Histone modification in MSC differentiation and aging.
Mesenchymal stem cells (MSCs) are progenitors of connective tissues, which have emerged as important tools for tissue engineering owing to their differentiation potential in various cell types. The therapeutic utility of MSCs hinges upon our understanding of the molecular mechanisms involved in cellular fate decisions. Thus, the elucidation of the regulation of MSC differentiation has attracted increasing attention in recent years. A variety of external cues contribute to the process of MSC differentiation, including chemical, physical, and biological factors. Among the multiple factors that are known to affect cell fate decisions, the epigenetic regulation of MSC differentiation has become a research hotspot. In this chapter, we summarize recent progress in the determination of the effects of epigenetic modification on the multilineage differentiation of MSCs.
- mesenchymal stromal cells
- post-translational modifications
- cellular fate decision
- epigenetic regulation
Mesenchymal stromal cells (MSCs) are progenitors of connective tissues, initially characterized as plastic adherent, fibroblastic cells, with the potential to differentiate into many types of cells, including predominantly osteoblasts (cells that secrete the matrix of the bones), chondrocytes (cells embedded in the lacunae of the cartilage matrix), and adipocytes (fat-storing cells), under appropriate conditions. MSC studies have progressed rapidly since the initial report of human MSC isolation from bone marrow. MSCs have been shown to reside within the connective tissues of most organs. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. Further, the fate decision of MSCs has also piqued the interest of scientists. During the last two decades, various signaling molecules important to MSC differentiation have been identified, and the epigenetic regulation of MSC differentiation has recently become a research hotspot.
The transformation process of MSCs from a self-renewing state to a specific lineage is always accompanied by changes in cell morphology and function, which are largely determined by the differential expression of genes. Specifically, genes related to self-renewal are turned off, and transcription of cell type-specific genes is activated. Epigenetic regulation refers to the phenotypic change through gene differential expression without DNA sequence alteration, including four main categories:  DNA methylation,  histone modifications, and  chromatin remodeling (nucleosome positioning); and  non-coding RNAs. It has been widely reported that epigenetic and post-translational modifications have a broad and far-reaching influence on MSC differentiation at multiple levels. Here, we provide an overview of the recent findings regarding the roles of epigenetic modification in the fate decision of MSCs.
2. DNA methylation
DNA methylation is an important epigenetic modification referring to the addition of a methyl (-CH3) group to the fifth carbon atom of a cytosine ring to form 5-methylcytosine (5-mC). The process is catalyzed by enzymes known as DNA methyltransferases (DNMTs). DNA methylation was the first epigenetic mark to be discovered, and it plays an important role in normal human growth, development, aging, tumorigenesis, and other genetic and epigenetic diseases. This epigenetic mark has the ability to turn genes on or off and can be inherited through cell division. Recent studies have suggested that methylation and demethylation of specific genes, such as
2.1 Osteogenic differentiation of MSCs and DNA methylation
According to numerous studies, DNA methylation is dynamically involved in the osteogenesis of MSCs. Generally, it may be considered that DNA methylation has a repressor role in the promoter regions with CpG islands, blocking gene expression. During osteogenic differentiation, demethylation was observed at specific CpG regions in the promoters of osteogenic lineage-specific genes, such as
2.2 Adipogenic differentiation of MSCs and DNA methylation
Adipogenesis is highly regulated by a sequential cascade of transcriptional events. Key transcriptional factors controlling adipogenesis include several CCAAT/enhancer-binding protein (C/EBP) family members, including C/EBPα, β, and δ, and the nuclear receptor peroxisome proliferator γ (PPARγ). On the other hand, a number of negative transcriptional factors have also been identified, including GATA2/3, chicken ovalbumin upstream promoter transcription factor (COUP-TF), interferon regulatory factors (IRFs), and Wnt family proteins.
2.3 Chondrogenic differentiation of MSCs and DNA methylation
DNA methylation and demethylation status also influence MSC chondrogenic differentiation. DNA methylation at specific CpGs has been shown to influence genes such as
3. Histone epigenetic modification
Histone modification, a common form of epigenetic regulation, refers to post-translational modifications that are added to the N-terminal tail of histones. Histone modification has been shown to play important roles in regulating cell-specific gene expression. So far, more than sixty different residues on core histones (H2A, H2B, H3, H4) with potential to be modified have been reported. These modifications made to histones, including acylation, methylation, phosphorylation, ubiquitination, and sumoylation, can impact gene expression by altering the chromatin structure or recruiting histone modifiers. Histone proteins function to package DNA, which wraps around the eight histones, into chromosomes. In general, it has been well established that histones on the promoter regions of master transcription factors associated with MSC cell fate commitment, such as
|Involved epigenetic histone modification||Factor||Mechanism||Result||Reference|
|Histone deacetylation||HDAC inhibitor||Stimulate the transcription of p21CIP1/WAF1 through enhancing the H3 and H4 acetylation||Arrest the cell cycle at the G2/M check point, inhibit adipogenic, chondrogenic, and neurogenic differentiation; promote osteogenesis|||
|Histone acetylation||Knockdown of PCAF (histone H3K9 acetyltransferase)||Insufficient to increase H3K9 acetylation at promoters of BMP2, BMP4, BMPR2B, and Runx2||INHIBIT adipogenic differentiation and promote osteogenic differentiation in MSCs; reduce the bone formation both in vitro and in vivo|||
|Histone acetylation||GCN5 knockdown||Insufficient to inhibit NF-κB signaling by mediating the proteasomal degradation of p65 (acetyl K310)||Inhibits osteogenic differentiation of MSCs|||
|Histone deacetylation||SIRT1 knockout||Insufficient to deacetylate β-catenin to promote its accumulation in the nucleus||Reduce differentiation towards osteoblasts, and chondrocytes|||
|Histone demethylation||Overexpression of KDM5A||Decrease H3K4me3 levels on promoters of Runx2 by demethylating H3K4me3||Inhibit osteogeninsis; lead to osteoporosis|||
|Histone methylation||G9a inhibitor||Unclear (correlate with PPARγ and C/EBPα expression)||Impair the proliferation but the anti-proliferative effect is not sustained; increase adipogenic potential and decrease osteogenic potential of MSCs|||
|Histone methylation||Downregulation of BMI1||Insufficient to recruit and stabilize PRC2 which trimethylate H3K27||Cellular senescence|||
|Histone methylation||Downregulation of EZH2||Insufficient to trimethylate H3K27 as catalytic subunit of PRC2 and keep a high extent of H3K27me3 to suppress p16INK4A-induced senescence||Cellular senescence|||
3.1 Histone acetylation modification
Histone acetylation is an epigenetic modification characterized by the addition of an acetyl group (COCH3) to histone proteins, specifically to lysine residues within the N-terminal tail. Histone acetylation is one of the most common epigenetic modifications, which leads to the neutralization of the positive charge on the histone proteins, weakening their interaction with DNA, and finally promoting the opening of chromatin structure and activating gene transcription. On the other hand, histone deacetylation is related to chromatin transcription inhibition. The level of histone acetylation is mainly regulated by histone acetylase (HAT) and histone deacetylase (HDAC).
The degree of histone acetylation of related regulatory genes can reflect the maintenance of stemness and the differentiation status of MSCs. During the process of osteogenic differentiation, the expression of osteogenic genes (such as
3.2 Histone methylation
Histone methylation is another common post-translational modification by which methyl groups are transferred to the amino acids of histone proteins that make up the nucleosomes. Histone methylation can occur at various sites in histone proteins, primarily on lysine and arginine residues, and it can be governed by multiple positive and negative regulators, even at a single site, to either activate or repress transcription. Histone methylation is regulated by histone methyltransferase (HMT) and histone demethylase (HDM), which can be monomethylated, dimethylated, or trimethylated.
The increase in methylation usually promotes the affinity of histones to DNA and increases the degree of transcriptional inhibition, such as H3K9 methylation and H3K27 methylation. H3K9 dimethylation and trimethylation are typical repressive histone modifications that mediate the formation of heterochromatic regions. It was reported that the knockdown of ESET, a H3K9 methyltransferase, causes an aberrant expression of Runx2 and finally leads to the impairment of osteogenic differentiation and bone defects in mice. On the other hand, the knockdown of EHMT1, a H3K9 specific methyltransferase, resulted in decreased H3K9me2 levels on the promoters of Runx2, thereby upregulating transcription in mouse tissues. With respect to the adipogenic differentiation of MSCs, it was found that the enrichment of H3K9me1 and H3K9me2 on the promoters of C/EBP and PPARγ was negatively associated with adipogenic differentiation. Lowering the H3K9 methylation levels in these regions by either H3K9 demethylase or HMT inhibitors ultimately promoted adipogenic differentiation. In addition, H3K9me3 levels in the promoter region of Sox9, as well as its target genes
4. Chromatin remodeling
Chromatin remodeling is the dynamic modification of chromatin architecture, which is an important mechanism for regulating gene expression. In eukaryotes, DNA is tightly wound into a complex called chromatin. Chromatin remodeling allows the access of tightly condensed DNA to various regulatory factors, such as transcription factors and components of DNA replication, so that specific genes can be expressed. The basic mechanism of chromatin remodeling depends on the three dynamic properties of nucleosomes: reconstruction, enzyme-induced covalent modification, and repositioning. In addition, the aforementioned histone modification is another important aspect of chromatin remodeling. Aberrations in chromatin remodeling proteins are associated with various human disorders and diseases. The major activities involved in nucleosome structure alterations use the energy supplied by ATP hydrolysis to affect nucleosomes. These enzymes are called ATP-dependent chromatin (or nucleosome) remodeling factors. The system involves four subfamilies of ATP-dependent chromatin remodeling complexes, namely switch/sucrose non-fermentable (SWI/SNF), nucleosome remodeler deacetylase (NuRD), INO80, and imitation switch (ISWI).
Several studies have demonstrated that functional SWI/SNF machinery plays an important role in regulating MSC tri-lineage differentiation by interacting with tissue-specific transcription factors and crosstalk with cell signaling pathways. Brahma-associated factor (BAF) complex subunits have been implicated in MSC osteo-lineage commitment. For example, depletion of BRG1 leads to constitutive osteo-lineage gene expression . BRM negatively regulates osteocalcin expression . Loss of the classical BAF restricted subunit Pbrm1/Arid2/Brd7 leads to reduced osteogenesis without compromising adipogenesis . It has also been reported that SWI/SNF-dependent chromatin remodeling is involved in MSC adipogenic differentiation. BRG1 overexpression was associated with promoted adipogenic differentiation, which was associated with a marked increase in the differentiation markers PPARγ and LPL . BAF45A was identified as an important regulator of adipogenic differentiation in human MSCs . In addition, other ATP-dependent chromatin remodelers, such as chromodomain helicase DNA binding (CHD) proteins, are also involved in MSC lineage commitment. CHD4 was reported to be implicated in chondrogenesis. Simon
5. Non-coding RNAs
The RNA world is divided into two classes: 1) RNAs that have coding potential (mRNAs) and 2) RNAs without coding potential, referred to as non-coding RNAs (ncRNAs). Although mRNAs have been extensively studied, ncRNAs span more than 98% of DNA transcripts. In the past, these molecules were considered as “evolutionary junk” but increasing evidence suggests that these molecules spatiotemporally regulate protein-coding gene expression in several molecular mechanisms. With improved RNA-sequencing techniques, in recent years, there have been great advances in identifying and understanding ncRNAs. Epigenetic ncRNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNA (piRNA), and long noncoding RNAs (lncRNA), have been reported to play key roles in the regulation of various diseases and biological processes, including cellular differentiation, proliferation, apoptosis, gene regulation, and cancer development.
5.1 Long non-coding RNA
lncRNA is a novel class of noncoding RNAs longer than 200 nt, which can regulate gene expression at the transcriptional and post-transcriptional levels. LncRNAs are mainly located in the cell nucleus or cytoplasm, affecting the status and fate of cells through different post-transcriptional mechanisms. Nuclear lncRNAs guide chromatin modifiers, such as DNA methyltransferase, histone methyltransferase, and heteronuclear ribosome protein, to a specific genetic locus and induce chromatin structure remodeling, which in turn regulates gene expression either positively or negatively. Cytoplasmic lncRNAs can either block the functional site or alter the structure and modification of specific proteins, thereby regulating the function and stabilization of these proteins, and ultimately alter the fate and function of cells. During the last decade, multiple studies have demonstrated that lncRNAs are widely involved in growth and development by controlling the fate of cells, including MSCs.
Studies have demonstrated the importance of lncRNAs in bone regeneration and bone formation. Many lncRNAs regulating the osteogenic differentiation of MSCs have been identified, including ANCR, AK141205, AK028326, DANCR, MALAT1, MEG3, MORD, and POIR; these either promote or inhibit osteogenic differentiation through diverse pathways. For example, MALAT1 promotes
MicroRNAs are the most abundant class of small ncRNAs with a length of 21–25 nt, and have been studied extensively. miRNAs are also involved in the epigenetic regulation of genes in both the cytoplasm and nucleus through different mechanisms. Their main action is the negative regulation of gene expression by specifically binding to a target mRNA through base complementary pairing and inducing its degradation or the inhibition of its translation.
Accumulating evidence indicates that miRNAs play an important role in the maintenance of stemness and differentiation of MSCs (Table 2). As mentioned above, lineage differentiation of MSCs is a complex biological process. For example, MSCs differentiate into osteogenic progenitor cells and subsequently osteoblasts, and then gradually become mature bone cells along with a variety of extracellular matrix mineralization. This process involves a large number of secretory and transcription factors. In addition, the differentiation and maturation of MSCs also involves signaling pathways such as WNT, BMP, and PI3K/Akt. The key effector molecules in these pathways can be regulated by miRNAs, which in turn affects MSC fate decisions. Recently, various miRNAs, including miR-20b, -29b, -30a-5p, -142-3p, -196a, -210, -746-5p, -2861, -3960, -335-5p, etc., have been reported to enhance osteogenic differentiation, whereas miR -23a, -26a, -30c, -34b, -34c, -125, -133a, -135a, -137, -141, -148, -200a, -204, -205, -206, -217, and -338 could impede osteogenic differentiation, and miR-143, -24, -31, -30c, and -642a-3p are involved in regulating adipogenesis. Oskowitz
|miR-23a||targets LRP5 and subsequently suppress the Wnt/β-catenin signaling pathway||Inhibit osteogenesis of MSCs|||
|miR-26a||in BMSCs: targets GSK3|
in ADSCs: targets Smad1 mainly and inhibits BMP signaling pathway
|Inhibit osteogenesis of ADSCs and promote osteogenesis of BMSCs|||
|miR-30c||reduces Runx2 protein||Inhibit osteogenesis of MSCs|||
|miR-20b||Activate the BMPs/Runx2 signaling pathway at four levels, which consists of repressing PPARγ, Bambi and Crim1||Promote ostegenesis||[54, 55]|
|miR-29b||activates the AKT/β-catenin signaling pathway by inhibiting PTEN expression||Promote osteogenesis of hADSCs|||
|miR-196a||targets HOXC8 (a negative regulator|
|Inhibit proliferation and promote osteogenesis of hDASCs|||
|miR-17-5p||Represses the Wnt signaling pathway effector Tcf7l2||Promote adipogenesis of BM-MSCs||[58, 59]|
|miR-21||Alters SMAD3 phosphorylation without affecting total levels of SMAD3 protein and modulate TGF-β signaling pathway||[59, 60]|
|miR-143||Directly represses MAP2K5 (a key member of the MAPKK family in the MAPK signaling pathway)||[59, 61]|
|miR-30a||Targets Runx2||Promote adipogenesis|||
|miR-642a-3p||unknown||In a high level in adipogenesis|||
“Epigenetics” was first used to define the complex interactions between the genome and the environment that are involved in the development and differentiation of organisms. Nowadays, the term refers to heritable alterations in gene expression that are not mediated at the DNA sequence level. Accumulating evidence has suggested that the processes of epigenetic modifications are crucial and largely responsible for the variable activation and repression of specific genes at specific time points during the lifespan of stem cells, allowing for the terminally differentiated phenotype. With the advances in biological and experimental technologies, a variety of epigenetic modifications involved in the cell fate determination of MSCs have been discovered in recent years. In addition to the types of epigenetic modifications introduced in the article, some researchers have suggested the role of histone phosphorylation, ubiquitination, and other modifications in the differentiation of MSCs. On this basis this information, drugs that effectively regulate these modifications have been developed to provide precise differentiation conditions for MSCs and make them more effective in clinical treatment. The disadvantage of epigenetic therapy using small molecule drugs is the lack of specificity, which needs to be further studied. In summary, epigenetic modifications play an important regulatory role in the cell fate determination of MSCs, but the precise function of these modifications in different MSC types, as well as the associated underlying mechanisms, remain to be thoroughly investigated. In-depth research in this field would provide important reference data for the differentiation mechanism research and clinical application of MSCs.
The authors were supported by the National Key Research and Development Program of China (2020YFA0113003, 2018YFC1004803) and the Fundamental Research Funds for the Central Universities.
Conflict of interest
The authors declare no competing financial interests.