Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

Yu-Kun Xia,Yu-Jie Tang,Meng-Li Zhang,Fang Liu,Yi-Rong Zeng,Yue Xiong,Cheng Zhang,Peng Liu,Zhen Yang,Kun-Liang Guan,Chen Yang,Jin-Ye Zhang,Chen-Xi He,Lei Song,Hao Zhang,Dan Ye,Chen Ding,Yi-Ping Sun,Pu Wang

doi:10.1007/s13238-020-00754-2

Abstract

Additional sex combs-like 1 (ASXL1) interacts with BRCA1-associated protein 1 (BAP1) deubiquitinase to oppose the polycomb repressive complex 1 (PRC1)-mediated histone H2A ubiquitylation. Germline BAP1 mutations are found in a spectrum of human malignancies, while ASXL1 mutations recurrently occur in myeloid neoplasm and are associated with poor prognosis. Nearly all ASXL1 mutations are heterozygous frameshift or nonsense mutations in the middle or to a less extent the C-terminal region, resulting in the production of C-terminally truncated mutant ASXL1 proteins. How ASXL1 regulates specific target genes and how the C-terminal truncation of ASXL1 promotes leukemogenesis are unclear. Here, we report that ASXL1 interacts with forkhead transcription factors FOXK1 and FOXK2 to regulate a subset of FOXK1/K2 target genes. We show that the C-terminally truncated mutant ASXL1 proteins are expressed at much higher levels than the wild-type protein in ASXL1 heterozygous leukemia cells, and lose the ability to interact with FOXK1/K2. Specific deletion of the mutant allele eliminates the expression of C-terminally truncated ASXL1 and increases the association of wild-type ASXL1 with BAP1, thereby restoring the expression of BAP1-ASXL1-FOXK1/K2 target genes, particularly those involved in glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways. In addition to FOXK1/K2, we also identify other DNA-binding transcription regulators including transcription factors (TFs) which interact with wild-type ASXL1, but not C-terminally truncated mutant. Our results suggest that ASXL1 mutations result in neomorphic alleles that contribute to leukemogenesis at least in part through dominantly inhibiting the wild-type ASXL1 from interacting with BAP1 and thereby impairing the function of ASXL1-BAP1-TF in regulating target genes and leukemia cell growth.

Highlights

Addition of sex combs (Asx) gene was originally discovered from genetic screen in Drosophila where Asx mutations augmented the phenotype of both trithorax (TrxG) and polycomb group (PcG) gene mutants (Sinclair et al, 1998)
By comparing the FOXK1 and FOXK2 chromatin immunoprecipitation assays with sequencing (ChIPseq) data, we found that 1,321 of 2,069 (63.8%) genes, which were affected by deletion of mutant Additional sex combs-like 1 (ASXL1) allele, C-terminally truncated ASXL1 mutant interferes with the BRCA1-associated protein 1 (BAP1)-ASXL1-FOXK1/K2 axis to down-regulate multiple tumor suppressor genes
ASXL1 is known to play a role in maintaining H3K27me3 levels by interacting with PRC2 complex, we found that the H3K27me3 levels at the FOXK1/K2 target gene promoters were comparable between ASXL1+/− and ASXL1+/N590 cells (Fig. S10), excluding the involvement of PRC2 and re-affirming that the expression of C-terminally truncated mutant ASXL1 proteins dominantly inhibits the function of wild-type ASXL1 at least in part by suppressing the recruitment of BAP1 to the promoters of FOXK1/K2 target genes and dysregulating gene expression through H2AK119 monoubiquitination

Summary

Introduction

Addition of sex combs (Asx) gene was originally discovered from genetic screen in Drosophila where Asx mutations augmented the phenotype of both trithorax (TrxG) and polycomb group (PcG) gene mutants (Sinclair et al, 1998). Subsequent discoveries that TrxG and PcG genes encode histone modifying enzymes link the function of Asx to epigenetic and transcriptional regulation. Mammalian ASXL proteins uniquely contain an N-terminal HARE-HTH domain (residues 1–94 in human ASXL1, known as ASXN domain) that was found in HB1, ASXL, restriction endonuclease and formed as winged helix-turn-helix (HTH) fold, and a DEUBAD (DEUBiquitinase adaptor) domain (residues 238–390, known as ASXH domain) shared with ubiquitin carboxyl-terminal hydrolase 37 (Uch37) and transcription factor NF-κB (Sanchez-Pulido et al, 2012). ASXL proteins do not contain a catalytic domain and instead, they participate in epigenetic regulation through interacting with other histone modifying enzymes. The major functional and physical partner of ASXL proteins is tumor suppressor BRCA1-associated protein 1 (BAP1), known as Calypso in Drosophila (Scheuermann et al, 2010; Dey et al, 2012), a UCH domain-containing deubiquitylase. A recent study found that there was a large overlap in genes downregulated between BAP1 knockout (KO) and ASXL1/2 double KO cells (Campagne et al, 2019), supporting the important role of ASXL proteins in mediating the function of BAP1

Objectives

Methods

Results

Conclusion