Site Specific Demethylation in the Promoter of Human Gamma-globin Gene Does Not Alleviate Methylation Mediated Suppression

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1987 Aug 1

PMID 3665878

Citations 35

Authors

E J Murray

F Grosveld

Affiliations

Soon will be listed here.

Abstract

Human gamma A-globin genes with different patterns of methylation were synthesized in vitro. These methylated constructs were transfected into mouse L cells and their expression analyzed by S1-nuclease protection assays. We show that methylation downstream of +92 does not affect transcription while methylation downstream of -210 abolishes transcription. To test which of the six CpG dinucleotides between -210 and +92 plays a role in DNA methylation mediated suppression of transcription, we generated mutant gamma-globin genes which lack the three CpGs 5' to the cap site, the three CpGs 3' to the cap site, or all six CpGs. After in vitro methylation, we show that removal of methylated residues 5' or 3' to the cap site did not alleviate suppression, whereas removal of both 5' and 3' methylated residues restored partial expression. These results indicate that CpG methylation at different positions can cause suppression of expression and they are inconsistent with the hypothesis that site-specific demethylations in the promoter region can alleviate suppression. We propose that a minimum length of methylation-free DNA in the promoter area is the only requirement for expression of this promoter by ubiquitous transcription factors, and discuss these results with reference to methylation-free islands.

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References

Sneider T, POTTER V . Methylation of mammalian DNA: studies on Novikoff hepatoma cells in tissue culture. J Mol Biol. 1969; 42(2):271-84. DOI: 10.1016/0022-2836(69)90043-6. View

Wang R, Zhang X, Khan R, Zhou Y, Huang L, Ehrlich M . Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs. Nucleic Acids Res. 1986; 14(24):9843-60. PMC: 341339. DOI: 10.1093/nar/14.24.9843. View

Waalwijk C, Flavell R . DNA methylation at a CCGG sequence in the large intron of the rabbit beta-globin gene: tissue-specific variations. Nucleic Acids Res. 1978; 5(12):4631-4. PMC: 342778. DOI: 10.1093/nar/5.12.4631. View

Van der Ploeg L, Flavell R . DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980; 19(4):947-58. DOI: 10.1016/0092-8674(80)90086-0. View

Slightom J, Blechl A, SMITHIES O . Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell. 1980; 21(3):627-38. DOI: 10.1016/0092-8674(80)90426-2. View

Shen C, Maniatis T . Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes. Proc Natl Acad Sci U S A. 1980; 77(11):6634-8. PMC: 350341. DOI: 10.1073/pnas.77.11.6634. View

Gruenbaum Y, Stein R, Cedar H, Razin A . Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 1981; 124(1):67-71. DOI: 10.1016/0014-5793(81)80055-5. View

Wasylyk B, Derbyshire R, Guy A, Molko D, Roget A, Teoule R . Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence. Proc Natl Acad Sci U S A. 1980; 77(12):7024-8. PMC: 350433. DOI: 10.1073/pnas.77.12.7024. View

Wallace R, Schold M, Johnson M, Dembek P, Itakura K . Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981; 9(15):3647-56. PMC: 327381. DOI: 10.1093/nar/9.15.3647. View

10.

Gruenbaum Y, Cedar H, Razin A . Substrate and sequence specificity of a eukaryotic DNA methylase. Nature. 1982; 295(5850):620-2. DOI: 10.1038/295620a0. View

11.

Shen S, Slightom J, SMITHIES O . A history of the human fetal globin gene duplication. Cell. 1981; 26(2 Pt 2):191-203. DOI: 10.1016/0092-8674(81)90302-0. View

12.

Stein R, Razin A, Cedar H . In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc Natl Acad Sci U S A. 1982; 79(11):3418-22. PMC: 346431. DOI: 10.1073/pnas.79.11.3418. View

13.

McKeon C, Ohkubo H, Pastan I, de Crombrugghe B . Unusual methylation pattern of the alpha 2 (l) collagen gene. Cell. 1982; 29(1):203-10. DOI: 10.1016/0092-8674(82)90104-0. View

14.

Groudine M, Weintraub H . Propagation of globin DNAase I-hypersensitive sites in absence of factors required for induction: a possible mechanism for determination. Cell. 1982; 30(1):131-9. DOI: 10.1016/0092-8674(82)90019-8. View

15.

Ott M, Sperling L, Cassio D, Levilliers J, Weiss M . Undermethylation at the 5' end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture. Cell. 1982; 30(3):825-33. DOI: 10.1016/0092-8674(82)90287-2. View

16.

Grosveld F, Lund T, Murray E, Mellor A, Dahl H, Flavell R . The construction of cosmid libraries which can be used to transform eukaryotic cells. Nucleic Acids Res. 1982; 10(21):6715-32. PMC: 326959. DOI: 10.1093/nar/10.21.6715. View

17.

Stein R, Razin A, Cedar H . Pattern of methylation of two genes coding for housekeeping functions. Proc Natl Acad Sci U S A. 1983; 80(9):2422-6. PMC: 393837. DOI: 10.1073/pnas.80.9.2422. View

18.

Cate R, Chick W, Gilbert W . Comparison of the methylation patterns of the two rat insulin genes. J Biol Chem. 1983; 258(10):6645-52. View

19.

Busslinger M, Hurst J, Flavell R . DNA methylation and the regulation of globin gene expression. Cell. 1983; 34(1):197-206. DOI: 10.1016/0092-8674(83)90150-2. View

20.

Munson L, Mandecki W, Caruthers M, Reznikoff W . Oligonucleotide mutagenesis of the lacPUV5 promoter. Nucleic Acids Res. 1984; 12(9):4011-7. PMC: 318807. DOI: 10.1093/nar/12.9.4011. View