Asxl1 ablation in mouse embryonic stem cells impairs neural differentiation without affecting self-renewal.

Additional sex comb-like1 (Asxl1) is known as a chromatin modulator that plays dual functions in transcriptional regulation depending on the cell type. Recent studies using Asxl1 knockout mice revealed that Asxl1 is important for the proliferation and differentiation of hematopoietic progenitor cells, and the development of organs. Although we previously reported Asxl1 as a Sox2 target gene, its function in embryonic stem cells (ESCs) remains largely unknown. For this purpose, we isolated ESCs from the blastocyst inner cell mass of Asxl1-/- mice. Asxl1 deficiency in ESCs exhibited no effect on cell proliferation, expression of core pluripotent transcription factors, or alkaline phosphatase activity, suggesting dispensability of Asxl1 for self-renewal of ESCs. By contrast, the differentiation of Asxl1-/- ESCs was significantly affected as shown by size reductions of embryoid bodies accompanied with apoptosis, aberrant expression of differentiation genes, downregulation of bivalent neurogenesis genes, and abnormal axon formation in neurons. Overall, our findings indicated that Asxl1 played a critical role in regulating genes associated with neural differentiation without affecting self-renewal of mouse ESCs.

Suppressive role of OGT-mediated O-GlcNAcylation of BAP1 in retinoic acid signaling.

BRCA1-associated protein 1 (BAP1) has been implicated in diverse biological functions, including tumor suppression. However, its regulation via glycosylation and its role in embryonic stem (ES) cells are poorly defined. BAP1 was recently reported to interact with O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). Here, we confirmed the physical interaction and investigated its functional significance. The O-GlcNAcylation of BAP1, which requires OGT, was examined in vivo and in vitro, and was proven using alloxan, an OGT inhibitor. OGT promoted the BAP1-induced repression of retinoic acid (RA)-induced RA receptor (RAR) activation. The repressive activity of BAP1 was relieved by alloxan but exacerbated by PUGNAc, an O-GlcNAcase (OGA) inhibitor. Finally, we addressed the role of O-GlcNAcylation in the RA-induced differentiation of murine ES cells. Alkaline phosphatase staining revealed the cooperation of RA and alloxan for impairing the pluripotency of ES cells. This cooperation was also observed by measuring the size of embryonic bodies and the expression of Sox2, a pluripotency marker. Overall, our data suggest that OGT-mediated O-GlcNAcylation of BAP1 prefers the maintenance of pluripotency, whereas its inhibition facilitates RA-induced differentiation in ES cells.

Role of Asxl1 in kidney podocyte development via its interaction with Wtip.

Additional sex comb-like (ASXL) family proteins are chromatin factors that function in transcriptional activation and repression. However, the underlying mechanisms and biological implications have not been well established. Here, we identified a LIM domain-containing protein, Wilms tumor 1-interacting protein (WTIP), as an ASXL1-binding partner. Biochemical assays confirmed an interaction between the murine homologs Asxl1 and Wtip. The suppressive role of Wtip in WT1 function and the expression of Wtip in kidney podocytes prompted us to investigate the role of Asxl1 in the kidney using Asxl1-null mice. In homozygous Asxl1(-/-) embryos, defects in kidney size and glomerular podocyte morphology were observed. Furthermore, up-regulation of Wt1/Wtip target genes was observed in the kidneys of Asxl1-null embryos. Overall, these findings implicate Asxl1 in the maintenance of podocyte structure via its association with Wtip and in the regulation of WT1 signaling during early kidney development.

Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.

The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4- FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.

Asxl1 exerts an antiproliferative effect on mouse lung maturation via epigenetic repression of the E2f1-Nmyc axis.

Although additional sex combs-like 1 (ASXL1) has been extensively described in hematologic malignancies, little is known about the molecular role of ASXL1 in organ development. Here, we show that Asxl1 ablation in mice results in postnatal lethality due to cyanosis, a respiratory failure. This lung defect is likely caused by higher proliferative potential and reduced expression of surfactant proteins, leading to reduced air space and defective lung maturation. By microarray analysis, we identified E2F1-responsive genes, including Nmyc, as targets repressed by Asxl1. Nmyc and Asxl1 are reciprocally expressed during the fetal development of normal mouse lungs, whereas Nmyc downregulation is impaired in Asxl1-deficient lungs. Together with E2F1 and ASXL1, host cell factor 1 (HCF-1), purified as an Asxl1-bound protein, is recruited to the E2F1-binding site of the Nmyc promoter. The interaction occurs between the C-terminal region of Asxl1 and the N-terminal Kelch domain of HCF-1. Trimethylation (me3) of histone H3 lysine 27 (H3K27) is enriched in the Nmyc promoter upon Asxl1 overexpression, whereas it is downregulated in Asxl1-deleted lung and -depleted A549 cells, similar to H3K9me3, another repressive histone marker. Overall, these findings suggest that Asxl1 modulates proliferation of lung epithelial cells via the epigenetic repression of Nmyc expression, deficiency of which may cause hyperplasia, leading to dyspnea.

Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation.

Although ASXL1 mutations are frequently found in human diseases, including myeloid leukemia, the cell proliferation-associated function of ASXL1 is largely unknown. Here, we explored the molecular mechanism underlying the growth defect found in Asxl1-deficient mouse embryonic fibroblasts (MEFs). We found that Asxl1, through amino acids 371 to 655, interacts with the kinase domain of AKT1. In Asxl1-null MEFs, IGF-1 was unable to induce AKT1 phosphorylation and activation; p27Kip1, which forms a ternary complex with ASXL1 and AKT1, therefore remained unphosphorylated. Hypophosphorylated p27Kip1 is able to enter the nucleus, where it prevents the phosphorylation of Rb; this ultimately leads to the down-regulation of E2F target genes as confirmed by microarray analysis. We also found that senescence-associated (SA) genes were upregulated and that SA ß-gal staining was increased in Asxl1 -/- MEFs. Further, the treatment of an AKT inhibitor not only stimulated nuclear accumulation of p27Kip1 leading to E2F inactivation, but also promoted senescence. Finally, Asxl1 disruption augmented the expression of p16Ink4a as result of the defect in Asxl1-Ezh2 cooperation. Overall, our study provides the first evidence that Asxl1 both activates the AKT-E2F pathway and cooperates with Ezh2 through direct interactions at early embryonic stages, reflecting that Asxl1 disruption causes cellular senescence.