Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Jan;5(1):214–225. doi: 10.1128/mcb.5.1.214

A domain of methylation change at the albumin locus in rat hepatoma cell variants.

A Orlofsky, L A Chasin
PMCID: PMC366696  PMID: 2984551

Abstract

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.

Full text

PDF
214

Images in this article

218Image
on p.218
219Image
on p.219
220Image
on p.220

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bird A., Taggart M., Macleod D. Loss of rDNA methylation accompanies the onset of ribosomal gene activity in early development of X. laevis. Cell. 1981 Nov;26(3 Pt 1):381–390. doi: 10.1016/0092-8674(81)90207-5. [DOI] [PubMed] [Google Scholar]
  3. Burch J. B., Weintraub H. Temporal order of chromatin structural changes associated with activation of the major chicken vitellogenin gene. Cell. 1983 May;33(1):65–76. doi: 10.1016/0092-8674(83)90335-5. [DOI] [PubMed] [Google Scholar]
  4. Busslinger M., Hurst J., Flavell R. A. DNA methylation and the regulation of globin gene expression. Cell. 1983 Aug;34(1):197–206. doi: 10.1016/0092-8674(83)90150-2. [DOI] [PubMed] [Google Scholar]
  5. Cassio D., Weiss M. C., Ott M. O., Sala-Trepat J. M., Friès J., Erdos T. Expression of the albumin gene in rat hepatoma cells and their dedifferentiated variants. Cell. 1981 Dec;27(2 Pt 1):351–358. doi: 10.1016/0092-8674(81)90418-9. [DOI] [PubMed] [Google Scholar]
  6. Cate R. L., Chick W., Gilbert W. Comparison of the methylation patterns of the two rat insulin genes. J Biol Chem. 1983 May 25;258(10):6645–6652. [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Compere S. J., Palmiter R. D. DNA methylation controls the inducibility of the mouse metallothionein-I gene lymphoid cells. Cell. 1981 Jul;25(1):233–240. doi: 10.1016/0092-8674(81)90248-8. [DOI] [PubMed] [Google Scholar]
  9. Cooper D. N., Taggart M. H., Bird A. P. Unmethylated domains in vertebrate DNA. Nucleic Acids Res. 1983 Feb 11;11(3):647–658. doi: 10.1093/nar/11.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deeley R. G., Udell D. S., Burns A. T., Gordon J. I., Goldberger R. F. Kinetics of avian vitellogenin messenger RNA induction. Comparison between primary and secondary response to estrogen. J Biol Chem. 1977 Nov 25;252(22):7913–7915. [PubMed] [Google Scholar]
  11. Derman E., Krauter K., Walling L., Weinberger C., Ray M., Darnell J. E., Jr Transcriptional control in the production of liver-specific mRNAs. Cell. 1981 Mar;23(3):731–739. doi: 10.1016/0092-8674(81)90436-0. [DOI] [PubMed] [Google Scholar]
  12. Deschatrette J., Weiss M. C. Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie. 1974;56(11-12):1603–1611. doi: 10.1016/s0300-9084(75)80286-0. [DOI] [PubMed] [Google Scholar]
  13. Deschatrette J., Weiss M. C. Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells. Somatic Cell Genet. 1975 Jul;1(3):279–292. doi: 10.1007/BF01538451. [DOI] [PubMed] [Google Scholar]
  14. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  15. Fradin A., Manley J. L., Prives C. L. Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5142–5146. doi: 10.1073/pnas.79.17.5142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ganschow R. Glucuronidase gene expression in somatic hybrids. Science. 1966 Jul 1;153(3731):84–85. doi: 10.1126/science.153.3731.84. [DOI] [PubMed] [Google Scholar]
  17. Gautsch J. W., Wilson M. C. Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression. Nature. 1983 Jan 6;301(5895):32–37. doi: 10.1038/301032a0. [DOI] [PubMed] [Google Scholar]
  18. Gerber-Huber S., May F. E., Westley B. R., Felber B. K., Hosbach H. A., Andres A. C., Ryffel G. U. In contrast to other Xenopus genes the estrogen-inducible vitellogenin genes are expressed when totally methylated. Cell. 1983 May;33(1):43–51. doi: 10.1016/0092-8674(83)90333-1. [DOI] [PubMed] [Google Scholar]
  19. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  20. Green H., Ephrussi B., Yoshida M., Hamerman D. Synthesis of collagen and hyaluronic acid by fibroblast hybrids. Proc Natl Acad Sci U S A. 1966 Jan;55(1):41–44. doi: 10.1073/pnas.55.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  22. Gruenbaum Y., Cedar H., Razin A. Substrate and sequence specificity of a eukaryotic DNA methylase. Nature. 1982 Feb 18;295(5850):620–622. doi: 10.1038/295620a0. [DOI] [PubMed] [Google Scholar]
  23. Harpold M. M., Evans R. M., Salditt-Georgieff M., Darnell J. E. Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 1979 Aug;17(4):1025–1035. doi: 10.1016/0092-8674(79)90341-6. [DOI] [PubMed] [Google Scholar]
  24. Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
  25. Kapuściński J., Skoczylas B. Simple and rapid fluorimetric method for DNA microassay. Anal Biochem. 1977 Nov;83(1):252–257. doi: 10.1016/0003-2697(77)90533-4. [DOI] [PubMed] [Google Scholar]
  26. Kruczek I., Doerfler W. The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression. EMBO J. 1982;1(4):409–414. doi: 10.1002/j.1460-2075.1982.tb01183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kunnath L., Locker J. Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes. EMBO J. 1983;2(3):317–324. doi: 10.1002/j.1460-2075.1983.tb01425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laurell C. B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem. 1966 Apr;15(1):45–52. doi: 10.1016/0003-2697(66)90246-6. [DOI] [PubMed] [Google Scholar]
  29. Levitt A., Axel R., Cedar H. Nick translation of active genes in intact nuclei. Dev Biol. 1979 Apr;69(2):496–505. doi: 10.1016/0012-1606(79)90307-5. [DOI] [PubMed] [Google Scholar]
  30. Mandel J. L., Chambon P. DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes. Nucleic Acids Res. 1979 Dec 20;7(8):2081–2103. doi: 10.1093/nar/7.8.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  32. Meijlink F. C., Philipsen J. N., Gruber M., Ab G. Methylation of the chicken vitellogenin gene: influence of estradiol administration. Nucleic Acids Res. 1983 Mar 11;11(5):1361–1373. doi: 10.1093/nar/11.5.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Naveh-Many T., Cedar H. Active gene sequences are undermethylated. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4246–4250. doi: 10.1073/pnas.78.7.4246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ott M. O., Sperling L., Cassio D., Levilliers J., Sala-Trepat J., Weiss M. C. 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 Oct;30(3):825–833. doi: 10.1016/0092-8674(82)90287-2. [DOI] [PubMed] [Google Scholar]
  35. Ott M. O., Sperling L., Weiss M. C. Albumin extinction without methylation of its gene. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1738–1741. doi: 10.1073/pnas.81.6.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rave N., Crkvenjakov R., Boedtker H. Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 1979 Aug 10;6(11):3559–3567. doi: 10.1093/nar/6.11.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  38. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  39. Riggs A. D., Jones P. A. 5-methylcytosine, gene regulation, and cancer. Adv Cancer Res. 1983;40:1–30. doi: 10.1016/s0065-230x(08)60678-8. [DOI] [PubMed] [Google Scholar]
  40. Rosenstraus M. J., Chasin L. A. Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity. Somatic Cell Genet. 1977 May;3(3):323–333. doi: 10.1007/BF01538750. [DOI] [PubMed] [Google Scholar]
  41. Sala-Trepat J. M., Dever J., Sargent T. D., Thomas K., Sell S., Bonner J. Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia. Biochemistry. 1979 May 29;18(11):2167–2178. doi: 10.1021/bi00578a006. [DOI] [PubMed] [Google Scholar]
  42. Sargent T. D., Jagodzinski L. L., Yang M., Bonner J. Fine structure and evolution of the rat serum albumin gene. Mol Cell Biol. 1981 Oct;1(10):871–883. doi: 10.1128/mcb.1.10.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sargent T. D., Wu J. R., Sala-Trepat J. M., Wallace R. B., Reyes A. A., Bonner J. The rat serum albumin gene: analysis of cloned sequences. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3256–3260. doi: 10.1073/pnas.76.7.3256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sargent T. D., Yang M., Bonner J. Nucleotide sequence of cloned rat serum albumin messenger RNA. Proc Natl Acad Sci U S A. 1981 Jan;78(1):243–246. doi: 10.1073/pnas.78.1.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Soreq H., Harpold M., Wilson M., Darnell J. E., Jr Rate of synthesis and concentration of specific mRNA sequences in cultured Chinese hamster ovary cells compared to liver cells. Biochem Biophys Res Commun. 1980 Jan 29;92(2):485–491. doi: 10.1016/0006-291x(80)90359-9. [DOI] [PubMed] [Google Scholar]
  46. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  47. Spradling A. C., Digan M. E., Mahowald A. P., Scott M., Craig E. A. Two clusters of genes for major chorion proteins of Drosophila melanogaster. Cell. 1980 Apr;19(4):905–914. doi: 10.1016/0092-8674(80)90082-3. [DOI] [PubMed] [Google Scholar]
  48. Stuhlmann H., Jähner D., Jaenisch R. Infectivity and methylation of retroviral genomes is correlated with expression in the animal. Cell. 1981 Oct;26(2 Pt 2):221–232. doi: 10.1016/0092-8674(81)90305-6. [DOI] [PubMed] [Google Scholar]
  49. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weintraub H., Larsen A., Groudine M. Alpha-Globin-gene switching during the development of chicken embryos: expression and chromosome structure. Cell. 1981 May;24(2):333–344. doi: 10.1016/0092-8674(81)90323-8. [DOI] [PubMed] [Google Scholar]
  52. Weiss M. C., Chaplain M. Expression of differentiated functions in hepatoma cell hybrids: reappearance of tyrosine aminotransferase inducibility after the loss of chromosomes. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3026–3030. doi: 10.1073/pnas.68.12.3026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. White R., Parker M. Developmental changes in DNA methylation around prostatic steroid-binding protein genes. J Biol Chem. 1983 Jul 25;258(14):8943–8948. [PubMed] [Google Scholar]
  54. Widman L. E., Chasin L. A. Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line. J Cell Physiol. 1982 Sep;112(3):316–326. doi: 10.1002/jcp.1041120303. [DOI] [PubMed] [Google Scholar]
  55. Widman L. E., Golden J. J., Chasin L. A. Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity. J Cell Physiol. 1979 Sep;100(3):391–400. doi: 10.1002/jcp.1041000302. [DOI] [PubMed] [Google Scholar]
  56. Wilks A. F., Cozens P. J., Mattaj I. W., Jost J. P. Estrogen induces a demethylation at the 5' end region of the chicken vitellogenin gene. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4252–4255. doi: 10.1073/pnas.79.14.4252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wolf J., Golden J. J., Chasin L. A. Rat serum albumin synthesis in variant rat hepatoma cells. J Cell Physiol. 1983 Jan;114(1):29–38. doi: 10.1002/jcp.1041140106. [DOI] [PubMed] [Google Scholar]
  58. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES