DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro

Abstract

Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-mediated knockdown of DOT1L impaired proliferation and survival of NSCs. DOT1L inhibition specifically induced genes that are activated during the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Chromatin-immunoprecipitation analyses revealed that two genes encoding for central molecules involved in the ER stress response, Atf4 and Ddit3 (Chop), are marked with H3K79 methylation. Interference with DOT1L activity resulted in transcriptional activation of both genes accompanied by decreased levels of H3K79 dimethylation. Although downstream effectors of the UPR, such as Ppp1r15a/Gadd34, Atf3, and Tnfrsf10b/ Dr5 were also transcriptionally activated, this most likely occurred in response to increased ATF4 expression rather than as a direct consequence of altered H3K79 methylation. While stem cells are particularly vulnerable to stress, the UPR and ER stress have not been extensively studied in these cells yet. Since activation of the ER stress program is also implicated in directing stem cells into differentiation or to maintain a proliferative status, the UPR must be tightly regulated. Our and published data suggest that histone modifications, including H3K4me3, H3K14ac, and H3K79me2, are implicated in the control of transcriptional activation of ER stress genes. In this context, the loss of H3K79me2 at the Atf4-and Ddit3-promoters appears to mark a point-of-no-return that activates the death program in NSCs. STEM CELLS 2016;34:233-245 SIGNIFICANCE STATEMENTPosttranslational histone modification control gene expression. They are means to pass on transcriptional information from one cell generation to another. We describe the impact of histone H3 dimethylation at lysine 79 (H3K79me2) in neural stem cells. Inhibiting the enzymatic activity mediating this modification leads to cell division defects and cell death by controlling expression of central transcription factors implicated in stress response. Histone methylations are a way to balance how stem cells can cope with the activation of the stress response, which can range from proliferation to differentiation and, in case of H3K79me2, to the control of cell death.

The FOXG1/FOXO/SMAD network balances proliferation and differentiation of cortical progenitors and activatesKcnh3expression in mature neurons

“…The procedure was carried out as previously described [53] with some modifications. CPCs were fixed with 1% freshly prepared PFA (Roth) for 5 min at RT.…”

Chemical probes targeting epigenetic proteins: Applications beyond oncology

“…When DOT1L is overexpressed, it is known to disrupt telomere silencing. Inhibition of DOT1L by SGC0946 118 or EPZ005676 75 in neural stem cells (NSCs) results in an increase in transcription of Atf4 and Ddit3 ; this was confirmed by a reduction of H3K79me2 at their promoters 119 . However, in 2 unrelated cell types, mouse embryonic stem cells and a leukemia cell line, the ER stress response genes were not induced after DOT1L inhibition.…”

Barcelona conference on epigenetics and cancer 2015: Coding and non-coding functions of the genome

“…Focusing on the MLL fusion partner AF9, the group of Tanja Vogel (University Freiburg, Germany) previously demonstrated how the recruitment of the histone methyltransferase DOT1L/ KMT4A leads to K79 methylation of histone H3 of target genes. 19 Presented by Nicole Hellbach, the Vogel lab provided data suggesting that DOT1L has an important role in regulating survival and proliferation of neural progenitor cells by keeping repressed several key effector genes of the unfolded protein response. 20…”

“…19 Presented by Nicole Hellbach, the Vogel lab provided data suggesting that DOT1L has an important role in regulating survival and proliferation of neural progenitor cells by keeping repressed several key effector genes of the unfolded protein response. 20…”