Changes in chromatin assembly drive embryonic stem cells to an early embryonic-like state
Embryonic stem cells are pluripotent because they lack the ability to form extra-embryonic tissue. In contrast, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling totipotent 2-cell stage embryos (2C-like cells) arise at very low frequency in embryonic stem cell cultures. We showed that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1.
HFSP Long-Term Fellow Takashi Ishiuchi and colleaguesauthored on Fri, 23 October 2015
Mammalian development starts from the zygote after fertilization. In mice, the zygote and each blastomere at the 2-cell stage embryo are fully totipotent because they can generate a complete organism from a single cell. Although these totipotent cells have been studied for a long time because of this attractive ability, it has not been known at all how this ability is acquired or maintained. In this study, we aimed to understand the molecular basis underlying totipotency by identifying specific features found exclusively in totipotent cells in vivo and in vitro. For this, we used 2-cell like (2C-like) cells as well as zygotes and 2-cell embryos as a model for totipotent cells, and compared them with embryonic stem (ES) cells, which are pluripotent. 2C-like cells are reported to be totipotent-like cells because they share a gene expression program that is highly similar to 2-cell embryos. They can be found as a small population (less than 1%) in ES cell cultures and express some transcripts specific to the 2-cell stage embryo such as the MERVL retrotransposon. We unexpectedly noticed that 2C-like cells lack DAPI-rich spots, known as chromocenters, in their nuclei, contrary to ES cells. This feature is actually unique to the totipotent zygote and 2-cell embryos. Therefore, we focused on this unique feature commonly found in both zygote/2-cell embryos and 2C-like cells.
Figure: Immunostaining of EGFP in ES cells under p150 (CAF-1 component) knockdown. 2C-like cells are marked by EGFP expression. EGFP-expressing 2C-like cells are increased by p150 knockdown.
To know whether this unique feature is indeed involved in totipotency, we aimed to induce this feature in ES cells. Because it was reported that the depletion of CAF-1, a molecular complex responsible for replication-dependent chromatin assembly, causes loss of chromocenters in ES cells, we successfully employed CAF-1 knockdown by siRNA to induce the loss of chromocenters. Interestingly, CAF-1 knockdown dramatically induced the emergence of 2C-like cells in culture. The number of 2C-like cells increased to 10-30 times higher than in controls. To understand how this happens, we performed rescue experiments using some deletion mutants of p150, the major subunit of the CAF-1 complex. This experiment revealed that the downregulation of chromatin assembly activity during DNA replication is the cause underlying induction of 2C-like cells.
Because CAF-1 has a fundamental role in replication-dependent chromatin assembly, CAF-1 knockdown can theoretically affect chromatin organization globally, and thereby gene expression. Therefore, we performed RNA-sequencing in CAF-1-knockdown cells to know whether genes/retrotransposons expressed in 2-cell embryos are specifically upregulated. This analysis clearly showed that the depletion of CAF-1 specifically upregulated transcripts uniquely expressed in 2-cell embryos. In particular, MERVL and major satellite that are specifically expressed in 2-cell embryos were the highest upregulated repeat elements, while other repeats such as LINE1 and IAP were not affected. Thus, we concluded that CAF-1 regulates the expression of 2-cell embryo-specific genes and retrotransposons in ES cells.
CAF-1 directly regulates chromatin organization through its chromatin assembly and histone deposition activity. Chromatin organization is known to be important for gene expression. Thus, we reasoned that chromatin organization might be affected by CAF-1 knockdown. To address this, we examined MNase accessibility in 2C-like cells induced by CAF-1 knockdown. This assay can address how much chromatin is open or closed. Compared to control ES cells, 2C-like cells induced by CAF-1 knockdown had a more open chromatin. This is consistent with the known role of CAF-1 to deposit histones onto DNA. Then, we wondered if this unique chromatin state in 2C-like cells has an important role in early development. To address this point, we performed somatic cell nuclear transfer (SCNT) with oocytes because it has been already shown that nuclei from early embryos are much better donors for efficient embryo development upon nuclear transfer compared to somatic cells. As we expected, induced, as well as spontaneously arising 2C-like cells, showed better development after SCNT compared to normal ES cells. These results suggest that the unique chromatin state induced by CAF-1 depletion has an important role in the embryo development. In this study we succeeded in inducing the unique molecular features of totipotent cells in vitro. Although this is quite far from inducing bona fide totipotent cells, we believe that repeated attempts of this kind of study is necessary to induce totipotent cells in the future.
Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Ishiuchi T, Enriquez-Gasca R, Mizutani E, Bošković A, Ziegler-Birling C, Rodriguez-Terrones D, Wakayama T, Vaquerizas JM, Torres-Padilla ME. 2015. Nat Struct Mol Biol, 22(9):662-71.