Received: June 27, Published: July 31, Epigenetic regulation mechanisms in stem cell differentiation. DOI: Download PDF. Stem cells give rise to almost different cell types that are present in a mammalian organism.
In spite of the fact that these distinct cell types have different functions and morphologies, they descend from a common ancestor cell and essentially share the same DNA. The underlying cause for the rise of specific cell types is not genetic differences; it is mostly due to how the genetic information is interpreted.
The epigenome, which consists of several degrees of regulatory mechanisms, dictates the gene expression profile of a certain type of cell.
Eukaryotic DNA is packaged into chromatin, folded and compacted which in turn affects its functionality as certain regions of DNA will not be accessible whereas some other regions will be easier to access for effect or proteins and modifiers to bind. The chromatin dynamics are modulated by several machineries including but not limited to histone PTMs and their variants, DNA methylation and RNA interference, which have crucial roles in regulating stem cell differentiation, cell fate determination and lineage specification.
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Pluripotent stem cells have the capability of differentiating into cells from all three germ layers endoderm, mesoderm and ectoderm; as well as an unlimited potency to self-renew, maintaining their stem cell identity. Embryonic stem cells that are derived from the inner cell mass of the mammalian blastocyst can give rise to a fully developed viable embryo with more than different cell types, which essentially share the same genetic information, namely the DNA.
The establishment of distinct cellular identities requires differential interpretation of this genetic information in the form of differential gene expression patterns, which is largely influenced by DNA accessibility and is modulated by mechanisms that are beyond DNA sequence, indicating epigenetic regulation.
In eukaryotes, DNA is compacted into a macromolecular structure called chromatin. The packaging of DNA into chromatin is done in a dynamic manner so that the DNA can still be accessible to carry out cellular functions such as replication, transcription and DNA repair. Metaphase chromosomes represent the most compact form of chromatin 2. Chromosomes are comprised of two structurally and functionally distinct domains; euchromatin represents the transcriptionally active, loosely packaged and gene-rich regions whereas heterochromatin is highly condensed and gene-poor.
Chromatin in stem cells is lightly packed and the actively transcribed genes are mainly the ones that are required for maintaining pluripotency. However, as they gradually lose their pluripotent characteristics and start differentiating, pluripotency genes are turned off and lineage specific gene expression takes over. Previous studies indicate a close connection between stem cell differentiation and widespread epigenome remodeling. Histones are major players of chromatin dynamics and transcriptional activity.
Specific histone post-translational modifications PTMs and incorporation of certain histone variants are associated with euchromatin and active transcription while others might be found at heterochromatic regions and repress gene expression. The tripartite structure of histones that are composed of a globular domain and unstructured N- or C-terminal tails allow these amino acids and also others to be subjected to several covalent modifications such as methylation, acetylation, phosphorylation.
The gene expression regulation during stem cell differentiation into lineage specific cells requires action of histone modifying enzymes. For instance, removal of certain histone acetylation marks by histone deacetylases is crucial for neuronal precursor cells to undergo neurogenesis. Studies indicate that increased levels of repressive histone marks such as H3K27me3 elevate levels of the polycomb group of protein complex PcG -associated BMI-1 B-lymphoma Mo-MLV insertion region-1 homolog , which results in decreased neural stem cell proliferation.
Heterochromatin usually has high levels of DNA methylation.
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The stem cell genome is highly hypomethylated whereas the global methylation levels generally increase as cells commit to a certain fate and differentiate. Non-coding RNAs have been linked with initiation of heterochromatin formation and transcriptional silencing. For instance, the long non-coding RNA Xist X-inactive specific transcript plays a crucial role in X chromosome inactivation. The expression patterns of small RNAs change among different tissues at different developmental time points; which in turn affects the expression levels of their downstream target genes.
Hence, the RNA molecules within the RNAi machinery play a major role in tissue-specific gene expression during development. If grown in Petri dishes, the P19 cells readily form aggregates and develop into EBs [ 13 ]. Mesoderm specification takes place at the early stage of EB formation, coinciding with expression of Brachyury T, a member of the T-box family of transcription factors [ 14 , 15 ].
However, EB formation per se does not result in myogenic differentiation, which requires additional regulatory signals. When cultured in the presence of signaling molecules, such as dimethyl sulfoxide DMSO or all- trans retinoic acid RA , during EB formation, the P19 cells differentiate into skeletal myocytes in a relative low frequency [ 16 , 17 ]. Treatment with combination of inducers, such as with both DMSO and RA, markedly enhances the myogenic conversion of P19 stem cells [ 18 ]. The differentiation of P19 stem cells is affected by the concentration of RA treatment.
The efficiency of P19 myogenic commitment is also affected by the timing and duration of RA treatments. Finally, the ability of P19 cells to undergo myogenesis is also influenced by other factors in the serum, and EB formation is a prerequisite for myogenic differentiation of the pluripotent P19 EC cells [ 24 ].
Effects of time course treatments on myogenic differentiation. A P19 stem cells were treated with DMSO and RA for the indicated times in Petri dishes during EB formation, maintained on coverslips for additional 5 days without any treatments and then stained for microscopic analysis. C Quantification of myocytes is presented as the fractions of cells stained positively for myosin heavy chain in relation to the total cell populations.
Error bars are the standard deviations of three independent experiments.
Most interestingly, recent studies have identified bexarotene, a selective ligand of retinoid X receptor RXR , to be an effective enhancer for the generation of skeletal myocytes by pluripotent stem cells [ 4 , 23 ]. Particularly, bexarotene enhances myogenic differentiation in a concentration dependent manner.
The range of working concentration is wide 10— nM and fits the kinetics of its affinity to RXR as a ligand [ 4 , 23 ]. More importantly, high concentrations of bexarotene do not inhibit the differentiation of P19 cells into skeletal myocytes [ 4 , 23 ], which is in marked contrast with the dose effects of RA on myogenic conversion [ 21 ].
Nevertheless, the below Kd narrow concentration range also applies to the enhancement effect of arotinoid acid, a selective ligand for retinoic acid receptor RAR , on myogenic specification [ 25 ]. In addition, the efficacy of bexarotene in P19 myogenic differentiation is comparable to RA and arotinoid acid [ 4 , 23 ]. Early events of embryonic myogenesis are also closely recapitulated by the differentiation of ES cells into skeletal muscle lineage [ 26 , 27 ]. RA is able to enhance myogenic differentiation of ES cells. More specifically, RA also affects the differentiation of ES cells into skeletal myocytes in a time- and concentration-dependant manner, similar manner as in pluripotent P19 EC cells.
However, when low concentrations of RA are administered at day 5—7 of differentiation, skeletal myogenesis is inhibited, whereas cardiomyogenesis is induced [ 28 ]. Since ES cells respond poorly to RA regarding myogenic differentiation, the effect of bexarotene on the differentiation of ES cells into skeletal muscle lineage thus becomes critical [ 24 ]. A hanging-drop procedure was used to form the EBs which leads to ES cell differentiation.
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However, bexarotene is about fivefold more efficient than RA and significantly increased the specification of skeletal muscle lineage [ 4 ]. Taken together, these data demonstrate that the RXR ligand is a more effective signaling molecule than RA to enhance the differentiation of ES cells into skeletal muscle lineage [ 4 ]. Vitamin A plays important roles in patterning and development during vertebrate embryogenesis [ 29 ].
Proper distribution and metabolism of vitamin A is fundamental for normal embryonic development and growth. Deficiency in vitamin A during early stage of embryogenesis results in congenital malformations affecting the patterning and development of many organ systems [ 30 ]. On the other hand, high concentrations of vitamin A or pharmacological concentration of RA, a potent derivative of vitamin A, have severe teratogenic consequences [ 31 ]. The diversified effects of RA are mediated by multiple levels of effectors, including enzymes that control the synthesis and degradation of RA, the cytoplasmic RA-binding proteins, and the nuclear receptors that are activated by RA [ 32 ].
The RARs are ligand-inducible transcription factors mediating the effects of RA on cellular activities [ 33 ]. Single subtype of RAR knockout mice is viable and appears normal, exhibiting few developmental defects [ 34 , 35 ]. Nevertheless, double RAR knockout mice present a wide range of developmental abnormalities which resemble the vitamin A deficiency syndrome [ 36 — 39 ].
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In fact, there appears to be a large degree of functional redundancy between RARs which play important roles in many distinct stages of embryonic patterning and organogenesis [ 33 ]. In this bimodal mode, ligand induction is through the RAR, wherein RXR is generally considered as a silent partner [ 44 ].
Mice with subtypes of RXRs knocked out are also well characterized. In addition, RXR is involved in an array of signaling cascades and has the capacity to converge multiple pathways as an liganded receptor [ 55 , 56 ]. Skeletal myogenesis is a complex process coordinated temporally by multiple myogenic regulatory factors including Myf5, MyoD, myogenin, and Mrf4 [ 57 , 58 ]. While Myf5 and MyoD initiate the expression of muscle-specific genes and commit the progenitor cells into the muscle lineage [ 59 — 61 ], myogenin and Mrf4 mainly regulate the late stage of differentiation, such as the fusion of myoblasts into myotubes [ 62 — 65 ].
At the upstream, Wnt signaling and Shh from the dorsal neural tube and notochord act as the positive regulators of Myf5 gene expression, whereas the expression of MyoD depends on the function of progenitor factor Pax3 and Myf5 [ 66 ]. Although both mesoderm factor Meox1 and Pax3 are important for myogenesis, overexpression of Mexo1 per se is not sufficient to induce P19 myogenic differentiation [ 67 , 68 ]. During P19 myogenic specification, Meox1 and Pax3 expression are upregulated by RA by day 4 of differentiation [ 4 , 22 , 23 ].
Similarly, Myf5 transcripts can also be detected by day 4 of differentiation following RA treatment [ 23 , 69 ]. Interestingly, bexarotene increases the transcript level of Meox1 with a greater efficiency than RA about twofold , whereas RA has a larger impact than bexarotene on gene expression of Pax3 and Myf5 [ 4 , 23 ]. In addition, the temporal gene expression pattern induced by bexarotene during P19 myogenic differentiation is similar to during myogenesis in vivo, and RXR ligand acts as an effective enhancer for the specification of muscle lineage [ 4 ].
It worth noting that bexarotene and RA have comparable efficacies at enhancing P19 myogenic differentiation [ 4 ]. While RA enhances skeletal myogenesis by expanding the progenitor population [ 22 ], bexarotene may affect germ layer fate determinations, particularly promoting mesoderm differentiation [ 4 ]. Intriguingly, bexarotene is a more efficient enhancer than RA for myogenesis in the ES cell system [ 4 , 23 ]. Similar as in the P19 stem cells, bexarotene augments Meox1 transcripts more potently than RA in ES cell system, whereas RA is more efficient at increasing Pax3 transcripts [ 4 , 23 ].
Nonetheless, bexarotene alone is able to induce the expression of early differentiation marker meox1, whereas RA requires additional signaling molecules to induce Meox1 expression.