative charge, and they can also act on gene expression and chromatin condensation via the recruitment or exclusion of proteins from chromatin. The question is whether these changes play a role in the organization of the mitotic chromosome structure.

Phosphorylation of histone H3 on serine 10 by Aurora B kinase is one of the most abundant PTM occurring in mitosis [97]. An additional four residues of histone H3 are phosphorylated in mitosis, including threonine 3 and 11 and serines 10 and 28.

Incomplete phosphorylation of histone H3 can lead to condensation and segregation of abnormal chromosomes because of the poor recruitment of condensin I [98, 99]. In early mitosis, the phosphorylation of threonine 3 on histone H3 (H3T3) by Haspin and phosphorylation of serine 10 on histone H3 (H3S10) by Aurora B recruits the deacetylase SIRT2 which in turn deacetylates lysine 16 on histone H4 (H4K16), leading to chromatin compaction [100, 101]. This mechanism has also been described in yeast [102]. In addition, phosphorylation modifications act on the attractive force between nucleosomes [102], which is generated by the binding of H4 tails [103, 104]. It results in the displacement of regulatory complexes from mitotic chromatin [96, 105, 106]. For example, phosphorylation on Ser10 of H3K9me3 causes the displacement of the heterochromatin protein HP1 from H3K9me2 or H3K9me3 sites.

Methylation of histones is associated with both the active and repressive marks on the chromatin [102]. For example, mono-di and trimethylation at Lys4 (H3K4me1, H3K4me2 and H3K4me3) are responsible for gene activation and retained on chromatin during mitosis. However, trimethylation of histone H3 at Lys27 (H3K27me3), and Lys9 (H3K9me) is instead associated with repressed genes. So far, these methylations are maintained during mitosis and are partly responsible for the epigenetic inheritance of cell identity [107, 108].

Acetylation of histones is generally associated with active transcription. Acetylation decreases during mitosis, but some active promoters retain these marks, for example, the acetylation at Lys27 (H3K27ac) on promoter and enhancers [16]. H3K27a seems to be involved in bookmarking enhancers and promoters’ genes critical for stem cell identity [16].

Ubiquitination to H2B is responsible for gene activation, which decreases in mitosis, whereas ubiquitination H2A is responsible for gene repression [109].

In mitosis, the histone modifications are also able to recruit specific regulatory proteins on the chromatin. For example, phosphorylation on histone H3T3 acts as a regulator together with Aurora B during mitosis, resulting in the correct attachment of the kinetochore to the microtubules and chromosome separation [110–112].

7.2. Transcription factors (TFs), key regulators of memory program for resetting transcription

NEBD and DNA structural changes result in the dispersion of RNA polymerases, transcription factors and chromatin remodeling complexes from chromatin, and thus reduction of transcription. Many transcription factors are subjected to post-translational modifications, e.g., phosphorylation, subsequently resulting in changes in the protein structure. These changes alter the activity of the transcription factors, preventing them from recognizing their targets and thus detaching from the DNA. For example, the transcription factor OCT1, once phosphorylated in mitosis, is no longer able to bind DNA and detaches [113]. Also, phosphorylation of transcription factors such as TFIIH, TFIID, and IKAROS leads to reduction of transcription [113–117]. In addition, the transcription factor SP1, which binds the human HSP70 promoter, shows reduced DNA-binding activity in mitotic cell extracts [118]. In contrast, the DNA-binding activities of three transcription factors bound to the Hsp70 promoter (C/EBP, GBP, and HSF1) were normal. This suggests that retention or displacement of transcription factors during mitosis could provide another mechanism for resetting transcription in the cell cycle. Note that phosphorylation not only causes protein structural changes, but it can also cause degradation via the ubiquitin degradation system, as in the case of EZH2. In mitosis, phosphorylation EZH2 by CDK1/Cyclin B leads to ubiquitination of EZH2 and subsequent degradation by the proteasome [119].

Other transcription factors, such as RUNX1/2, GATA1, FOXA1, TBP, HNF1B, bind to the chromosomes during mitosis to maintain the memory program [22, 120–125]. So far, it has been demonstrated that the degree of localization of TFs is correlated with some interphase TF features, such as the subnuclear localization and the charge of the DNA-binding domain [126]. These factors are of great importance to gene reactivation during early G1. For instance, the mitotic retention of Brd4 leads to accelerated decondensation and resumption of post-mitotic transcription [127]. Moreover, Brd4-deficient mouse embryonic fibroblasts exhibit delayed gene expression in early G1 [128].

7.3. Nucleosome re-positioning, an alternative bookmarking mechanism

The control of nucleosome positioning is another regulatory mechanism controlling the mitotic bookmarking. In mitosis, nucleosomes occupy the transcriptional start sites (TSSs), preventing the binding of the transcriptional machinery, and thus reducing the gene expression. When mitosis ends, nucleosomes recover their initial position to disclose the TSS [129].

7.4. Mitotic transmission of phenotype-specific mRNAs in cancer cells

Beyond chromosome-related bookmarking mechanisms, cells may complement this genomic flagging of genes to maintain phenotype identity by the targeted transmission of mRNAs as a complementary level of translational control. For example, the osteogenic phenotype of osteosarcoma cells is directly linked to the expression of RUNX2. Osteosarcoma cells apply a non-genomic ‘mRNA stocking mechanism’ in which cells produce maximal levels of RUNX2 mRNA, but not protein, prior to mitotic cell division [130]. RUNX2 mRNA then partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Somatic transmission of RUNX2 mRNAs in these bone cancer cells ensures that RUNX2 levels can be rapidly replenished during early G1 by translational rather than transcriptional activation to sustain its mitotic bookmarking function during interphase in osteosarcoma cells.

8. Perturbation of chromosomal rearrangements in cancer

As mentioned above, both condensin I and condensin II are essential for chromosome condensation during mitosis. Equally, condensins regulate the spatial genome organization, replication, and transcription [131]. Therefore, it is well known that condensins are indispensable for maintaining genome stability. For example, depletion of condensin II provokes genome instability by damaging DNA throughout the genome and by impairing telomere function [132]. Condensin depletion can induce breakage–fusion–bridge (BFB) cycles and large chromosomal rearrangements [133]. Additionally, TCGA analysis shows that condensins are mutated in many cancers [134]. Most of these mutations are missense and are susceptible to driving sublethal effects on chromosome structure. Together, these results suggest that condensin inhibition may be a potential trigger involved in the initiation and progression of tumorigenesis because of perturbation of the mitotic chromosome condensation, genome instability, and telomere dysfunction.

9. Transcription factor fusion proteins as therapeutic targets

Transcription factor fusions have been identified as a trigger of various subtypes of leukemias. Examples include AML1–ETO and CBFB fused with SMMHC in acute myeloid leukemia. It has been shown that these protein fusions prevent differentiation, assuming a more stem cell-like state [135]. In addition, some of these protein fusions have been shown to cause significant changes in DNA repair genes [136], leading to more mutations and uncontrolled proliferation as well as clonal heterogeneity of leukemia. This finding suggests that transcription factor fusion proteins are a trigger for other genomic alterations, making these chimeric proteins desirable targets for drug development.

A recent approach called PROTACs (or PROteolysis Targeting Chimeras) has been employed to degrade transcription factors in cells with high accuracy [137–139]. PROTACs consist of two-headed small molecules that selectively join the target protein with an E3 ubiquitin ligase. This results in ubiquitylation and proteasome-mediated destruction of the target. Unlike traditional pharmacological approaches, this approach allows a single molecule to degrade multiple targets and thus represents a key area for future therapy.

10. Conclusion

Epigenetic control of the cell cycle is contributing insight into mechanisms that govern the regulation of proliferation. DNA application and mitosis are mediated by remodeling of the genome and the architecturally organized regulatory machinery that orchestrates cell cycle progression and molecular processes that are obligatory for the fidelity of the proliferation process. Cancer-compromised cell cycle control functions prominently in the onset and progression of tumorigenesis. Evidence is accruing for epigenetic involvement in breaches of physiological control for stringent balances between competency for cell division and alignment of checkpoint mechanisms that support competency for cell survival.