Myeloid Elf-1-like Factor, an ETS Transcription Factor, Up-regulates Lysozyme Transcription in Epithelial Cells through Interaction with Promyelocytic Leukemia Protein

Abstract

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348 -517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian twohybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.The mammalian cell nucleus contains a large number of specialized nuclear domains or compartments. Each nuclear domain has been reported to relate with various physiological processes, such as transcription, DNA replication and pre-mRNA splicing (1). PML 1 nuclear body (also called nuclear domain 10 and promyelocytic leukemia protein oncogenic domain) is one of the nuclear domains (1-3). PML NBs have been reported to vary in size from 0.2 to 1.0 m in diameter, and a typical mammalian nucleus contains 10 -30 of these nuclear bodies (2, 4). Two major components, PML and Sp100, are considered to build the framework of PML nuclear bodies (5). PML was identified as part of a fusion protein with retinoic acid receptor ␣, resulting from the t(15;17) chromosomal translocation expressed in acute promyelocytic leukemia (6, 7). Sp100 was first characterized as an autoantigen in certain autoimmune disorders (8). Although PML and Sp100 are core components of PML nuclear bodies, numerous proteins, such as p53, SUMO-1, CBP, HDAC, Daxx, pRB, and HIPK2, seem to transiently localize to PML nuclear bodies (4, 9 -12). A variety of functions has been suggested for PML nuclear bodies, including tumor suppression and transcriptional regulation through the titration, modification, and compartmentalization of proteins (4, 9). The ETS transcription factor family has more than 30 members, and a number of these factors have been shown to play roles in important physiological processes, including cellular proliferation, differentiation, development, hematopoiesis, angiogenesis, and transform...

The Transcription Factor MEF/Elf4 Is Dually Modulated by p53-MDM2 Axis and MEF-MDM2 Autoregulatory Mechanism

“…observation considering that MEF is known to be constitutively nuclear localizing and is barely detected in the cytoplasm (18,40) (Fig. 6, A and C).…”

“…Immunoprecipitation was performed as essentially reported in Ref. 18. Briefly, HCT116 cells were washed with cold PBS(Ϫ) twice and cross-linked with 1 mM dithiobis[succinimidylpropionate] for 30 min at room temperature.…”

“…The nuclear lysate was collected for analysis. Equal amounts of proteins were subjected to Western blot analysis using the protocol described previously (18,27). Immunoprecipitation was performed as essentially reported in Ref.…”

“…Luciferase activity was determined 48 h after transfection using the dual-luciferase reporter assay system (Promega) and a luminometer (TD-20/20; Promega) according to the manufacturer's instructions. Luciferase activity for each sample was normalized to the Renilla reporter as described previously (18).…”

“…MEF activity is enhanced upon interaction with promyelocytic leukemia protein that induces accumulation of MEF in the promyelocytic leukemia nuclear bodies (17,18). MEF is phosphorylated by the cyclin A-cdk2 complex, ubiquitinated by Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF Skp2 , and degraded by proteasome at the G 1 /S phase transition (19).…”