Characterization of Multimerization Domains of Chimeric Transcription Factors in Leukemia
Authors: Kawamata Norihiko , Minata Mutsuko , Wang Xin , Koeffler H. Phillip , Itakura Keiich 
Published: October 2019

Abstract 1276

A number of genes encoding transcription factors are rearranged in leukemia. These rearrangements frequently generate chimeric transcription factors involving their DNA binding domain fused to multimerization domain from their partner genes. We have previously reported that multimerization is a key element for the dominant-negative action of these chimeric transcription factors over the wild-type transcription factors (Kawamata N. et al, Oncogene 2012). To analyze functional properties of these multimerization domains in the chimeric transcription factors, we focus on four chimeric transcription factors: PAX5-C20orf112, PAX5-ETV6, RUNX1-ETO, and CBFb-MYH11. C20orf112 has a helical domain; ETV6 has SAM domain; ETO has the NHR2 domain; MYH11 has a myosin tail domain; each causing multimerization. At first, we fused these multimerization domains to red-color fluorescence protein (mCherry) and expressed these in 293 cells to examine their localization pattern in living cells. Interesting, mCherry-NHR2 was predominantly localized in the cytoplasm although the molecular weight of mCherry-NHR2 was less than 60kDa which usually allows penetration into the nucleus by diffusion. In contrast, mCherry-SAM and mCherry-Myosin-Tail were dominantly localized in the nucleus; mCherry-C20helix was localized in both the nucleus and the cytoplasm. To characterize the function of NHR2 domain further, we fused core peptide sequence of NHR2 domain (32 amino acid) to mCherry; it continued to be localized in the cytoplasm. Then, we have inserted the DNA binding domain of RUNX1 (RUNT domain) to mCherry-NHR2 (mCherry-RUNT-NHR2) and this fusion protein predominantly localized in the nucleus. These data suggested that NHR2 may have a nucleus export signal (NES) and be continuously exported from nucleus and that the RUNT domain is required for nuclear localization of this fusion protein. To analyze the multimerization property of these four different potential multimerizing domains, Bi-molecule Fluorescence Complementation (BiFC) was used. Yellow-Fluorescence Protein (YFP) was divided into two fragments (N-terminal YFP: N-YFP and C-terminal YFP: C-YFP) and fused to these multimerization domains. Interestingly, C20helix, Myosin-tail, and SAM domains, each displayed complementation of fluorescence color in the nucleus demonstrating that multimerization occurred in the living cells. However, NHR2 did not show complementation of fluorescence in the cytoplasm suggesting that no multimerization of NHR2 occurred in the cytoplasm. In contrast, co-expression of N-YFP-RUNT-NHR2 and C-YFP-RUNT-NHR2 produced complementation of fluorescence in the nucleus. These data suggest that RUNT domain is needed for both stable localization of this protein in the nucleus and stable multimerization. Although a number of multimerization domains are involved in chimeric transcription factors in cancerous cells, our data suggest that each multimerization domain has multiple functional properties other than multimerization; and strength of multimerization is different in each multimerization domain, which sometimes requires DNA binding to generate stable multimerization. Our data support identification of small molecules to inhibit multimeriation of these chimeric transcription factors in cancer cells as a novel therapeutic approach

Disclosures:

No relevant conflicts of interest to declare.