UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity

Abstract

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development. I n vitro, embryonic stem (ES) cells are capable of differentiating into three germ layers, ectoderm, endoderm, and mesoderm, which mimics the early stage of embryonic development in vivo (1). Transcription factor Brachyury (T) is highly expressed in the primitive streak during gastrulation and is required for mesoderm formation (2). Mutation of Brachyury gene in mice causes defective formation of posterior mesoderm, failure of notochord morphogenesis, and embryonic death around 10 d of gestation (2). Brachyury expression is directly regulated by Wnt/β-catenin signaling in mesoderm and in ES cells. Wnt/β-catenin signaling promotes de-phosphorylation of β-catenin, which enters nucleus and binds transcription factor LEF1/TCF1 to activate Brachyury expression (3, 4). Brachyury also positively regulates Wnt/β-catenin signaling. Such a positive auto-regulatory loop between Brachyury and Wnt/β-catenin signaling maintains the mesodermal progenitor cells and is essential for posterior mesoderm development in vertebrates (5).The Polycomb Repressive Complex 2 (PRC2) is critical for the proper differentiation of ES cells. PRC2 is localized on a large number of developmental regulator genes in ES cells. Disruption of PRC2 in ES cells markedly decreases the global levels of H3K27 di-and trimethylation (H3K27me2 and H3K27me3) and leads to up-regulation of many developmental regulator genes (6, 7).UTX belongs to a subfamily of JmjC domain-containing proteins that also includes UTY and JMJD3. Pr...

Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition

“…ESCs were maintained on MEF feeder cells and differentiated into EBs as described previously (18). ESCs were split off feeder cells and cultured on gelatincoated dishes for growth assay, colony formation assay, and AP staining as decribed (18).…”

Inhibition of KDM6 activity during murine ES cell differentiation induces DNA damage

“…Knockout (KO) embryos of KDM6A fail to activate expression of the early mesodermal marker brachyury (gene symbol T ), and embryos show severe defects in the development of the notochord, hematopoietic and cardiac tissues . The KDM6A-KO phenotype is partially rescued by UTY in male embryos or by expression of a catalytically inactive KDM6A protein, suggesting a demethylase-independent function of KDM6A Morales Torres et al, 2013;Thieme et al, 2013;Wang et al, 2012). So far it is unknown to what extent the demethylase activities of KDM6A and KDM6B can compensate for each other.…”

Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche

“…For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development (Lan et al, 2007), and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development (Vandamme et al, 2012), including gonad development (Agger et al, 2007). Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation (Wang et al, 2012), and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency (Mansour et al, 2012). These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming.…”