ES Cell Cycle Progression and Differentiation Require the Action of the Histone Methyltransferase Dot1L

Abstract

Mouse embryonic stem cells (ESCs) proliferate with rapid cell cycle kinetics but without loss of pluripotency. The histone methyltransferase Dot1L is responsible for methylation of histone H3 at lysine 79 (H3K79me). We investigated whether ESCs require Dot1L for proper stem cell behavior. ESCs deficient in Dot1L tolerate a nearly complete loss of H3K79 methylation without a substantial impact on proliferation or morphology. However, shortly after differentiation is induced, Dot1L-deficient cells cease proliferating and arrest in G2/M-phase of the cell cycle, with increased levels of aneuploidy. In addition, many aberrant mitotic spindles occur in Dot1L-deficient cells. Surprisingly, these mitotic and cell cycle defects fail to trigger apoptosis, indicating that mouse ESCs lack stringent cell cycle checkpoint control during initial stages of differentiation. Transcriptome analysis indicates that Dot1L deficiency causes the misregulation of a select set of genes, including many with known roles in cell cycle control and cellular proliferation as well as markers of endoderm differentiation. The data indicate a requirement for Dot1L function for early stages of ESC differentiation where Dot1L is necessary for faithful execution of mitosis and proper transcription of many genes throughout the

DOT1L regulates dystrophin expression and is critical for cardiac function

“…Results shown in Figure 2F demonstrate that the percentage of proliferating cells (ratio of Ki-67-positive nuclei to total nuclei, multiplied by 100) is significantly increased in the CKO hearts compared with the control, which may contribute to the observed increase in the CKO heart mass. We note that this increased cell proliferation in the DOT1L-deficient heart is in contrast to previous studies showing a requirement for DOT1L in embryonic stem cell cycle progression (Jones et al 2008;Barry et al 2009), suggesting cell type specificity. Taken together, the above data support the notion that CKO hearts exhibit multiple phenotypes similar to those observed in DCM.…”

Deficiency of H3K79 Histone Methyltransferase Dot1-like Protein (DOT1L) Inhibits Cell Proliferation

“…H3K79me2 is enriched in the open reading frames and promoters of G 1 /S-regulated genes in yeast (42). In addition, a subset of genes related to cell growth and cell cycle has also been reported to be up-or down-regulated in DOT1L-deficient ES cells (27). As in the differential transcriptome pattern in DOT1L-deleted yeast and ES cells, the cancer cells treated with DOT1L siRNA might undergo transcriptional changes in the genes related to cell cycle regulation.…”

“…This abnormal mitotic regulation induced by DOT1L deficiency consequently results in mitotic arrest (27,29). However, although DOT1L-deficient cancer cells showed similar phenotypes such as multipolar mitotic spindles and the resulting aneuploidy, these cells showed irreversible arrest at G 1 phase rather than at the mitotic phase, suggesting that DOT1L-deficient cancer cells bypassed the spindle assembly checkpoint.…”

“…Mammalian DOT1L is also known to control proliferation and differentiation in embryonic stem (ES) cells. DOT1L deficiency causes the accumulation of hyperploid cells and defects in differentiation (27). Moreover, it has been reported that Grappa, the fly ortholog of Dot1, and DOT1L regulate embryogenesis in Drosophila and mice, respectively (28 -31).…”

The diverse functions of Dot1 and H3K79 methylation

“…Although DOT1L-depleted ES cells retain their proliferation and pluripotency DOT1 and H3K79 methylation capacity, they displayed severe proliferation defects upon RA-induced in vitro differentiation, resulting in G2/M accumulation (Barry et al 2009). Interestingly, both undifferentiated and differentiated DOT1L knockdown ES cells exhibited aneuploidy, with the phenotype being more pronounced upon differentiation (Barry et al 2009). Consistent with this, DOT1L knockout ES cells also displayed a G2/M cell cycle defect and aneuploidy (Jones et al 2008).…”