Human differentiation-related gene NDRG1 is a Myc downstream-regulated gene that is repressed by Myc on the core promoter region
Introduction
The up-regulation of mouse ndrg1 was initially discovered in N-Myc knockout mouse embryos (Shimono et al., 1999). Consequently, ndrg1 is described as an N-Myc downstream-regulated gene. The mouse ndrg family consists of 3 members: ndrg1, ndrg2, and ndrg3. The amino acid sequences of the ndrg2 and ndrg3 proteins are homologous to ndrg1, exhibiting identities of 54% and 64%, respectively (Okuda and Kondoh, 1999, Zhou et al., 2001). Both c-Myc and N-Myc can repress the expression of mouse ndrg1 (Shimono et al., 1999). The human NDRG family is composed of 4 members: NDRG1, NDRG2, NDRG3, and NDRG4 (Qu et al., 2002, Zhao et al., 2001, Deng et al., 2003, Kovacevic and Richardson, 2006). The members of the human NDRG family share approximately 57–65% homology at the amino acid level.
We previously identified human NDRG2 (AF 159092) and demonstrated that this gene is a candidate tumor suppressor gene (Deng et al., 2003). Further experiments demonstrated that Myc represses human NDRG2 via a Miz-1-dependent interaction with the core promoter of NDRG2 (Zhang et al., 2006). In view of the high degree of homology between Ndrg1 and Ndrg2, we were eager to investigate how Myc regulates NDRG1, and to determine whether NDRG1 is regulated by Myc in a manner similar to NDRG2. Although Leo Kretzner has reported that Myc transcriptionally represses mouse ndrg1 (Li and Kretzner, 2003), the exact regulatory mechanism of human NDRG1 remains obscure.
Human Ndrg1, as a differentiation-related gene, also referred to as Drg1, Cap43, and RTP/rit42 (Kovacevic and Richardson, 2006; Kurdistani et al., 1998, Zhou et al., 1998), exhibits 94% identity with mouse ndrg1. Previous work has demonstrated that nickel and hypoxia can induce NDRG1 expression in an HIF-1-dependent manner (Salnikow et al., 2002). The expression of NDRG1 increases in human leukemia and intestinal cells after induced differentiation (van Belzen et al., 1997). NDRG1 also participates in the process of tumor suppression and metastasis inhibition (Bandyopadhyay et al., 2003). Moreover, ndrg1-deficient mice exhibit a progressive demyelinating disorder of the peripheral nervous system, indicating the important function of ndrg1 in maintaining normal nervous system function. The transcription factor p53 can up-regulate NDRG1 via transcriptional activation. Further, NDRG1 is necessary, although not sufficient, for p53-mediated apoptosis (Stein et al., 2004). Taken together, these observations indicate that NDRG1 is induced under a number of stress and pathological conditions. Although the first discovered member of the NDRG family, the regulatory mechanism of human NDRG1 has to date remained unclear.
Myc, a well-known transcription factor, belongs to the BR-HLH-Zip protein family. Myc is composed of 2 N-terminal conserved transactivation domain Myc Boxes, Mb I and Mb II, also known as C-terminal basic helix-loop-helix and leucine zipper regions (BR-HLH-Zip) (Dang, 1991, Blackwood and Eisenman, 1991). The Myc family includes several members; however, only c-Myc, N-Myc, and L-Myc contribute to the genesis of a wide variety of human tumors (Bishop, 1982, Cole, 1986). Myc exerts its biological functions, at least partly, through the transcriptional regulation of its target genes. The best understood function of Myc is the ability to activate target genes. Myc recognizes the consensus E-box (CACGTG) sequence as part of a heterodimeric complex with its partner protein, Max. The complex recruits several co-activators to DNA and thus contributes to promoter activation. While the activation of genes by Myc is well understood, recent data suggests that the transcriptional repression of target genes might also be an important function of Myc (Wanzel et al., 2003). To date, there have been at least 3 alternative models put forward to explain the mechanism of repression by Myc. However, most data supports the view that Myc is recruited to the core promoters of target genes through protein–protein interactions and indirect binding to DNA. Candidate proteins that have been proposed to recruit Myc to core promoters are TFII-I, YY-1, Sp-1, Miz-1, and NF-Y (Wanzel et al., 2003, Keiko et al., 2003 Wu et al., 2003).
In the present study, we initially demonstrated an inverse regulatory relationship between Myc and NDRG1 gene expression events during induced differentiation and in response to serum growth stimuli. Further, the ectopic expression of Myc represses the expression of NDRG1. However, a protein synthesis inhibitor was able to reverse the inhibition of Myc. Finally, in common with NDRG2, human NDRG1 can be transcriptionally down-regulated by Myc via indirect interaction with the NDRG1 core promoter.
Section snippets
Plasmids
The human NDRG1 promoter was amplified from the BAC clone CTC-458A3 (kindly provided by Dr. Gaiping Wen) using Advantage-GC Genomic Polymerase (Clontech Corporation, Palo Alto, CA). The polymerase chain reaction (PCR) product was cloned into the pGL3-basic vector (Promega, WI) in order to generate an NDRG1/luciferase reporter plasmid. A series of mutants of the human NDRG1 promoter were generated using the PCR method. The identity of each of the newly constructed plasmids was confirmed by
Ndrg1 expression was down-regulated during the processes of cell differentiation and proliferation
It has been previously demonstrated that Myc reduction can be induced during the differentiation of leukemia cells (Tsuneoka et al., 2002, Facchini and Penn, 1998). In order to explore the possible relationship between Myc and the regulation of human NDRG1 expression, we induced the differentiation of leukemia cells U937 and HL-60 with ATRA and analyzed the resulting changes in protein expression. As a marker in monocytes, the expression of CD18 increases rapidly, along with the ATRA-induced
Discussion
As an essential oncogene, Myc controls the cell proliferation and differentiation program by the transcriptional regulation of its target genes (Eisenman, 2001). The expression of mouse ndrg1 is repressed by N-Myc and c-Myc (Shimono et al., 1999), implying that ndrg1 is a potential downstream target gene of Myc. It is possible that the CAC repeat sequence of the core region of mouse ndrg1 is responsible for Myc binding (Shimono et al., 1999). Human Ndrg1, the differentiation-related gene,
Acknowledgments
This work was supported by grants from the National Key Basic Research & Development Program of China (No. 2002CB513007), the National High Technology Research and Development Program of China (863 Program) (No. 2006AA02Z194), the Program for Scholars and Innovative Research Team in University (PCSIRT0459) and the National Natural Science Foundation of China (No. 30700416, No. 30700918, No. 30670452, No. 30600161, No. 30570676 and No. 06G092).
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These authors contribute equally to this work.