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. 1996 Jul;16(7):3245–3254. doi: 10.1128/mcb.16.7.3245

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

V Ngô 1, D Gourdji 1, J N Laverrière 1
PMCID: PMC231318  PMID: 8668139

Abstract

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

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Selected References

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  1. Bach I., Rhodes S. J., Pearse R. V., 2nd, Heinzel T., Gloss B., Scully K. M., Sawchenko P. E., Rosenfeld M. G. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2720–2724. doi: 10.1073/pnas.92.7.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bodner M., Castrillo J. L., Theill L. E., Deerinck T., Ellisman M., Karin M. The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell. 1988 Nov 4;55(3):505–518. doi: 10.1016/0092-8674(88)90037-2. [DOI] [PubMed] [Google Scholar]
  3. Borrelli E., Heyman R. A., Arias C., Sawchenko P. E., Evans R. M. Transgenic mice with inducible dwarfism. Nature. 1989 Jun 15;339(6225):538–541. doi: 10.1038/339538a0. [DOI] [PubMed] [Google Scholar]
  4. Boyes J., Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell. 1991 Mar 22;64(6):1123–1134. doi: 10.1016/0092-8674(91)90267-3. [DOI] [PubMed] [Google Scholar]
  5. Boyes J., Bird A. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. EMBO J. 1992 Jan;11(1):327–333. doi: 10.1002/j.1460-2075.1992.tb05055.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford A. P., Conrad K. E., Wasylyk C., Wasylyk B., Gutierrez-Hartmann A. Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain. Mol Cell Biol. 1995 May;15(5):2849–2857. doi: 10.1128/mcb.15.5.2849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brandeis M., Frank D., Keshet I., Siegfried Z., Mendelsohn M., Nemes A., Temper V., Razin A., Cedar H. Sp1 elements protect a CpG island from de novo methylation. Nature. 1994 Sep 29;371(6496):435–438. doi: 10.1038/371435a0. [DOI] [PubMed] [Google Scholar]
  8. Bryans M., Kass S., Seivwright C., Adams R. L. Vector methylation inhibits transcription from the SV40 early promoter. FEBS Lett. 1992 Aug 31;309(1):97–102. doi: 10.1016/0014-5793(92)80748-6. [DOI] [PubMed] [Google Scholar]
  9. Cherington P. V., Tashjian A. H., Jr Clonal analysis of a 5-azacytidine-induced specific increase in growth hormone production by rat pituitary cells. Endocrinology. 1983 Jul;113(1):418–420. doi: 10.1210/endo-113-1-418. [DOI] [PubMed] [Google Scholar]
  10. Choi Y. C., Chae C. B. DNA hypomethylation and germ cell-specific expression of testis-specific H2B histone gene. J Biol Chem. 1991 Oct 25;266(30):20504–20511. [PubMed] [Google Scholar]
  11. Clark S. J., Harrison J., Paul C. L., Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994 Aug 11;22(15):2990–2997. doi: 10.1093/nar/22.15.2990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Day R. N., Koike S., Sakai M., Muramatsu M., Maurer R. A. Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol. 1990 Dec;4(12):1964–1971. doi: 10.1210/mend-4-12-1964. [DOI] [PubMed] [Google Scholar]
  13. Eden S., Cedar H. Role of DNA methylation in the regulation of transcription. Curr Opin Genet Dev. 1994 Apr;4(2):255–259. doi: 10.1016/s0959-437x(05)80052-8. [DOI] [PubMed] [Google Scholar]
  14. Ehrlich M., Ehrlich K. C. Effect of DNA methylation on the binding of vertebrate and plant proteins to DNA. EXS. 1993;64:145–168. doi: 10.1007/978-3-0348-9118-9_7. [DOI] [PubMed] [Google Scholar]
  15. Elsholtz H. P., Albert V. R., Treacy M. N., Rosenfeld M. G. A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression. Genes Dev. 1990 Jan;4(1):43–51. doi: 10.1101/gad.4.1.43. [DOI] [PubMed] [Google Scholar]
  16. Feil R., Charlton J., Bird A. P., Walter J., Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25;22(4):695–696. doi: 10.1093/nar/22.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frommer M., McDonald L. E., Millar D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Glass C. K., Franco R., Weinberger C., Albert V. R., Evans R. M., Rosenfeld M. G. A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. Nature. 1987 Oct 22;329(6141):738–741. doi: 10.1038/329738a0. [DOI] [PubMed] [Google Scholar]
  19. Gourdji D., Laverrière J. N. The rat prolactin gene: a target for tissue-specific and hormone-dependent transcription factors. Mol Cell Endocrinol. 1994 Apr;100(1-2):133–142. doi: 10.1016/0303-7207(94)90292-5. [DOI] [PubMed] [Google Scholar]
  20. Gutierrez-Hartmann A. INSIGHT: Pit-1/GHF-1: a pituitary-specific transcription factor linking general signaling pathways to cell-specific gene expression. Mol Endocrinol. 1994 Nov;8(11):1447–1449. doi: 10.1210/mend.8.11.7877613. [DOI] [PubMed] [Google Scholar]
  21. Gutierrez-Hartmann A., Siddiqui S., Loukin S. Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5211–5215. doi: 10.1073/pnas.84.15.5211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holliday R. Epigenetic inheritance based on DNA methylation. EXS. 1993;64:452–468. doi: 10.1007/978-3-0348-9118-9_20. [DOI] [PubMed] [Google Scholar]
  23. Hornsby P. J., Yang L. Q., Raju S. G., Cheng C. Y. Changes in gene expression and DNA methylation in adrenocortical cells senescing in culture. Mutat Res. 1991 Mar-Nov;256(2-6):105–113. doi: 10.1016/0921-8734(91)90004-u. [DOI] [PubMed] [Google Scholar]
  24. Hornstra I. K., Yang T. P. High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors. Mol Cell Biol. 1994 Feb;14(2):1419–1430. doi: 10.1128/mcb.14.2.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hsieh C. L. Dependence of transcriptional repression on CpG methylation density. Mol Cell Biol. 1994 Aug;14(8):5487–5494. doi: 10.1128/mcb.14.8.5487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ivarie R. D., Schacter B. S., O'Farrell P. H. The level of expression of the rat growth hormone gene in liver tumor cells is at least eight orders of magnitude less than that in anterior pituitary cells. Mol Cell Biol. 1983 Aug;3(8):1460–1467. doi: 10.1128/mcb.3.8.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jackson S. M., Keech C. A., Williamson D. J., Gutierrez-Hartmann A. Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells. Mol Cell Biol. 1992 Jun;12(6):2708–2719. doi: 10.1128/mcb.12.6.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnson C. A., Goddard J. P., Adams R. L. The effect of histone H1 and DNA methylation on transcription. Biochem J. 1995 Feb 1;305(Pt 3):791–798. doi: 10.1042/bj3050791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jost J. P., Saluz H. P., Pawlak A. Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site. Nucleic Acids Res. 1991 Oct 25;19(20):5771–5775. doi: 10.1093/nar/19.20.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kass S. U., Goddard J. P., Adams R. L. Inactive chromatin spreads from a focus of methylation. Mol Cell Biol. 1993 Dec;13(12):7372–7379. doi: 10.1128/mcb.13.12.7372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Komura J., Okada T., Ono T. Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells. Biochim Biophys Acta. 1995 Jan 2;1260(1):73–78. doi: 10.1016/0167-4781(94)00180-b. [DOI] [PubMed] [Google Scholar]
  32. Larsen P. R., Harney J. W., Moore D. D. Repression mediates cell-type-specific expression of the rat growth hormone gene. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8283–8287. doi: 10.1073/pnas.83.21.8283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laverriere J. N., Morin A., Tixier-Vidal A., Truong A. T., Gourdji D., Martial J. A. Inverse control of prolactin and growth hormone gene expression: effect of thyroliberin on transcription and RNA stabilization. EMBO J. 1983;2(9):1493–1499. doi: 10.1002/j.1460-2075.1983.tb01613.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Laverriere J. N., Muller M., Buisson N., Tougard C., Tixier-Vidal A., Martial J. A., Gourdji D. Differential implication of deoxyribonucleic acid methylation in rat prolactin and rat growth hormone gene expressions: a comparison between rat pituitary cell strains. Endocrinology. 1986 Jan;118(1):198–206. doi: 10.1210/endo-118-1-198. [DOI] [PubMed] [Google Scholar]
  35. Laverrière J. N., Richard J. L., Buisson N., Martial J. A., Tixier-Vidal A., Gourdji D. Thyroliberin and dihydropyridines modulate prolactin gene expression through interacting pathways in GH3 cells. Neuroendocrinology. 1989 Dec;50(6):693–701. doi: 10.1159/000125301. [DOI] [PubMed] [Google Scholar]
  36. Levine A., Cantoni G. L., Razin A. Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10119–10123. doi: 10.1073/pnas.89.21.10119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Levine A., Yeivin A., Ben-Asher E., Aloni Y., Razin A. Histone H1-mediated inhibition of transcription initiation of methylated templates in vitro. J Biol Chem. 1993 Oct 15;268(29):21754–21759. [PubMed] [Google Scholar]
  38. Liang J., Kim K. E., Schoderbek W. E., Maurer R. A. Characterization of a non-tissue-specific, 3',5'-cyclic adenosine monophosphate-responsive element in the proximal region of the rat prolactin gene. Mol Endocrinol. 1992 Jun;6(6):885–892. doi: 10.1210/mend.6.6.1386649. [DOI] [PubMed] [Google Scholar]
  39. Lipkin S. M., När A. M., Kalla K. A., Sack R. A., Rosenfeld M. G. Identification of a novel zinc finger protein binding a conserved element critical for Pit-1-dependent growth hormone gene expression. Genes Dev. 1993 Sep;7(9):1674–1687. doi: 10.1101/gad.7.9.1674. [DOI] [PubMed] [Google Scholar]
  40. Mangalam H. J., Albert V. R., Ingraham H. A., Kapiloff M., Wilson L., Nelson C., Elsholtz H., Rosenfeld M. G. A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally. Genes Dev. 1989 Jul;3(7):946–958. doi: 10.1101/gad.3.7.946. [DOI] [PubMed] [Google Scholar]
  41. Meehan R. R., Lewis J. D., Bird A. P. Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. Nucleic Acids Res. 1992 Oct 11;20(19):5085–5092. doi: 10.1093/nar/20.19.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Meyer P., Niedenhof I., ten Lohuis M. Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida. EMBO J. 1994 May 1;13(9):2084–2088. doi: 10.1002/j.1460-2075.1994.tb06483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nelson C., Albert V. R., Elsholtz H. P., Lu L. I., Rosenfeld M. G. Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science. 1988 Mar 18;239(4846):1400–1405. doi: 10.1126/science.2831625. [DOI] [PubMed] [Google Scholar]
  44. Ngô V. M., Laverrière J. N., Gourdji D. CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines. Mol Cell Endocrinol. 1995 Feb 27;108(1-2):95–105. doi: 10.1016/0303-7207(94)03462-3. [DOI] [PubMed] [Google Scholar]
  45. Ngô V., Laverrière J. N., Gourdji D. Binding capacity and cis-acting efficiency of DNA regulatory sequences can be distinguished in an in vivo competition assay. Nucleic Acids Res. 1993 Dec 11;21(24):5795–5796. doi: 10.1093/nar/21.24.5795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Okimura Y., Howard P. W., Maurer R. A. Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of Pit-1. Mol Endocrinol. 1994 Nov;8(11):1559–1565. doi: 10.1210/mend.8.11.7877624. [DOI] [PubMed] [Google Scholar]
  47. Pan W. T., Liu Q. R., Bancroft C. Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins. J Biol Chem. 1990 Apr 25;265(12):7022–7028. [PubMed] [Google Scholar]
  48. Park J. G., Chapman V. M. CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene. Mol Cell Biol. 1994 Dec;14(12):7975–7983. doi: 10.1128/mcb.14.12.7975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pfeifer G. P., Steigerwald S. D., Hansen R. S., Gartler S. M., Riggs A. D. Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8252–8256. doi: 10.1073/pnas.87.21.8252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Pichon B., Christophe-Hobertus C., Vassart G., Christophe D. Unmethylated thyroglobulin promoter may be repressed by methylation of flanking DNA sequences. Biochem J. 1994 Mar 15;298(Pt 3):537–541. doi: 10.1042/bj2980537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rajnarayan S., Chiono M., Alexander L. M., Gutierrez-Hartmann A. Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms. Mol Endocrinol. 1995 Apr;9(4):502–512. doi: 10.1210/mend.9.4.7659093. [DOI] [PubMed] [Google Scholar]
  52. Reddy P. M., Stamatoyannopoulos G., Papayannopoulou T., Shen C. K. Genomic footprinting and sequencing of human beta-globin locus. Tissue specificity and cell line artifact. J Biol Chem. 1994 Mar 18;269(11):8287–8295. [PubMed] [Google Scholar]
  53. Rosenfeld M. G. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev. 1991 Jun;5(6):897–907. doi: 10.1101/gad.5.6.897. [DOI] [PubMed] [Google Scholar]
  54. Roy R. J., Vallières L., Leclerc S., Guérin S. L. The rat growth hormone proximal silencer contains a novel DNA-binding site for multiple nuclear proteins that represses basal promoter activity. Eur J Biochem. 1994 Oct 1;225(1):419–432. doi: 10.1111/j.1432-1033.1994.00419.x. [DOI] [PubMed] [Google Scholar]
  55. Saluz H. P., Jiricny J., Jost J. P. Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7167–7171. doi: 10.1073/pnas.83.19.7167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Santoro R., D'Erme M., Mastrantonio S., Reale A., Marenzi S., Saluz H. P., Strom R., Caiafa P. Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation. Biochem J. 1995 Feb 1;305(Pt 3):739–744. doi: 10.1042/bj3050739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schaufele F., Cassill J. A., West B. L., Reudelhuber T. Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter. J Biol Chem. 1990 Aug 25;265(24):14592–14598. [PubMed] [Google Scholar]
  58. Seidah N. G., Barale J. C., Marcinkiewicz M., Mattei M. G., Day R., Chrétien M. The mouse homeoprotein mLIM-3 is expressed early in cells derived from the neuroepithelium and persists in adult pituitary. DNA Cell Biol. 1994 Dec;13(12):1163–1180. doi: 10.1089/dna.1994.13.1163. [DOI] [PubMed] [Google Scholar]
  59. Shmookler Reis R. J., Goldstein S. Variability of DNA methylation patterns during serial passage of human diploid fibroblasts. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3949–3953. doi: 10.1073/pnas.79.13.3949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Silva A. J., Ward K., White R. Mosaic methylation in clonal tissue. Dev Biol. 1993 Apr;156(2):391–398. doi: 10.1006/dbio.1993.1086. [DOI] [PubMed] [Google Scholar]
  61. Simmons D. M., Voss J. W., Ingraham H. A., Holloway J. M., Broide R. S., Rosenfeld M. G., Swanson L. W. Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev. 1990 May;4(5):695–711. doi: 10.1101/gad.4.5.695. [DOI] [PubMed] [Google Scholar]
  62. Szyf M., Rouleau J., Theberge J., Bozovic V. Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation. J Biol Chem. 1992 Jun 25;267(18):12831–12836. [PubMed] [Google Scholar]
  63. Tate P. H., Bird A. P. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev. 1993 Apr;3(2):226–231. doi: 10.1016/0959-437x(93)90027-m. [DOI] [PubMed] [Google Scholar]
  64. Theill L. E., Karin M. Transcriptional control of GH expression and anterior pituitary development. Endocr Rev. 1993 Dec;14(6):670–689. doi: 10.1210/edrv-14-6-670. [DOI] [PubMed] [Google Scholar]
  65. Toth M., Lichtenberg U., Doerfler W. Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns. Proc Natl Acad Sci U S A. 1989 May;86(10):3728–3732. doi: 10.1073/pnas.86.10.3728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Voss J. W., Wilson L., Rosenfeld M. G. POU-domain proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can cooperate to induce expression of the prolactin promoter. Genes Dev. 1991 Jul;5(7):1309–1320. doi: 10.1101/gad.5.7.1309. [DOI] [PubMed] [Google Scholar]
  67. Zhang Z. X., Kumar V., Rivera R. T., Pasion S. G., Chisholm J., Biswas D. K. Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation. DNA. 1989 Oct;8(8):605–613. doi: 10.1089/dna.1989.8.605. [DOI] [PubMed] [Google Scholar]

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