ZNF384-Fusion Proteins Have High Affinity to EP300, Which Increases Their Transcriptional Activities
Authors: Yamamoto Hideyuki , Hayakawa Fumihiko , Yasuda Takahiko , Minamikawa Yuka , Tange Naoyuki , Hirano Daiki , Kojima Yuki , Morishita Takanobu , Tsuzuki Shinobu , Mano Hiroyuki , Naoe Tomoki , Kiyoi Hitoshi 
Published: October 2019

Abstract
ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions.
Figure legends
Figure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts.

Disclosures
Naoe: Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.