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Cancer Sci
2018 Feb 01;1092:395-402. doi: 10.1111/cas.13466.
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Regulation of c-MYC transcriptional activity by transforming growth factor-beta 1-stimulated clone 22.
Zheng L, Suzuki H, Nakajo Y, Nakano A, Kato M.
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c-MYC stimulates cell proliferation through the suppression of cyclin-dependent kinase (CDK) inhibitors including P15 (CDKN2B) and P21 (CDKN1A). It also activates E-box-mediated transcription of various target genes including telomerase reverse transcriptase (TERT) that is involved in cellular immortality and tumorigenesis. Transforming growth factor-beta 1 (TGF-β1)-stimulated clone 22 (TSC-22/TSC22D1) encodes a highly conserved leucine zipper protein that is induced by various stimuli, including TGF-β. TSC-22 inhibits cell growth in mammalian cells and in Xenopus embryos. However, underlying mechanisms of growth inhibition by TSC-22 remain unclear. Here, we show that TSC-22 physically interacts with c-MYC to inhibit the recruitment of c-MYC on the P15 (CDKN2B) and P21 (CDKN1A) promoters, effectively inhibiting c-MYC-mediated suppression of P15 (CDKN2B) and also P21 (CDKN1A) promoter activities. In contrast, TSC-22 enhances c-MYC-mediated activation of the TERT promoter. Additionally, the expression of TSC-22 in embryonic stem cells inhibits cell growth without affecting its pluripotency-related gene expression. These results indicate that TSC-22 differentially regulates c-MYC-mediated transcriptional activity to regulate cell proliferation.
Figure 1.
TSC‐22 inhibits cell proliferation. A, Immunoblot analysis showing expression of TSC‐22, c‐MYC, and α‐TUBULIN in FLAG‐TSC‐22‐expressing HaCaT cells (clones #11 and #17), as indicated. B, Cell growth of Mock and FLAG‐TSC‐22‐expressing HaCaT cells (clones #11 and #17). Mean ± SD, n = 3. *P < .05, **P < .01 (on days 5 and 6). C‐E, Mouse ES cells (MGZ5 cells) were transfected with Mock, FLAG‐TSC‐22, and FLAG‐ID2 expression plasmids, as indicated, transferred to 6‐cm dishes and cultured for 6 d in the presence of puromycin. C, Morphology of colonies transfected with Mock, FLAG‐TSC‐22, and FLAG‐ID2 cDNAs, as indicated. D, Whole view of the colonies in 6‐cm dishes stained with crystal violet. E, Number of colonies. Mean ± SD. n = 3
Figure 2.
TSC‐22 interacts with c‐MYC in HaCaT cells. A, Interaction of TSC‐22 and c‐MYC in Mock‐ and FLAG‐TSC‐22‐expressing HaCaT cells (clone #17). Cell lysates were immunoprecipitated with anti‐FLAG antibody (IP), followed by immunoblotting using antibodies as indicated (Blot). IgG (H), immunoglobulin heavy chain used for IP. B, Localization of TSC‐22 in FLAG‐TSC‐22‐expressing HaCaT cells (clone #17). Cells were cultured on coverslips and stained with anti‐TSC‐22 and c‐MYC antibodies. Nuclei were counterstained with DAPI
Figure 3. Effects of TSC‐22 on c‐MYC‐mediated transcriptional activation and repression. A, ChIP analysis of c‐MYC binding to the P15, P21, and TERT promoters in Mock‐ and FLAG‐TSC‐22‐expressing HaCaT cells (clone #17). Control IgG was used as a negative control. B‐D, 293T cells were transfected with the luciferase reporter constructs; (B) P15 (CDKN2B)‐luc, (C) WWP (CDKN1A)‐luc, and (D) hTERT E‐box2‐luc, along with various combinations of c‐MYC and TSC‐22 expression plasmids, and luciferase activity was measured. Mean ± SD. n = 3. E, Expression of P15, P21, and TERT
mRNA in Mock‐ and FLAG‐TSC‐22‐expressing HaCaT cells (clone #11 and #17). β‐actin was used as a loading control
Figure 4. Domains required for TSC‐22 and c‐MYC interaction. A,C,D,E, 293T cells were transfected with expression plasmids, as indicated. Cell lysates were immunoprecipitated with anti‐FLAG antibody (IP), followed by immunoblotting using antibodies as indicated (Blot). A, Leucine zipper domain (LZ) of c‐MYC is required for TSC‐22 and c‐MYC interaction. IgG (H), IgG heavy chain used for IP. B, Schematic representation of the structures of TSC‐22 wild‐type (WT), deletion mutants of leucine zipper domain (ΔLZ), TGF‐β1 stimulated clone 22 box (ΔTSC), N‐terminal part (ΔN), C‐terminal part (ΔC), and four leucine‐to‐alanine mutations in the LZ domain (4LA). C, Interaction of c‐MYC and WT or deletion mutants of TSC‐22. N‐terminal part and LZ domain of TSC‐22 are required for TSC‐22‐c‐MYC interaction. D, Homodimer formation of FLAG‐TSC‐22 WT and HA‐TSC‐22 WT or HA‐TSC‐22 4LA, as indicated. E, Heterodimer formation of HA‐c‐MYC and FLAG‐TSC‐22 WT or FLAG‐TSC‐22 4LA, as indicated. F, P15‐luc activity suppressive effects of c‐MYC are canceled by TSC‐22 WT but not by TSC‐22 ΔLZ. 293T cells were transfected with the luciferase construct P15 (CDKN2B)‐luc along with various combinations of the indicated expression plasmids, and luciferase activity was measured. Mean ± SD. n = 3. *P < .05, **P < .01
Figure 5. Effects of TSC‐22 on c‐MYC‐MAX and c‐MYC‐MIZ‐1 heterodimer formation. A‐C, 293T cells were transfected with expression plasmids, as indicated. Cell lysates were immunoprecipitated with anti‐FLAG antibody (IP), followed by immunoblotting using antibodies as indicated (Blot). IgG (H) and IgG (L), IgG heavy chain and light chain used for IP, respectively. A, Interaction of TSC‐22 with c‐MYC but not with MIZ‐1 and MAX. B, TSC‐22 enhances c‐MYC‐MAX interaction and MAX competes with TSC‐22 for c‐MYC binding. C, TSC‐22 suppresses c‐MYC‐MIZ‐1 interaction
Bouchard,
Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.
1999, Pubmed
Bouchard,
Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.
1999,
Pubmed Claassen,
A role for transcriptional repression of p21CIP1 by c-Myc in overcoming transforming growth factor beta -induced cell-cycle arrest.
2000,
Pubmed Clevers,
The cancer stem cell: premises, promises and challenges.
2011,
Pubmed Desbarats,
Discrimination between different E-box-binding proteins at an endogenous target gene of c-myc.
1996,
Pubmed Dohrmann,
Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.
1999,
Pubmed el-Deiry,
WAF1, a potential mediator of p53 tumor suppression.
1993,
Pubmed Grandori,
c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I.
2005,
Pubmed Hashiguchi,
Role of TSC-22 during early embryogenesis in Xenopus laevis.
2004,
Pubmed
,
Xenbase Jones,
Rapid cytoplasmic turnover of c-myc mRNA: requirement of the 3' untranslated sequences.
1987,
Pubmed Kelly,
Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor.
1983,
Pubmed Kester,
Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity.
1999,
Pubmed Kohl,
Transposition and amplification of oncogene-related sequences in human neuroblastomas.
1983,
Pubmed Kondo,
A role for Id in the regulation of TGF-beta-induced epithelial-mesenchymal transdifferentiation.
2004,
Pubmed Lasorella,
Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins.
2000,
Pubmed Li,
Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site.
1995,
Pubmed Lüscher,
New light on Myc and Myb. Part I. Myc.
1990,
Pubmed Mukherjee,
Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and trans-acting dominant mutants.
1992,
Pubmed Nair,
X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.
2003,
Pubmed Nakamura,
Transforming growth factor-β-stimulated clone-22 is a negative-feedback regulator of Ras / Raf signaling: Implications for tumorigenesis.
2012,
Pubmed Nakashiro,
Down-regulation of TSC-22 (transforming growth factor beta-stimulated clone 22) markedly enhances the growth of a human salivary gland cancer cell line in vitro and in vivo.
1998,
Pubmed Nau,
L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer.
,
Pubmed Niwa,
Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells.
2002,
Pubmed Ogawa,
Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells.
2007,
Pubmed Rentsch,
Differential expression of TGFbeta-stimulated clone 22 in normal prostate and prostate cancer.
2006,
Pubmed Shibanuma,
Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.
1992,
Pubmed Shostak,
Downregulation of putative tumor suppressor gene TSC-22 in human brain tumors.
2003,
Pubmed Staller,
Repression of p15INK4b expression by Myc through association with Miz-1.
2001,
Pubmed Suzuki,
c-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements.
2004,
Pubmed Taub,
Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells.
1982,
Pubmed Wu,
Direct activation of TERT transcription by c-MYC.
1999,
Pubmed Wu,
Myc represses differentiation-induced p21CIP1 expression via Miz-1-dependent interaction with the p21 core promoter.
2003,
Pubmed Yagi,
c-myc is a downstream target of the Smad pathway.
2002,
Pubmed Ying,
BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.
2003,
Pubmed Yu,
TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia.
2009,
Pubmed Zheng,
Regulation of c-MYC transcriptional activity by transforming growth factor-beta 1-stimulated clone 22.
2018,
Pubmed
,
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