Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage

Abstract

Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2-and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.

X-Linked Histone H3K27 Demethylase Kdm6a Regulates Sexually Dimorphic Differentiation of Hypothalamic Neurons

“…First, we validated the effectiveness of GSK-J4 blocking Kdm6 demethylase activity in primary cultures of hypothalamic neurons from wild-type CD1 mice, performing a dose/response curve and evaluating the H3K27me3 levels by Western blot. In agreement with times and concentrations previously reported by others [49], treatment of cultures for 24 h with GSK-J4 1.8 μM showed the highest H3K27me3 levels (Fig. 4a) without affecting cell viability, proving to be effective in inhibiting the Kdm6 enzymatic activity.…”

Effect of histone demethylase KDM5A on the odontogenic differentiation of human dental pulp cells

“…Methylation at arginine residues, and di-/tri-Methylation at the H3K4, H3K36, and H3K79 are normally characterized as active marks, while methylation at the H3K9, H3K27, and H4K20 residues remain controversial [16,17]. The establishment and maintenance of histone methylation are dynamically regulated by site-specific histone methyltransferases (HMTs) and histone demethylases (HDMs), which play important roles in multiple cellular processes, including cell proliferation, differentiation, senescence, DNA damage response, oncogenesis and individual development [18][19][20][21]. During murine tooth germ development, covalent histone modifications characterized by H3K4me3 and H3K27me3 can be detected in the first molars of mouse embryos.…”

KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects

“…Moreover, introduction of KDM6A variants, especially truncated variants, elicited elevated phospho-γH2A.X levels ( Figure S11). Phospho-γH2A.X is activated during the DNA damage stress response (23). Enrichment of proteins involved in DNA repair and stress response (DDR) appeared in our MS-data analysis from three different urothelial cancer cell lines with stably or transiently transfected KDM6A WT ( Figure S9).…”

“…activity in differentiating embryonal stem cells has been linked to DNA damage response pathways by colocalization with γH2A.X positive foci (23). In addition, as an oxygen-dependent enzyme KDM6A serves as a sensor to control chromatin and cell fate (24).…”

“…Enrichment of proteins involved in DNA repair and stress response (DDR) appeared in our MS-data analysis from three different urothelial cancer cell lines with stably or transiently transfected KDM6A WT (Figure S9). Notably, KDM6A activity in differentiating embryonal stem cells has been linked to DNA damage response pathways by colocalization with γH2A.X positive foci (23). In addition, as an oxygen-dependent enzyme KDM6A serves as a sensor to control chromatin and cell fate (24).…”