Embryonic stem cells are capable of self-renewing and differentiating to the desired fate depending on their position in the body. Stem cell homeostasis is maintained through epigenetic mechanisms that are highly dynamic in regulating the chromatin structure as well as specific gene transcription programs. Epigenetics has been used to refer to changes in gene expression, which are heritable through modifications not affecting the DNA sequence.
The mammalian epigenome undergoes global remodeling during early stem cell development that requires commitment of cells to be restricted to the desired lineage. There has been multiple evidence suggesting that the maintenance of the lineage commitment of stem cells is controlled by epigenetic mechanisms such as DNA methylation, histone modifications and regulation of ATP-dependent remolding of chromatin structure. Based on the histone code hypothesis, distinct covalent histone modifications can lead to functionally distinct chromatin structures that influence the cell's fate.
This regulation of chromatin through epigenetic modifications is a molecular mechanism that determines whether the cell continues to differentiate into the desired fate. A research study by Lee et al. examined the effects of epigenetic modifications on the chromatin structure and the modulation of these epigenetic markers during stem cell differentiation through in vitro differentiation of murine embryonic stem (ES) cells.
Experimental background
Embryonic stem cells exhibit dramatic and complex alterations to both global and site-specific chromatin structures. Lee et al. performed an experiment to determine the importance of deacetylation and acetylation for stem cell differentiation by looking at global acetylation and methylation levels at certain site-specific modification in histone sites H3K9 and H3K4. Gene expression at these histones regulated by epigenetic modifications is critical in restricting the embryonic stem cell to desired cell lineages and developing cellular memory.
For mammalian cells, the maintenance of cytosine methylation is catalyzed by DNA methyltransferases and any disruption to these methyltransferases will cause a lethal phenotype to the embryo. Cytosine methylation is examined at H3K9, which is associated with inactive heterochromatin and occurs mainly at CpG dinucleotides while global acetylation is examined at H3K4, which is associated with active euchromatin. The mammalian zygotic genome undergoes active and passive global cytosine demethylation following fertilization that reaches a minimal point of 20% CpG methylation at the blastocyst stage to which is then followed by a wave of methylation that reprograms the chromatin structure in order to restore global levels of CpG methylation to 60%. Embryonic stem cells containing reduced or elevation levels of methylation are viable but unable to differentiate and therefore require critical regulation of cytosine methylation for mammalian development.
Effects of global histone modifications during embryonic stem cell differentiation Histones modifications in chromatin were analyzed at various time intervals (along a 6-day period) following the initiation of in vitro embryonic stem cell differentiation. The removal of leukemia inhibitory factor (LIF) triggers differentiation. Representative data of the histone modifications at the specific sites after LIF removal, assessed using Western blotting, confirms strong deacetylation at the H3K4 and H3K9 positions on histone H3 after one day, followed by a small increase in acetylation by day two.
The methylation of histone H3K4 also decreased after one day of LIF removal but showed a rebound between days 2–4 of differentiation, finally ending with a decrease in methylation on day five. These results indicate a decrease in the level of active euchromatin epigenetic marks upon initiation of embryonic stem cell differentiation which is then followed immediately by reprogramming of the epigenome.
Histone modifications of H3K9 position show a decrease in di- and tri-methylation of undifferentiated embryonic stem cells and had a gradual increase in methylation during the six-day time course of in vitro differentiation, which indicated that there is a global increase of inactive heterochromatin levels at this histone mark.