Epigenetic regulation−The guardian of cellular homeostasis and lineage commitment
1Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, India
2Department of Life Sciences, Sharda University, Greater Noida, Uttar Pradesh, 201310, India
3Department of Biotechnology, Campus–Bhimtal, Kumaun University, Nainital, Uttarakhand, 263136, India
4Department of Botany, Mahatma Gandhi Central University, Motihari, Bihar, 845401, India
*Address correspondence to: Kavitha Govarthanan, firstname.lastname@example.org; Ram Prasad, email@example.com
Received: 27 September 2020; Accepted: 06 January 2021
Abstract: Stem cells constitute the source of cells that replenishes the worn out or damaged cells in our tissue and enable the tissue to carry out the destined function. Tissue-specific stem cells are compartmentalized in a niche, which keeps the stem cells under quiescent condition. Thus, understanding the molecular events driving the successful differentiation of stem cells into several lineages is essential for its better manipulation of human applications. Given the developmental aspects of the cell, the cellular function is greatly dependent on the epigenomics signature that in turn governs the expression profile of the cell. The stable inheritance of the epigenome is crucial for the development, modulation, and maintenance of the cell and its complex tissue-specific function. Emerging evidence suggesting that stem cell chromatin comprises a specialized state in which self-renewing genes and its downstream lineage-specific genes are kept paralleled poised for activation. Thus, the epigenetic regulatory network and pathway dictate lineage commitment and differentiation. It mainly modifies the chromatin landscape to facilitate euchromatin and heterochromatin architecture, which in turn alters the accessibility of transcription factors to the gene loci. DNA methylation and histone marks are the two widely studied epigenetic modifications regulating the transcriptome profile of a specific lineage. Abnormalities in the epigenetic landscape lead to diseases or disorders. Here, we emphasize the prominence of the epigenetic network and its regulation in normal tissue functioning and in the diseased state. Furthermore, we highlighted the emerging role of epigenetic modifiers in lineage differentiation and epigenetic markers as novel druggable targets for cancer therapy.
Keywords: Epigenetics; Chromatin Genome; Methylation; Inheritance; Modulation
The concept of genomic equivalence states that all cells in an organism carry the same genetic material, but the expression profiles may vary according to its destined function. Further, in order to facilitate the differential expression profile, epigenetics plays a central role in modulating the sequential changes in the chromatin landscape that finally leads to the specific chromatin signature for a particular lineage. Epigenetics is classically defined as “the branch of biology which deals with the cross-talk between genes and its products that finally leads to the phenotype into existence” (Waddington, 1942). The current biologist defines epigenetics as collective information about the chromatin landscape resulting in a specific transcriptome profile of cells without the involving changes in the primary DNA sequence (Russo et al., 1996). In a simpler way, it can be described as an alteration in phenotype without alteration in genotype. In the complex mammalian system, epigenetics modulates the chromatin configuration upon which the expression profile of the genes varies. If any aberrations or changes happen in the epigenetic network, then it may lead to the development of a variety of human diseases. Therefore, a stable inheritance of epigenetic state is very crucial for the development, modulation, and maintenance of cells and their complex tissue-specific function.
Chromatin is a multimeric complex made up of domains of histone proteins on which the DNA is tightly wound and well packaged within the cell. An epigenome is a complimentary term connected with the chemical changes occurring in the cytosine moiety of DNA base sequence in the chromatin without altering the sequence of the DNA. This change can be often transferred down to the progeny via transgenerational epigenetic inheritance (Waterland and Michels, 2007). If there is any change in the epigenome, then it may result in the modification of chromatin organization leading to an alteration in the genome’s function (Bannister and Kouzarides, 2011). The epigenome involves gene expression regulation, embryonic and fetal development, tissue differentiation, genomic imprinting, suppression of transposable elements, and inactivation of X-chromosome (Fedoriw et al., 2012; Li et al., 2012). In contrast, the underlying genome remains fundamentally inert within an individual, whereas the epigenome may be constantly altered by cues such as age, diet, and also in the diseased states. The epigenome is highly influenced by environmental factors and external cues, thus studying its fundamental mechanism may help us to enrich our knowledge in stem cell identity, fine-tuning of stem cell differentiation, and other developmental mechanisms related to tissue functioning. In eukaryotes, the gene regulation is tightly bound by the specific epigenetic signatures where the expression and repression of a specific gene are distinct in a particular cell type. Major epigenetic contributing events widely studied in gene regulation are methylation of DNA and histone protein modifications. (Russo et al., 1996; Avgustinova and Benitah, 2016). This current review talks about the highlights of epigenetic variations occurring in the chromatin landscape resulting in the maintenance of cellular homeostasis and differentiation. Also, several epigenetic markers as new druggable targets will be discussed in this review for effective killing of tumorigenic stem cells or CSCs.
Epigenetic Modifications in Chromatin Architecture
DNA packaging and chromatin assembly are tightly organized phenomena that determine the transcriptome profile of cell type. Thus, the cellular identity and its homeostasis principally depend on the epigenetic modifications that widely occur throughout the genome. The conventional chromatin modifications that occur in the mammalian genome are methylation of DNA and modifications in the core histone residues. In DNA methylation, the methyl groups donated from S-adenosyl methionine (SAM) are covalently added cytosine or adenine residues of DNA by the DNMTase family of enzymes. Methyl group is added to the 5th position of cytosine residue in CpG dinucleotide is the most studied and highly heritable modification suggested to maintain the stable genomic integrity (Razin and Shemer, 1995; Lachner and Jenuwein, 2002). The methylated form of cytosine causes the spatial hindrance to the binding of a transcription factor to the promoter region and thereby represses the gene expression. DNA methylation analysis confers stable information about the transcripts, and it is found to be directly coupled with cellular differentiation (Jaenisch and Bird, 2003). Methylation of DNA at the promoter region dictates the transcriptome profile of the cell by impeding the interaction of transcriptional machinery with the gene. Further, the binding of methyl-CpG-binding domain proteins (MDBs) to the methylated DNA, in turn, attracts the ancillary proteins like histone deacetylase and other chromatin remodeling proteins. Therefore, forming an inactive chromatin state called heterochromatin and ultimately hypothesized to involve in the key regulatory processes like genomic imprinting, X-chromosome inactivation, transposable elements repression, cellular senescence, and cancer initiation and progression. Thus, methylation positioned at the gene promoter region modifies the function of the DNA, thereby repressing the gene transcription (Wainwright and Scaffidi, 2007).
Histone core modifications
Histones are proteins made up of basic amino acids and involved in the formation of nucleosome core as octamer complex containing a C-terminal globular domain and N-terminal tail (Luger et al., 1997). Several covalent post-translational alterations, comprising methylation, phosphorylation acetylation, ubiquitination, and sumoylation, occur on particular residues of N-terminal tail regions of histone. These modifications are indispensable in regulating the key cellular processes, including transcription and repair mechanisms (Kouzarides, 2007). Hence, it has been hypothesized that the complementary modifications on histone residues are encoded as “histone code” and it contributes to epigenetic memory inside a cell (Jenuwein and Allis, 2001). Chromatin accessibility is tightly regulated by recruiting non-histone effector proteins that act as a block in decoding the message determined by its modification. In contrast to DNA methylation, histone modifications on the specific type on the specific determine the activation or repression of the downstream molecular events. The mammalian cell is made up of chromatin that presents in two forms, either as euchromatin (open for gene transcription) or heterochromatin (closed for gene transcription), and it is solely dependent on the post-translational modifications on histone.
Presence of heavily acetylated histone residues (H3K9ac, H4K12ac (Hebbes et al., 1988; Liang et al., 2004), and the histone core is enriched with H3K4Me3 (Bernstein et al., 2002) and linked with histone variant H3.3 (Ahmad and Henikoff, 2002; Mcittrick et al., 2004) sorts the Euchromatin. However, heterochromatin is typically contained with repressive methyl marks H3K27Me3, H3K9Me2 (Zhang and Reinberg, 2001, Umlauf et al., 2004; Dong and Weng, 2013). A huge panel of active and repressive histone marks have been identified, forming a complex regulatory network indispensable for various cellular activities (Bernstein et al., 2007). Hence, the compact wrapping of eukaryotic DNA in the form of chromatin determines the accessibility of DNA.
The chromatin remodelers are ATP-dependent multi-enzyme complexes involving in the process of chromatin opening using an ATP dependent manner. According to the configuration and order of the ATPase subunits, the nucleosome remodelers are classified into four classes, such as the SWI/SNF family, the ISWI family, the CHD family and the INO80 family (Munoz et al., 2012). BRM/BAF are human analogs of SWI/SNF classes of remodelers having a role in both activation and repression of genes performing key functions in development (Lessard and Crabtree, 2010). Studies about double conditional knock out of the BAF155 and BAF170 core units in mice showed BAF complex could globally modulate key chromatin marks H3K27Me2 and 3 by direct regulation of Utx and jmjd3, an H3K27 demethylase. Additionally, loss of BAF complexes impaired forebrain development and embryogenesis by upregulating H3K27Me3 (Nguyen et al., 2016). Additionally, knock-out experiments pertaining to BA170 in BAF complex showed loss of pluripotency whereas overexpression showed impaired differentiation to mesoderm and endoderm lineage (Wade et al., 2015).
Non-coding RNAs (ncRNAs)
In the central dogma of molecular biology, the information flow from DNA to protein contributes through RNA, which functions to code for a protein with a destined specific function. However, a few exceptions to this paradigm are still present in which RNAs do not code for proteins, which in turn function in the regulation and processing of other RNAs (mRNAs, tRNAs, and rRNAs). It includes in a process such as splicing (snRNAs), nucleotide modification (snoRNAs), processing pre-tRNAs (RNase P, a ribozyme). Other small ncRNAs like miRNAs and siRNAs that involve in gene regulation by targeting mRNAs. LncRNAs are > 200 nucleotides to 2 kb sequence of non-coding transcripts (Carninci et al., 2005; Dinger et al., 2009; Perkel, 2013). These are putative non-coding RNAs lacking an elusive open reading frame (ORF) of 300 nucleotides or longer. (Pang et al., 2006). The hierarchy of epigenetic regulation with respect from the genomic DNA to the transcriptome mRNA is shown in detail in Fig. 1.
Epigenetics of Stem Cells
DNA methylation role in stem cell fate
DNA methylation occurs mainly in the CpG dinucleotide region, which is found in clusters called CpG islands. 60% of human gene promoter has CpG islands, which is unmethylated in stem cells and get methylated, leads to tissue-specific expression during early embryonic development or in different specific tissue types in adults. Stem cells unveil a unique gene expression profile that governs cell fate during lineage commitment and differentiation (Straussman et al., 2009). DNA methylation array could give key evidence about hypo-methylation (transcriptionally open state) and hyper-methylation (transcriptionally repressed state) of various target genes (Fouse et al., 2008). DNA methylation negatively controls the gene expression via recruitment of MBD, which in turn recruits histone-modifying and chromatin-remodeling complexes to the specific methylated site. Recruitment of various proteins disables the availability of promoter region for transcription factors, which suppress the gene expression (Protela and Esteller, 2010). GATA2, TAL1, and LMO2 are the oncogenes and myeloid key transcription factors responsible for myeloid lineage commitment.
These genes were highly methylated during lymphoid differentiation demonstrating DNA methylation may not only interfere with gene expression but also block the binding of unwanted myeloid transcription factors, which are of oncogenic nature. DNA methylation parallelly inhibits the activation of TF gene loci, as well as block the binding of TFs across the whole genome to make sure the two-tier epigenetic barrier in regulating the expression of myeloid TFs in lymphoid cells. Thus, a high-resolution global DNA Methylation mapping proved the gain of DNA methylation and its direct correlation in loss of gene expression during the course of differentiation (Shen, 2009). This also suggested that these epigenetic switches were performing the role of surveillance mainly to prevent the aberrant expression of stem cell-related genes after differentiation.
The differentiation potential of MSCs to adipocytes was directly correlated with its long-term culture and demonstrated the methylated status of LEP promoter upon adipogenic stimulation (Digirolamo et al., 1999; Noer et al., 2007; Banfi et al., 2008). Another study suggested that hypermethylation of ADIPOQ in late passages restricted the adipogenic differentiation (Lara-Castro et al., 2007). The core hypermethylated regions in the genome of mesenchymal progenitors showed a common epigenetic marker suggesting that MSCs obtained from adipose tissue, bone marrow and skeletal muscle were having a common origin (Hupkes et al., 2011; Sorrenson et al., 2010; Hakelein et al., 2014). This enabled us to understand that cellular identity is mainly defined by its epigenetic state (switch) and gets modified according to the lineage-committed during differentiation.
The promoter methylation status of NKX 2.5 and sFRP4 in umbilical cord MSCs displayed that both promoters underwent demethylation and further validated with upregulated expression at mRNA level upon cardiac stimulation (Bhuvanalakshmi et al., 2017). Hyper methylated CD31 promoter region showed a restricted differentiation potential to endothelial lineage in MSCs derived from adipose tissue (Bonquest et al., 2007). Any aberrant change in the DNA methylation signature at the enhancer region could lead to inappropriate gene expression and delayed differentiation in intestinal stem cells (Sheaffer et al., 2014). Similarly, a restricted differentiation potential of the C2C12 myoblast cell line to adipogenic and osteogenic lineage was observed due to the promoter hypermethylation (Hupkes et al., 2011). The differentiated methylation patterns were found to be established already in MSCs at its progenitor state, and also the differentiation potential of MSCs directly coupled with the methylation profile of the lineage-specific markers, where hypermethylation representing a barrier to differentiation (Sorrenson et al., 2010).
Histone core modification role in stem cell fate
A specific mark on histone protein contributes to its post-translational modifications governs gene expression patterns and differentiation potential in stem cells (Meissner, 2010; Fisher and Fisher, 2011). Global genome-wide analysis revealed that the presence of ‘‘bivalent or poised’’ chromatin domains in many developmental genes exhibiting both ‘‘active’’ (H3K4me3) and ‘‘repressive’’ (H3K27me3) marks on histone proteins (Azuara et al., 2006; Bernstein, 2006). This intermediate bivalent marked state is anticipated to permit for the further précised and sequential gene expression. Acetylation of lysine (K) residues in H3 (Histone-3) is considered as an active mark on the euchromatin near those genes that are actively transcribed.
PcG protein complex maintains the gene expression of many cells during development. PcG proteins like EZH (EZH1/EZH2), EED and SUZ 12 along with methyltransferase, PRC2 acts on histone H3 lysine 27 (H3K27), are very essential for maintenance and control of pluripotency. PRC2, along with jumonji protein, acts as a master regulatory switch by which this protein complex rapidly reprograms the epigenome either by repression or subsequent activation via H3K27me3 (Shen et al., 2009). A specialized chromatin signature is essential for the proper conversion of pluripotent ESCs to multipotent ESCs in which a bivalent chromatin structure was documented in developmental and pluripotent genes. It maintained a gene in a transcriptional open state, which allows the instant transcription activation of specific genes upon induction with differentiation factors and shutting of pluripotency genes (Aranda et al., 2009).
The master regulatory pluripotency triads such as SOX2, OCT4, and NANOG determine the stemness and differentiation potential of embryonic stem cells (Silva et al., 2008; Han et al., 2010). However, MSCs are also shown to express these factors at primary culture conditions and its expression level declines markedly upon successive or repeated passaging (Greco et al., 2007; Li et al., 2011; Yannarelli et al., 2013). Also, the osteogenic and adipogenic genes were expressed at a considerably increased level due to spontaneous differentiation cultured over a longer period of time (Tsai, 2010). However, spheroid conditions increased levels of pluripotent genes were observed with declined expression levels of the osteoblast and adipocyte-specific genes cultured over for several passages. Deposition of acetylation marks in H3K9 and K14 residues in pluripotent genes were consistently observed in in vitro MSCs aging experiments. However, promoter DNA methylation levels of pluripotent master regulatory genes have no correlation with the expression levels. It is then substantiated with another report showing that promoter DNA methylation level has no role in dictating the transcription levels of Oct4 and Nanog in human Wharton’s jelly and bone marrow MSCs model system. Thus, the histone modifications in the promoter’s region of specific genes are to be expected to play a crucial role in channelizing MSC’s stemness and potency (Tan, 2008; Yu et al., 2011).
Histone lysine demethylase (KDM2A) was shown to regulate MSCs proliferation and osteo-/dentinogenic differentiation. Knock-in/Knock out studies on KDM2A has enhanced the SCAP differentiation potential into the adipogenic and chondrogenic lineage. Also demonstrated knockdown of KDM2A, showed cofactor BCOR has considerably increased expression of Sox2 and Nanog by depositing H3K4Me3 marks in the Sox2 and Nanog loci (Dong et al., 2013). Other findings revealed that KDM2A along BCOR showed an increased deposition of histone marks K4/36 methylation in Epiregulin (EREG) gene promoter, thereby inhibited the osteo-/dentinogenic differentiation potential of human MSCs (Du et al., 2013). RNF40 ubiquitinated Histone H2B (H2Bub1) genes, which further triggered the changeover to an active chromatin signature by resolving H3K4Me3/H3K27Me3 bivalent controlled state on the lineage-specific induction of differentiation markers. Thus, RNF40 mediated ubiquitination has significantly increased during hMSCs differentiation into various lineage-committed precursor cells (Karpiuk et al., 2012).
Comprehensive epigenomic profiling between CD44+ and CD24+ in breast epithelial cells showed the crosstalk between K27 and DNA methylation correlated with higher expression of CD24+ genes independent of the K27 mark. But CD44+ cells showed CD44 high dependence of K27 marks. Thus, suggesting a presumed strategy independent of gene body methylation and gene expression and further correlated with an increase of promoter K27 marks (Maruyama et al., 2011).
WGBS of large partially methylated domains study showed signature similar to DPSCs as compared to 30–40% with ICM. DPSCs showed a similar methylation profile with neuronal stem cell lines and placenta-derived cells, as demonstrated by principal component analysis (Dunaway et al., 2017). It was also identified that the loss of chromatin-modifying enzyme HDAC-1 affects early cardiovascular differentiation in mESCs and iPSCs (Hoxha et al., 2012). During the course of differentiation, iPSCs generated by reprogramming erases somatic epigenetic signatures from silent pluripotent loci and establishes alternative epigenetic marks. Nanog and Esrrb loci are considered as the early essential pluripotent loci preceding the induction of methylcytosine and hydroxyl methylcytosine Parp1 and Tet2. Hence Tet2 and Parp1 are needed for activating chromatin state at pluripotent loci and promotes the opening of oct4 promoter for reprogramming (Doege et al., 2012).
The ChIP-on-chip assay revealed that promoters of RUNX, MSX2, and DLK5, early mineralization genes provided with H3K4Me3 active marks whereas repressive marks H3K9Me3 or H3K27Me3 augmented in OSX, IBSP, and BGLAP gene promoters. It also mediated the suppression of dental family genes (DSPP and DMP1 genes) in dental follicular (DF) cells and not in dental pulp (DP) cells. (Gopinathan et al., 2013). Histone demethylase KDM6B (JMJD3) epigenetically regulated by removing H3K27Me3 marks from promoters of osteogenic commitment. In odontogenic lineage, KDM6B was recruited to BMP2 promoters and facilitating the removal/silencing of odontogenic master transcription gene (Xu et al., 2013). A recent study investigation on odontogenic commitment in dental MSC differentiation showed that the ultimate balance between H3K27Me3 and H3K4Me3 marks mediated by JMJD3 and MLL co-activator complex finally regulate transcription activities of Wnt5A during differentiation (Zhou, 2018).
miRNAs role in stem cell fate
Precise chromatin configuration leads to appropriate gene expression which ensures proper stem cell and progenitor differentiation, lineage commitment. miRNAs are demonstrated to act mainly in RNA silencing and post-transcriptional via base-pairing with complementary sequences within mRNA molecules, which triggers the degradation of mRNA strand (Bartel, 2004). MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in normal biological processes and are commonly dysregulated in human diseases. Cells express different levels of numerous miRNAs that can target at various stages of differentiation or sustaining pluripotency. Given recent studies supported the critical regulatory roles of miRNAs in the stemness and commitment potential of normal and tumor-inducing stem cells. Hence, miRNA signature profiling is very useful for identifying the biomarkers at various developmental stages of specific cell types and is also used for cellular identity. Tab. 1 summarizes various miRNAs and their targeted cells related to their involvement in several regulatory processes of development.
Epigenetics of cancer stem cells
Each mammalian cell differs from the other in the differentiated state but still retains a similar genome, which was inherited from the common precursor ESC. These cells have the potential to de-differentiate and acquire their totipotent character in a specific milieu. However, this process is determined by the expurgation of diverse epigenetic states in the chromatin through various covalent modifications in DNA and histone leads to change of fate by reprogramming. The initiation of CSCs also involves the parallel route during cancer triggering might be hypothesized based on epigenetic reprogramming in which downregulation of differentiation-specific genes and upregulation of stemness property, thereby eventually escapes the natural cell death process. The major cellular event that drives the carcinogenesis is reprogramming of the epigenome initiated by a series of cellular signaling cascades, finally culminating in gaining and maintenance of stem cell properties (Shukla and Meeran, 2014).
The CSCs are a subpopulation of cells present in the tumor niche, which undergo changes in Methylome and chromatin signature that finally transform into CSCs. During the initial phase of cancer initiation, the epigenetic modifiers might facilitate opening up target oncogenic DNA sites by requisite over-expression of oncogenic factors.
Through epigenetic analysis, various druggable targets are identified and targeted. Some of which are currently in clinical trials. In Fig. 2, the role of epigenetic modifiers in regulating cellular function under the imbalanced state is reversed that finally leads to the transformation of the normal somatic stem cells or progenitor cells into a highly aggressive cancer stem cell.
The expression of repressed tumor-promoting factors and silencing tumor-suppressing genes were correlated with the downregulation of DNMT enzyme (Meeran et al., 2011; Meeran et al., 2012; Wang, 2013). Hypermethylation of tumor suppressor genes like DKK1, ASCL2, APCDD1, AXIN2, and LGR5A has correlated with poor tumor prognosis in colorectal cancer (CRC) and its elevated levels showed good prognosis in CSCs showed effective treatment in CRC patients (De Sousa et al., 2011). In acute myeloid leukemia (AML), hyper-methylation of tumor suppressor genes is correlated with the prognosis of tumor progression (Deneberg et al., 2011). A previous report demonstrated the occurrence of higher hypomethylation in breast CSCs than in non-CSC populations at differentially methylated regions (DMRs) and correlated with cancer prognosis (El Helou et al., 2014). More hypomethylation in DMR in breast CSCs showed poor prognosis when compared to the non-CSCs population. Knockdown of DNMT showed reduced stem cell properties, which again confirmed that epigenetic imbalances drive carcinogenesis and DNMT1, could be a candidate target for treating breast cancers.
Wnt pathway was epigenetically regulated by Brahma-related gene 1 (BRG1), which is the main tumor-initiating factor in triggering intestinal cancer, and its downregulation prevents adenoma development and decreased TIC population. Also, BRG is involved in leukemia maintenance, as BRG-AML cells are more sensitive to treatments than the BRG+ cells (Holik et al., 2014). Prevalent mutations in BRG1 are observed in the 30–40% non-small cell lung cancer, and thus highlighted BRG1as a significant key regulator in lung tumor development (Medina et al., 2008; Wu, 2012). BRG1 down-regulated Oct4 and Sox2 gene targets and promote self-renewable potential in B-cell lymphomas. Also, the high incidence of Mo-MLV insertion in BMI-1and EZH2 regions of the PcG family have also been linked with poor prediction in many different cancers (Ho et al., 2009; Kidder et al., 2009).
BMI-1 maintains the stemness of CSCs and also intricate in triggering various types of cancers. Overexpression of BMI-1 induced stemness property in CSCs which in turn augmented tumor initiation (Molofsky et al., 2003; Siddique and Saleem, 2012; Proctor et al., 2013). Mutation in chromatin remodeling complex (nearly 20 %) mainly SWI/SNF plays a role in triggering carcinogenesis (Lee and Roberts, 2013). Target abolition of the PRC-1 and PRC-2 complexes components were revealed to have dissimilar effects on the in vitro re-programming competence in the pluripotent cells and somatic cells. Knockdown of H3K27 methyltransferase leads to reduced re-programming competence in pluripotent and somatic cells. Also, downregulation of SUV39H1, DOT1L, and transcription factor YY1 was established to trigger pluripotency (Onder et al., 2012).
Efficient silencing of appropriate chromatin remodeling complexes in differentiated cell types induces pluripotency. Any modifications in the transcriptome profiling of chromatin remodelers are proficient in initiating tumor genesis. Hence, regulating chromatin complexes modifies the capability to induce CSCs phenotype. Ectopic expression of these complexes could cause repression of tumor suppressors or expression of oncogenic promoter genes. EZH2 expression was found higher in side-populations as observed in breast and pancreatic cancer lines than in non-CSC populations. Also, a recent report showed that knockout of EZH2 resulted in decreased CSCs incidence, which supplementary endorses EZH2 as a useful CSC marker and targeting protein for therapeutic purposes. Regulatory genes are often silent in ES cells (Bernstein, 2006). Early developmental studies in fruit flies show that the bithorax locus is co-occupied by Polycomb repressive group proteins (PcG) and trithorax G proteins (trxG), where the trxG protein is essential for gene induction. Chip assays on ES cells revealed that H3K4 methylation within bivalent domains and trxG protein may help the methylated H3K4 (active state) regions in murine ESC cell line. The presence of bothactive and repressed states co-exist at the same locus on the same chromosome.
A chip on-chip studies showed the overlapping of more H3K27 trimethylated (repressed state) regions with few such specific modifications where both active and inactive marks co-exist in the particular gene promoter region is called “Chromatin Bivalency,” which is considered as a unique feature frequently found in the domains of developmental regulatory genes poised for induction. In human primary T cells, both H3K27 and H3K4 methylation co-exhibit in the HOXB7 promoter region, which is as compared analogy to the proposed role of bivalent chromatin in ES cells (Roh et al., 2006). Thus, a similar mechanism is also observed for priming the dynamic gene expression in naive T cells upon antigen triggering.
The well-known PcG group of proteins has two main histone modifiers, PRC1 and PRC2, respectively. PRC1 complexes (BMI1, RING1A, RING1B, PHC) are capable of catalyzing mono-ubiquitination of lysine residues on Histone2A proteins. Any aberrations in PRC1, specifically RING1B, exhibited embryonic lethality in in vivo mouse embryo studies; however, other group members of PRC1 knock-out studies showed a severe developmental defect rather than embryonic lethality. Another familiar member of PcG proteins is PRC2 (SUZ12, EED, EZH2), which is known to catalyze di- or tri-methylation in H3K27 residues. Like PRC1, PRC2 knock out experiments also showed embryonic lethality. Thus, suggesting that PcG complex proteins are very crucial for normal embryo development (Arisan et al., 2005; Chase and Cross, 2011; Yoo and Hennighausen, 2012; Zingg, 2015).
High mobility nuclear proteins regulate the expression of many developmental genes via the formation of chromatin remodeling complexes on the distant promoter regulatory landscapes. Research on knock-in and knock-out model of HMGA2 showed its role in modulating cardiomyogenesis via gain of function induces cardiomyogenesis, and its siRNA mediated knockdown hindered the differentiation of embryonal carcinoma cell line PC19CC6 to cardiomyocyte lineage. Another study also demonstrated HMGA2 along with Smad transcription factor in response with bone morphogenetic protein (BMP) coordinately upregulated NKX 2.5 promoter activity. In Xenopus laevis embryo, morpholino or dominant-negative HMGA2 experiments have been shown to hinder normal heart formation. Thus, HMGA2 can act as an optimistic controller of the Nkx2.5 gene, and its expression is indispensable for in vivo heart development (Monzen et al., 2008).
Active DNA demethylation is the prerequisite obligation for cells to recover self-renewal property and reverts to their pluripotent state. This can be achieved progressively by modification of 5-methyl cytosine to thymine or 5-hydroxy methylcytosine by Ten-Eleven Translocation proteins (TET) and activation-induced deamination (AID), respectively (Klungland and Robertson, 2016). miR combo (miR-1, 133, 208, and 499) were shown to reprogram cardiac fibroblasts into cardiomyocytes directly. It was found that histone methyltransferase and demethylase regulating H3K27 trimethylation were shown to be modified in miR combo treated fibroblasts. Similarly, cardiac TFs showed a decreased H3K27Me3 mark in chip-qRT PCR reaction. Also, knockdown of H3K27 demethylase KDM6A and KDM6B restored the level of H3K27Me3 and inhibited cardiac gene expression in miR combo treated fibroblast (Dal-Pra et al., 2017).
EZH2 in stem cell fate determination
The characteristic property of pluripotency in ESCs is highly dependent on the PcG proteins, where it tends to maintain the balance between repressing markers and pluripotent specific markers by repressing the early differentiation marker genes and maintains the pluripotency genes. Initial days of fate commitments, deposition of PCR2 mediated histone H3K27Me3 makes these cells trigger the early differentiation marker genes, but still, PcG proteins suppress the late differentiation genes for a specific lineage. Consistent high expression of EZH2 in ESCs and early mouse development, which determines the pluripotent state upon declining its level differentiation is triggered. Also, EZH2 is abundantly expressed in progenitor cells of the epidermis region; nevertheless, its level declines upon commitment. Additionally, EZH2 was shown to maintain the multipotency in Mesoderm derived stem cells like myeloid and lymphoid progenitors, muscle progenitors, and neural progenitors.
EZH2 cover-expressed in the HSCs preserves the long-term self-renewing potential, which prevents HSCs depletion after serial transformation. Increased EZH2 expression blocked muscle differentiation from myoblasts due to histone lysine methyltransferase (HKMT) activity in its SET domain. NSCs also expressed high EZH2 level, and further commitment to astrocytes its level declined. Reduced differentiation potential into astrocytes in NSCs on ectopic expression of EZH2 further substantiated the role of EZH2 in preserving pluripotent or multipotent property of stem cells (Birve, 2001; Czermin et al., 2002; Chambers et al., 2003; Erhardt, 2003; Caretti et al., 2004; Cao and Zhang, 2004; Boiani and Scholer, 2005; Boyer et al., 2005; Pasini et al., 2007; Aloia et al., 2013). Transcriptional reprogramming of bone marrow MSCs to hepatocytes mainly depends on the deposition of activation marks (H3K4me3, H3K9Ac) and depletion of repressive marks (H3K9Me3, H3K27Me3) at the promoter binding site of hepatic transcription factors. However, the repressive H3K27 methylation was belligerently regulated by EZH2 and JMJD3, and the promoter activation of epigenetically poised hepatic genes was preceded by restricted nuclear reprogramming (Kochat et al., 2017).
It is well demonstrated that complex coordinated networking between epigenetic mediators and chromatin landscapes facilitates the expression of Glial gene expression and favors glial fate determination in Neuronal Stem Cells (NSCs). Whole-genome bisulfite sequencing analysis data demonstrated that distinct epigenetic signatures leading to three different neuronal sub-populations in NSCs, such as self-renewing neurons stem cells, progenitors consistently expressing neuronal markers actively, and cells switched from neurogenic to gliogenic phase, respectively (Nakagawa et al., 2020). Nuclear factor-1 binding motif nfia expression is highly correlated with the active neurogenesis and facilitates the demethylation of genes specific for astrocyte generation. Similarly, miR153 guides the astrogenesis via targeting the expression of nfia and nfib (Tsuyama et al., 2015).
Small molecule inhibitor-based fate determination
Molecular events associated with lineage decisions are the contemporary area of research in normal stem cells for its better manipulation in clinical use. A large number of evidences is available to depict chemical-based approaches, as a versatile tool in controlling the stem cell properties and their fate, such as stemness, lineage differentiation, reprogramming and regeneration. Our previous study about methylation profiling of cardiac-specific gene (CSG) promoter in human Wharton’s jelly derived MSCs at single-nucleotide resolution mapping (GATA4, SERCA, NKX 2.5, TBX5, MYH6, and MYL7) suggested that no DNA methylation level hindrances were found in native MSCs which underscored that the functional restriction to become competent cardiomyocyte is not due to DNA methylation. Hypo-methylation in CSGs suggested that WJ-MSCs exhibit a permissive methylome for cardiomyocyte lineage differentiation (Govarthanan et al., 2020). Further fine-tuning of differentiation protocol with other small molecule inhibitors like Histone deacetylase inhibitors, voltage channel agonists may yield the fully matured cardiomyocyte differentiation in WJ-MSCs.
CHIR 99021 is one such molecule that is an agonist of the Wnt pathway, widely used to sustain pluripotency in ESCs, induce reprogramming in somatic cells along with few Yamanaka factors, and lineage differentiation in MSCs (Ring et al., 2003; Ying et al., 2008; Li et al., 2015; Cao et al., 2016; Narcisi et al., 2016). An interesting study using WJ-MSCs pretreated with CHIR 99021 showed an increased state of potency, exhibiting enhanced differentiation capabilities with de-methylated OCT 4 promoter region. Thus, suggesting MSCs treated with CHIR 99021 can be potent, alternative sources of stem cells that are well suited to cell-based regenerative therapy (Govarthanan et al., 2020). These crucial evidences have laid down a strong foundation to employ the use of small-molecule inhibitors successfully in the process of fine-tuning the dedifferentiation towards the development of new biological therapies.
Sharma and Bhonde (2020) suggested that the age of stem cells could have a direct impact on cell-based therapy. Effectively, the more the passage number which concomitantly decreases the proliferative state of the cells and finally leads to senescence. This is mainly due to the varying degree of hypermethylation pattern and it is known to cause a direct effect on cell cycle control, DNA replication and repair, differentiation potential, etc. (Sharma and Bhonde, 2020). Intriguingly, several bioactive molecules collectively named “epi-drug” are presently employed in various clinical trials for devising potential cancer management treatments. Similarly, previous drugs shown good efficacy in reversing the aberrant disease-associated epigenetic status such as SWI/SNF, Polycomb, MLL-fusion proteins, jumonji-C domain encompassing histone demethylase, ten eleven translocation are actively recommended for drug repurposing and this strategy mostly improves the transition of epi-drugs towards clinical applications (Chiacchiera et al., 2020).
Current trends in epigenetic research areas
From the above-cited references and reports, it is well apparent that epigenetics is either directly or indirectly involved in the lineage commitment, identity, differentiation potential of the cell. On the other hand, it is playing a predominant role in cancer initiation, tumor progression and dissemination. We found much of the work has been extensively done from 2010 to 2016, and this accelerated us to understand the contribution of the epigenetic regulatory network in the above-mentioned areas of research. Due to this, many avenues of cancer research and basic fundamental research in stem cell biology are started employing epigenetic modifiers in disease management and in vitro differentiation cues respectively. A recent study by Pan et al., 2020 showed lineage-specific gastrointestinal adenocarcinoma-specific master regulatory transcription factor HNF4A promoted cancer proliferation and survival in cancer cells. Here, the group has employed a novel computational algorithm called enhancer linking by methylation/expression relationships (ELMER) to map the key transcription factor associated with the tumor initiation progression, etc. Thus, the group has demonstrated HNF4A could be a common potential target for gastrointestinal groups of carcinomas.
A study by Cakouros et al. (2019) speculated that hydroxyl methylation and enzyme regulating hydroxyl methylation such as DNA hydroxylase TET family of enzymes played a crucial role in bone repair and remodeling via regulating the osteogenesis process. This group showed TET1 enzyme inhibited osteogenesis and adipogenesis via indirect recruitment of epigenetic modifiers like SIN3A and EZH2, while TET2 directly promoted osteogenesis. Additionally, the relevance of TET1 and TET2 enzymes were found to be present in downregulated levels in osteoporosis, therefore targeting TET1 seems to be an ideal target for the new therapeutic strategies to prevent bone loss.
Interestingly screening of small molecules having the potential to rescue MSCs senescence-related concerns under in vitro conditions and boosting its plasticity are currently identified as novel methods to reverse the MSCs aging in aged patients and employed for regenerative therapies further. Here, small molecules such as Gemcitabine and Chidamide hugely 5.9- and 2.3- fold increased osteogenic differentiation potential of aged donors of hMSCs. It also increased the differentiation potential via 2.4- and 2.6- fold in late passaged osteogenic differentiation induced MSCs, respectively (Dhaliwal et al., 2018).
Limitations of epigenetic study
The generation of iPSCs has revelutionized the avenues of stem cell biology, still the concept of reprogramming is not fully understood. Hitherto, the two well-known proposed models of reprogramming such as the elite and stochastic were widely accepted. However, it is still under debate about the mechanism of reprogramming. The elite model proposes that not all the cells were conducive for reprogramming, this often correlated with the reprogramming efficiencies of the source cells employed for reprogramming studies. Whereas the stochastic model proposes that every cell inherently has the potential to undergo the process of reprogramming and become iPSCs (Yamanaka, 2009; Wakao et al., 2011). In any case, the efficiency of cellular reprogramming is highly dependent on its epigenetic state. Therefore, the current advanced reprogramming methods have started to incorporate the small molecule inhibitors targeting epigenetic modifiers, thus greatly influencing its reprogramming efficiencies. However, the probability and the actual phenomenon occurring in the genome and epigenome of the cell undergoing the reprogramming is still unknown. In addition, the status of histone modifications was also found to be associated with the transformation of cells. Therefore, forthcoming studies on the cells having conducive epigenome for reprogramming may give us a clear picture of the factors that hinder the other subset of the population from not responding to the transformation protocols.
Epigenetics plays a significant regulatory role in determining stem cell linage and cellular differentiation. Its predominant function has also been recognized in recruiting the appropriate transcriptional machinery during embryonic development and adult tissue homeostasis. Specialized chromatin structure dictates the unique expression profiles of stem cells intact and regulates its differentiation into various downstream lineages. Numerous epigenetic modifications occur concomitantly during the differentiation of MSCs to respective cell types. However, the knowledge about the epigenetic regulatory mechanism in relation to differentiation towards specific cell lineage is limited. Further investigations of the epigenetic profiling may help us in better understanding the systematic derivation of the physiologically competent cell types for exploitation in the field of various other regenerative therapy pursuits.
The MSCs have been used in preclinical models for various bone and cartilage tissue engineering. The development of tissue-engineered products has given considerable promising use for rebuilding damaged or diseased tissues. Epigenetic regulatory mechanisms are likely to enhance scientific hold on transcriptional regulation, especially critical for stem cells, their potential for self-renewal and differentiation. Classification based on gene expression profiles could help us in segregating stem cells into pluripotent stem cells, multipotent stem cells, and multipotent adult stem cells. Analysis of histone modifications mediated by PcG proteins and promoter histone methylation of a gene have demonstrated that certain marks on the histone bodies are necessary for the self-renewing stem cell populations, and its subsequent loss could deliberately lead to differentiation of a specific lineage. DNA methylation may often correlate with the restricted differentiation potential towards the specific lineage.
Research on the chromatin signature and cellular behavior would be more useful in fishing out the long-term self-renewing potential cells for transplantation and regenerative therapy. Overall, a combination of DNA methylation at gene promoter region and histone core modification marks in gene promoter and gene body contributes to the epigenetic regulations in stem cell state and determines the degree of differentiation impending from pluripotent stem cells to multipotent stem cells and progenitors. Similarly, the epigenetic road map will give us a clear picture of normal and cancerous chromatin organization or architectural difference, which will contribute to identifying new potential druggable targets for cancer treatment regime in the future.
Acknowledgement: KG is thankful to UGC, PKG and BZE to MHRD for their fellowships. All authors are grateful to IIT-Madras, Kumaun University, Mahatma Gandhi Central University, and Department of Life Sciences, SBSR, Sharda University for infrastructure and facility.
Author’s Contributions: KG wrote the manuscript, and responsible for the overall direction of the review, PKG contributed in manuscript writing, editing and graphical designing of figures, BZE, RG & SP contributed in review compilation and manuscript editing, RP contributed for overall final editing and proof read of the manuscript.
Ethics Statement: This article does not contain any studies with animals performed by any of the authors.
Funding Statement: The author(s) received no specific funding for this study.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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