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Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression

Abstract

Microsatellite instability (MSI) is caused by the dysfunction of mismatch repair genes, such as hMLH1, hMSH2. Loss of hMLH1 expression and methylation of CpG sites in hMLH1 promoter are frequently present in sporadic colorectal cancer with MSI. In this study, by transient transfection assay with constructs containing different lengths of hMLH1 promoter and a luciferase reporter gene, we located a proximal region of hMLH1 promoter, which plays a main role in regulating the gene. The fact that luciferase activities were high in all host cell lines regardless of their hMLH1 expression levels indicates that the transcription machinery is intact even in non-expressing cells. When hMLH1 promoter was in vitro methylated before transfection, the luciferase activities in the transfectants were significantly reduced. This observation indicates that methylation causes the inhibition of hMLH1 promoter activity. By electrophoretic mobility shift assay (EMSA), we identified a CCAAT box in this region, which specifically bound transcription factor CBF. Mutations in CCAAT box not only inhibited its binding to CBF factor, but also reduced its ability to drive the expression of luciferase gene. The role of CBF in activating transcription was further substantiated by inhibition of promoter activity with a plasmid expressing a dominant negative CBF-B mutant. Methylation at a CpG site two base pairs upstream of the CCAAT box inhibited the binding of CBF to CCAAT box. We conclude that methylation of an adjacent CpG site inhibits binding of the CBF transcription to the corresponding CCAAT box, and is one of the causes of hMLH1 gene silencing in colon cancer cells.

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References

  • Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin J-P, Jarvinen H, Powell SM, Jen J, Hamilton SR, Petersen GH, Kinzler KW, Vogelstein B, de la Chapelle A . 1993 Science 260: 812–816

  • Andrews NC, Faller DV . 1991 Nucl. Acids Res. 19: 2499

  • Cunningham JM, Chrislensen ER, Tester D, Kim C-Y, Roche PC, Burgart LJ, Thibodeau SN . 1998 Cancer Res. 58: 3455–3460

  • Currie RA . 1997 J. Biol. Chem. 272: 30880–30888

  • Deng G, Chen A, Hong J, Chae HS, Kim YS . 1999 Cancer Res. 59: 2029–2033

  • Dobrovic A, Simpfendorfer D . 1997 Cancer Res. 57: 3347–3350

  • Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, Samid D, Duan DSR, Gnarra JR, Linehan WM, Baylin SB . 1994 Proc. Natl. Acad. Sci. USA 91: 9700–9704

  • Herman JG, Merlo A, Mao L, Lapidus RG, Issa J-PJ, Davidson NE, Sidransky D, Baylin SB . 1995 Cancer Res. 55: 4525–4530

  • Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa J-PJ, Markowitz S, Willson JKV, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB . 1998 Proc. Natl. Acad. Sci. USA 95: 6870–6875

  • Hiltunen MO, Alhonen L, Koistinaho J, Myohanen S, Paakkonen M, Marin S, Kosma VM, Janne J . 1997 Int. J. Cancer 70: 644–648

  • Hu Q, Maity SN . 2000 J. Biol. Chem. 275: 4435–4444

  • Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB . 1994 Nat. Genet. 7: 536–540

  • Jones PA, Laird PW . 1999 Nat. Genet. 21: 163–166

  • Kanai Y, Ushijima S, Hui AM, Ochiai A, Tsuda H, Sakamoto M, Hirohashi S . 1997 Int. J. Cancer 71: 355–359

  • Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner R . 1997 Cancer Res. 57: 808–811

  • Kinzler KA, Vogelstein B . 1996 Cell 87: 159–170

  • Kominato Y, Tsuchiya T, Hata N, Takizawa H, Yamamoto F . 1997 J. Biol. Chem. 272: 25890–25898

  • Mantovani R . 1998 Nucl. Acids Res. 26: 1135–1143

  • Mantovani R, Li X-Y, Pessara U, van Huisjduijnen RH, Benoist C, Mathis D . 1994 J. Biol. Chem. 269: 20340–20346

  • Marziali G, Perrotti E, Ilari R, Testa U, Caccia EM, Battistini A . 1997 Mol. Cell. Biol. 17: 1387–1395

  • Mathey-Prevot B, Andrews NC, Murphy HS, Kreissan SG, Nathan DG . 1990 Proc. Natl. Acad. Sci. USA 87: 5046–5050

  • Orita T, Shimozaki K, Murakami H, Nagata S . 1997 J. Biol. Chem. 272: 23216–23223

  • Papadopoulos N, Nicolaides NC, Wei Y-F, Ruben SM, Carter KC, Rosen CA, Haseltine WA, Fleischmann RD, Fraser CM, Adams MD, Venter JC, Hamilton SR, Petersen GM, Watson P, Lynch HT, Peltomaki P, Mecklin J-P, de la Chapelle A, Kinzler KW, Vogelstein B . 1994 Science 263: 1625–1629

  • Qian XC, Brent TP . 1997 Cancer Res. 57: 3672–3677

  • Sinha S, Kim I-S, Sohn K-Y, de Crombrugghe B, Maity SN . 1996 Mol. Cell. Biol. 16: 328–337

  • Stirzaker C, Millar DS, Paul CL, Warnecke PM, Harrison J, Vincent PC, Frommer M, Clark SJ . 1997 Cancer Res. 57: 2229–2237

  • Tanaka T, Shimada Y, Harada H, Shinoda M, Hatooka S, Imamura M, Ishizaki K . 1998 Cancer Res. 58: 3429–3434

  • Tate PH, Bird AP . 1993 Curr. Opin. Genet. Dev. 3: 226–231

  • Thibodeau SN, Bren G, Schaid D . 1993 Science 260: 816–819

  • Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM, Moslein G, Baker SM, Liskay RM, Burgart LJ, Honchel R, Halling KC . 1996 Cancer Res. 56: 4836–4840

  • Veigl ML, Kasturi L, Olechnowicz J, Ma A, Lutterbaugh JD, Periyasamy S, Li G, Drummond J, Modrich PL, Sedwick WD, Markowitz SD . 1998 Proc. Natl. Acad. Sci. USA 95: 8698–8702

  • Xiong Z, Laird PW . 1997 Nucl. Acids Res. 25: 2532–2534

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Acknowledgements

We thank Drs Suzanne Crawley, Roger Erickson and James Gum for helpful discussions, suggestions and editorial assistance during the course of this work. We also thank Drs Sanker N Maity and Qianghua Hu for providing us with the plasmid carrying a dominant negative CBF mutant. This work was supported by the Theodora Betz Foundation, the Department of Veteran Affairs Medical Research Service and by Grant 24321 from the US Public Health Service, National Cancer Institute.

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Correspondence to Guoren Deng.

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Deng, G., Chen, A., Pong, E. et al. Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression. Oncogene 20, 7120–7127 (2001). https://doi.org/10.1038/sj.onc.1204891

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Keywords

  • colorectal cancer
  • hMLH1 gene
  • methylation
  • transcription factor CBF

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