A synthetic lethality screen reveals ING5 as a genetic dependency of catalytically dead Set1A/COMPASS in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.

Epigenetic targeted therapy of stabilized BAP1 in ASXL1 gain-of-function mutated leukemia.

Mutations of ASXL1, encoding a component of the BAP1 histone H2A deubiquitinase complex, occur in human myeloid neoplasms and are uniformly associated with poor prognosis. However, the precise molecular mechanisms through which ASXL1 mutations alter BAP1 activity and drive leukemogenesis remain unclear. Here we demonstrate that cancer-associated frameshift mutations in ASXL1, which were originally proposed to act as destabilizing loss-of-function mutations, in fact encode stable truncated gain-of-function proteins. Truncated ASXL1 increases BAP1 protein stability, enhances BAP1 recruitment to chromatin and promotes the expression of a pro-leukemic transcriptional signature. Through a biochemical screen, we identified BAP1 catalytic inhibitors that inhibit truncated-ASXL1-driven leukemic gene expression and impair tumor progression in vivo. This study represents a breakthrough in our understanding of the molecular mechanisms of ASXL1 mutations in leukemia pathogenesis and identifies small-molecular catalytic inhibitors of BAP1 as a potential targeted therapy for leukemia.

Coordinated regulation of cellular identity-associated H3K4me3 breadth by the COMPASS family.

Set1A and Set1B, two members of the COMPASS family of methyltransferases that methylate the histone H3 lysine 4 (H3K4) residue, have been accredited as primary depositors of global H3K4 trimethylation (H3K4me3) in mammalian cells. Our previous studies in mouse embryonic stem cells (ESCs) demonstrated that deleting the enzymatic SET domain of Set1A does not perturb bulk H3K4me3, indicating possible compensatory roles played by other COMPASS methyltransferases. Here, we generated a series of ESC lines harboring compounding mutations of COMPASS methyltransferases. We find that Set1B is functionally redundant to Set1A in implementing H3K4me3 at highly expressed genes, while Mll2 deposits H3K4me3 at less transcriptionally active promoters. While Set1A-B/COMPASS is responsible for broad H3K4me3 peaks, Mll2/COMPASS establishes H3K4me3 with narrow breadth. Additionally, Mll2 helps preserve global H3K4me3 levels and peak breadth in the absence of Set1A-B activity. Our results illustrate the biological flexibility of such enzymes in regulating transcription in a context-dependent manner to maintain stem cell identity.

Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb and DNA methylation.

The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.

ß-Catenin/Tcf7l2-dependent transcriptional regulation of GLUT1 gene expression by Zic family proteins in colon cancer.

The zinc finger of the cerebellum (ZIC) proteins has been implicated to function in normal tissue development. Recent studies have described the critical functions of Zic proteins in cancers and the potential tumor-suppressive functions in colon cancer development and progression. To elucidate the functional roles of Zic proteins in colorectal cancer, we knocked out the Zic5 gene and analyzed the chromatin localization pattern and transcriptional regulation of target gene expression. We found that Zic5 regulates glucose metabolism, and Zic5 knockout is accompanied by an increased glycolytic state and tolerance to a low-glucose condition. Furthermore, loss of ß-catenin or TCF7l2 diminishes the chromatin binding of Zic5 globally. Our studies suggest that the Wnt/ß-catenin signaling pathway has a strong influence on the function of Zic proteins and glucose metabolism in colorectal cancers through GLUT1. Interfering Wnt/-catenin-Zic5 axis-regulated aerobic glycolysis represents a potentially effective strategy to selectively target colon cancer cells.

CATACOMB: An endogenous inducible gene that antagonizes H3K27 methylation activity of Polycomb repressive complex 2 via an H3K27M-like mechanism.

Using biochemical characterization of fusion proteins associated with endometrial stromal sarcoma, we identified JAZF1 as a new subunit of the NuA4 acetyltransferase complex and CXORF67 as a subunit of the Polycomb Repressive Complex 2 (PRC2). Since CXORF67's interaction with PRC2 leads to decreased PRC2-dependent H3K27me2/3 deposition, we propose a new name for this gene: CATACOMB (catalytic antagonist of Polycomb; official gene name: EZHIP ). We map CATACOMB's inhibitory function to a short highly conserved region and identify a single methionine residue essential for diminution of H3K27me2/3 levels. Remarkably, the amino acid sequence surrounding this critical methionine resembles the oncogenic histone H3 Lys27-to-methionine (H3K27M) mutation found in high-grade pediatric gliomas. As CATACOMB expression is regulated through DNA methylation/demethylation, we propose CATACOMB as the potential interlocutor between DNA methylation and PRC2 activity. We raise the possibility that similar regulatory mechanisms could exist for other methyltransferase complexes such as Trithorax/COMPASS.

The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response.

FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.

TET2 coactivates gene expression through demethylation of enhancers.

The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress.

PURPOSE: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). METHODS AND MATERIALS: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. RESULTS: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. CONCLUSIONS: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.