A novel EVI1 gene family, MEL1, lacking a PR domain (MEL1S) is expressed mainly in t(1;3)(p36;q21)-positive AML and blocks G-CSF–induced myeloid differentiation

Abstract

We have identified a novel gene MEL1 (MDS1/EVI1-like gene 1) encoding a zinc finger protein near the breakpoint of t(1; 3)(p36;q21)-positive human acute myeloid leukemia (AML) cells. Here, we studied the structure, expression pattern, and function of MEL1 in leukemia cells. In this study, we have identified 3 transcription start sites, 1 in exon 1 and 2 in exon 2, and 2 kinds of translation products, 170 kDa (MEL1) and 150 kDa (MEL1S). Notably, the 150-kDa band of MEL1S was detected mainly in the t(1;3)(p36;q21)-positive AML cells. By immunoblot analysis and proteolytic mapping, it is suggested that the 150-kDa band of MEL1S in the leukemia cells is translated from the internal initiation codon ATG597 in exon 4 and is mostly lacking the amino-terminal PR domain of MEL1. By the cyclic amplification and selection of targets (CASTing) method for identifying consensus sequences, it was shown that the consensus sequences of MEL1 were included in 2 different consensus sequences for DNA-binding domain 1 and 2 (D1-CONS and D2-CONS) of EVI1. In reporter gene assays, MEL1S activated transcription via binding to D2-CONS; however, the fusion of MEL1 or MEL1S to GAL4 DNA-binding domain (DBD) made them GAL4 binding sitedependent transcriptional repressors. IntroductionThe PRDI-BF1-RIZ1 homologous (PR) domain is a newly recognized amino-terminal module 1 and was first noted for the homologous 100-amino acid region shared between positive regulatory domain I binding factor 1 (PRDI-BF1) (PRDM1) 2 and retinoblastoma-interacting zinc finger protein (RIZ) (PRDM2). 1 PR domain members (PRDMs) are also known to be in the suppressor of var, enhancer of zeste and trithorax (SET) domain superfamily. 3 RIZ is isolated by protein-protein interaction with the retinoblastoma tumor suppressor protein (Rb). 1 Consistent with a potential role in the Rb pathway, RIZ may play an important role in the pathogenesis of human cancer. Interestingly, RIZ produces 2 different gene products, a full-length RIZ1 and a short-form RIZ2 lacking a PR domain at the amino-terminus, which is generated by an alternative transcript derived from an internal promoter. 4 Both transcripts are widely expressed in many organs. However, recent study revealed that the expression rate of RIZ1 is decreased or several point mutations in the RIZ1 coding region exist in many kinds of tumors, suggesting that RIZ1 with a PR domain is a candidate tumor suppressor gene. 5,6 Murine ecotropic virus integration 1 site (Evi-1) zinc finger protein was isolated from common sites of viral integration in murine myeloid leukemia. 7 The human homolog, EVI1 at chromosomal position 3q26, is also transcriptionally activated by several recurrent chromosomal aberrations in acute myeloid leukemia (AML). 8 The most frequent rearrangements are t(3;3)(q21;q26) and inv(3)(q21q26) associated with AML (3q21q26 syndrome) and myelodysplastic syndrome (MDS). 8 A PR domain was later found in the MDS1/EVI1 gene product, which is derived from one of the EVI1 alternative transcripts. 9 The genomic region of ...

Sumoylation of MEL1S at lysine 568 and its interaction with CtBP facilitates its repressor activity and the blockade of G-CSF-induced myeloid differentiation

“…Combining G-CSF with TSA treatment overcomes the blockade of G-CSF-induced differentiation in murine L-G3 cells induced by MEL1S expression We previously reported that the short form of MEL1 (MEL1S), a transcription factor that is specifically expressed in acute myeloid leukemia with the t(1;3) translocation, could block G-CSF-induced myeloid differentiation of L-G3, an IL-3-dependent myeloid leukemia cell line (Nishikata et al, 2003). Because MEL1S works as a transcriptional repressor in hematopoietic cells, including L-G3 cells ( Supplementary Figure 1), we initially investigated whether treatment of L-G3 cells with the HDAC inhibitor TSA prevents the blockade of G-CSF-induced differentiation by MEL1S expression.…”

“…To construct human MEL1S expression vectors, a FLAG-or a hemagglutinin (HA)-epitope tagged, short-form MEL1 lacking an N-terminal PR domain (MEL1S) (Mochizuki et al, 2000;Nishikata et al, 2003) was placed under the control of a cytomegalovirus (CMV) promoter in a mammalian expression vector pCMV26 (Sigma-Aldrich, St Louis, MO, USA) (pFLAG-MEL1S or pHA-MEL1S). To construct the human MEL1 expression vector, a HA-epitope tagged, long-form MEL1 with a PR domain (Mochizuki et al, 2000;Nishikata et al, 2003) was inserted into the pCMV26 (pHA-MEL1).…”

“…To construct the human MEL1 expression vector, a HA-epitope tagged, long-form MEL1 with a PR domain (Mochizuki et al, 2000;Nishikata et al, 2003) was inserted into the pCMV26 (pHA-MEL1). Using the In-Fusion cloning kit (Clontech, Mountain View, CA, USA), deletion mutants of MEL1S, including MEL1S/DDBD1 (DBD1, containing the 1-7 zinc-finger repeats, aa 40-264), MEL1S/DPRR (proline-rich region, aa 265-522), MEL1S/DCID (CID, aa 523-644), MEL1S/DRD (repression domain, aa 645-766), MEL1S/DDBD2 (DBD2, containing the 8-10 zinc-finger repeats, aa 767-887) and MEL1S/DCT (C-terminal region containing acidic domain, aa 893-1072), were generated by PCR amplification of human MEL1S complementary DNA lacking each domain structure (Figure 2a).…”

“…We previously identified the MDS1/ EVI1-like gene 1 (MEL1) as a member of the EVI1 gene family and also as a PR domain family member (PRDM16); this gene is located near the chromosomal breakpoint at chromosome 1p36 of t(1;3)(p36;q21) (Mochizuki et al, 2000;Shimizu et al, 2000). PRdomain deficient forms of EVI1 and MEL1 (MEL1S) are upregulated in both types of leukemia (Mochizuki et al, 2000;Nishikata et al, 2003). We discovered that the overexpression of MEL1S in murine interleukin-3 (IL-3)-dependent myeloid L-G3 cells blocked the granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF) (Nishikata et al, 2003).…”

“…PRdomain deficient forms of EVI1 and MEL1 (MEL1S) are upregulated in both types of leukemia (Mochizuki et al, 2000;Nishikata et al, 2003). We discovered that the overexpression of MEL1S in murine interleukin-3 (IL-3)-dependent myeloid L-G3 cells blocked the granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF) (Nishikata et al, 2003). Using the GAL4 DNA-binding domain (DBD) fused to MEL1 and MEL1S, we also identified that both MEL1 and MEL1S function as transcriptional repressors (Nishikata et al, 2003).…”