Overview of Stem Cell Epigenetics
Stem cell is considered to have two distinctive characteristics: self-renewal and pluripotency or multipotency. Its homeostasis and differentiation are partly maintained by epigenetic mechanisms that are highly dynamic in regulating the chromatin structure and specific gene transcription. Epigenetics means changes in gene expression which are heritable by the modifications that don’t affect DNA sequence. The stem cell epigenetic landscape is indispensable for setting up different degrees as well as conveying specialized gene expression patterns. So it defines the molecular basis of pluripotency, reprogramming and early human development. There are several epigenetic mechanisms included: post-translational modifications of histones (histone PTMs) and incorporation of histone variants, changes in DNA methylation, ATP-dependent chromatin remodeling and the implementation of RNAi pathways and non-protein coding RNAs. These different pathways orchestrate and collaborate together to establish the unique epigenetic states and to drive the final outcome of the transcriptional hierarchy mediated by transcriptional factors. Any perturbation of these epigenetic components may lead to changes to local chromatin configuration and nuclear architecture within the stem cell which will collapse the self-renewal circuitry and trigger the loss of stemness by promoting differentiation. It is believed that further investigation of stem cell epigenetics will promise to provide more innovational ideas in the diagnosis and treatment of a wide array of human diseases.
Figure 1. Epigenetic response to extrinsic signals occurs through the transcriptional factors network.
DNA methylation, as a classic example of epigenetic inheritance of gene expression, is not surprisingly playing a key role in stem cell function. DNA methyltransferase (DNMT) family directs and preserves the DNA methylation patterns, and the effects of DNA methylation are regulated by the recruitment of the “reader” methyl-CpG-binding domain (MBD) family, including proteins such as MBD2, MBD3, MBD4, MeCP2 and KAISO, which block the binding of transcriptional factors to their cognate response elements. After the genome-wide demethylation during embryonic development, genes which are essential for stem cell renewal are activated, indicating that previously existing DNA methylation must be erased, especially at the promoters of genes which are indispensable for pluripotency, such as NANOG and OCT3/4. Some reported that DNA methylation at genes which are essential for stem cell renewal are related to coding sequences, instead of gene promoters.
Ten-11 translocation family proteins, TET1-3, is able to convert 5-mC to 5-hydroxymethyl-cytosine (5-hmC). It is reported that 5-Hydroxymethylcytosine levels are high in mESCs and hESCs. The modification from mC to the hmC suggests that a hydroxylated methyl group could be an intermediate for oxidative demethylation or a stable modification. Furthermore, 5hmC can be oxidized by all three TETs to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be excised by thymine DNA glycosylase (TDG), triggering subsequent base excision repair and indicating a potential role for active demethylation. So 5hmC may function in the pluripotency establishment and differentiation. In addition, it is involved in the differentiation process. Loss of all three TET enzymes limit the normal differentiation of ESCs, and Tet null ESCs have almost no contribution to developing embryos and cannot support development.
Histone acetyltransferases (HATs/KAT) catalyze the acetylation of histones H3 and H4, and the removal of acetyl-group is achieved by the histone deacetylases (HDACs). It is known that HATs and HDACs are integral components of transcriptional co-activator and co-repressor complexes respectively. Otherwise, they also play a role in the ES cell differentiation and adult stem cell function. For example, the differentiation of ES cell is prevented by the inhibition of HDACs, indicating that histone deacetylation is part of cell-type specification. Likewise, differentiation of ES cell also requires the histone acetyltransferase KAT3B (p300). Despite the role in the modification of histones, HDACs and HATs also function in the dynamic acetylation/deacetylation of key regulatory ES cell differentiation modulators, such as members of the SOX family, TGF-β family, WNT and NOTCH.
In order to maintain the stable self-renewal of ESCs, the mechanisms that inhibit their differentiation and promote their proliferation must be transmitted to their daughter cells, which indicate the significance of the “epigenetic memory” mechanisms in this process. Stem cells maintain their identity through this “epigenetic memory”, even when exposed to extracellular environments that induce other cell fate. Historically, it is explained that the methylation of promoter DNA function in this cellular inheritance. However, with more research, it is said that a lot of other mechanisms are also involved in it including of the histone methylation and demethylation.
In order to maintain pluripotency, human and mouse ES cells have mechanisms for dynamic repression of genes regulating developmental pathways in such a way that this repression can be epigenetically maintained through cell division. The PcG complex, as an epigenetic modifier, can perform this function. The PcG complex is an evolutionary conserved family of chromatin regulators which is best known for their function in establishing and maintaining the silent state of homeotic gene expression during embryonic development. Mammalian PcG proteins assemble at least three biochemically distinct complexes PRC1, PhoRC and PRC2. PRC2 (polycomb repressive complex 2) plays a role in stabilizing repressive chromatin structure through the function of chromatin modifiers, such as enhancer of zeste (EZH2), embryonic ectoderm development protein (EED), and suppressor of zeste 12 (SUZ12), all of which are histone methyltransferase responsible for depositing H3K27me2 and H3K27me3 marks onto chromatin. As for PRC1, nucleosomes containing H3K27me3 can recruit the PRC1 complex containing PHC, CBX, BMI1 and RING1A, RING1B (RNF2) and MEL-18 (PCGF2) via the affinity of the chromodomain containing proteins to these PTMs. PRC1 functions in the establishing of high-order chromatin structures. It is reported that the E3-ligase activity of the RING1A and RING1B proteins present in the PRC1 complex can mono-ubiquitinate H2AK199. This E3-ligase activity is likely to be stimulated by the PRC1 subunit: BMI1 and MEL-18 (PCGF2).
Trithorax group (TrxG) protein complexes mediate the transcriptional activity of self-renewal genes, such as OCT3/4, SOX2 and KLF. In contrast to PcG complexes, TrxG complexes mediate the deposition of histone PTMs which is the mark of active transcription, such as H3K4me3.
Histone demethylases are integrated in the transcriptional factor regulatory networks in ES cells. For example, JARID2, also known as Jumonji (JMJ), the target of seven core regulators in ES cells, is high expressed in ES cells. However, it is downregulated in the whole embryonic body at the onset of differentiation. In addition, histone methylases and demethylases can be interconnected with DNA methylation, which provides for the balance of histone PTMs in stem cells.
One of the most well studied complexes of ATP-dependent chromatin remodelers is the SWI/SNF complex (mSWI/SNF in mammals, also known as BAF). The 9-12 subunits of mSWI/SNF are gene families and are assembled together with one of the two mutually exclusive catalytic ATPase subunits, brahma homolog (BRM, also known as SMARCA2) or BRM/SWI2-related gene 1 (BRG1,also known as SMARCA4). Variations in the mSWI/SNF subunit composition function in the targeting, assembly and regulation of lineage-specific pathways during ES cell differentiation. It acts as a transcriptional repressor on a number of differentiation specific genes in ES cells.
CDH1 (chromodomain helicase DNA binding protein 1) functions as substrate recognition component of the transcription regulatory histone acetylation (HAT) complex SAGA. It is reported that CHD1 can be used as a molecular adaptor, recruiting SNF2, the FACT complex and the PAF complex to H3K4me2/3, which is required to maintain the open chromatin in ES cells, thus providing for pluripotency.
miRNAs, a class of small, ~21nt, non-coding RNAs, play a crucial role in post-transcriptional gene expression by the regulation of mRNA stability and specific protein abundance. Up to now, miRNA’s function in ES cells can be concluded into three mechanisms: 1) participation in the maintenance of stem cell self-renewal and pluripotency through the inhibition of negative factors controlling these events; 2) initiation of stem cell differentiation through the inhibition of master pluripotency factors; 3) maintenance of lineage definition by restricting the expression of genes from other lineages.
It is reported that Polycomb group complex PRC2 use non-coding RNA co-factors as sequence specific guides to direct Polycomb group complexes to their cognate binding sites within the genome. So long ncRNAs can represent a “flexible scaffold”, mediating interactions between DNA and protein complexes.
Several evidences suggest that master pluripotency regulators such as OCT3/4 and NANOG might be involved in the regulation of transcriptional activity of ES cell-specific non-coding RNAs,
Relations with Diseases
Different kinds of stem cells will contribute to different diseases. And any disorder of the epigenetic regulation of stem cell may cause a lethal result. So it is important to elucidate the relationship between stem cell epigenetics and diseases. Therefore, more strategies can be used to treat certain diseases. Here are some examples of the relationship between stem cell epigenetics and diseases. Differentiated neural stem cells can be used as a potential treatment for AD (Alzheimer’s Disease). Because these NSCs undergo epigenetic changes which control both the intrinsic and extrinsic signals before they get specialized, which interact and depend on each other to push the NSCs into mature neurons and glial cells. Recently, it is reported that the Zuotin-related factor 1, ZRF1, has been identified as an epigenetic regulator of gene transcription in stem cell and cancer. On the one hand, ZRF1 plays an important role in oncogene-induced senescence (OIS) by activating the INK4/ARF locus, thus working as a tumor suppressor; on the other hand, it can promote leukemogenesis in acute myeloid leukemia (AML) by a polycomb-independent fashion.