Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 May 1;113(3):497–506. doi: 10.1083/jcb.113.3.497

Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein

PMCID: PMC2288975  PMID: 1849905

Abstract

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6- transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Affolter M., Côté J., Renaud J., Ruiz-Carrillo A. Regulation of histone and beta A-globin gene expression during differentiation of chicken erythroid cells. Mol Cell Biol. 1987 Oct;7(10):3663–3672. doi: 10.1128/mcb.7.10.3663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Appels R., Bolund L., Gato S., Ringertz N. R. The kinetics of protein uptake by chick erythrocyte nuclei during reactivation in chick-mammalian heterokaryons. Exp Cell Res. 1974 Mar 30;85(1):182–190. doi: 10.1016/0014-4827(74)90229-8. [DOI] [PubMed] [Google Scholar]
  3. Benchaibi M., Mallet F., Thoraval P., Savatier P., Xiao J. H., Verdier G., Samarut J., Nigon V. Avian retroviral vectors derived from avian defective leukemia virus: role of the translational context of the inserted gene on efficiency of the vectors. Virology. 1989 Mar;169(1):15–26. doi: 10.1016/0042-6822(89)90036-6. [DOI] [PubMed] [Google Scholar]
  4. Bergman M. G., Wawra E., Winge M. Chicken histone H5 inhibits transcription and replication when introduced into proliferating cells by microinjection. J Cell Sci. 1988 Oct;91(Pt 2):201–209. doi: 10.1242/jcs.91.2.201. [DOI] [PubMed] [Google Scholar]
  5. Briand G., Kmiecik D., Sautiere P., Wouters D., Borie-Loy O., Biserte G., Mazen A., Champagne M. Chicken erythrocyte histone H5. IV. Sequence of the carboxy-termined half of the molecule (96 residues) and complete sequence. FEBS Lett. 1980 Apr 7;112(2):147–151. doi: 10.1016/0014-5793(80)80167-0. [DOI] [PubMed] [Google Scholar]
  6. Cullen B. R., Raymond K., Ju G. Functional analysis of the transcription control region located within the avian retroviral long terminal repeat. Mol Cell Biol. 1985 Mar;5(3):438–447. doi: 10.1128/mcb.5.3.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Emerman M., Temin H. M. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell. 1984 Dec;39(3 Pt 2):449–467. [PubMed] [Google Scholar]
  8. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  9. Gandrillon O., Jurdic P., Benchaibi M., Xiao J. H., Ghysdael J., Samarut J. Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo. Cell. 1987 Jun 5;49(5):687–697. doi: 10.1016/0092-8674(87)90545-9. [DOI] [PubMed] [Google Scholar]
  10. Garcia M., Wellinger R., Vessaz A., Diggelmann H. A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas. EMBO J. 1986 Jan;5(1):127–134. doi: 10.1002/j.1460-2075.1986.tb04186.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  12. Hwang L. H., Gilboa E. Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection. J Virol. 1984 May;50(2):417–424. doi: 10.1128/jvi.50.2.417-424.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karin M., Richards R. I. Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature. 1982 Oct 28;299(5886):797–802. doi: 10.1038/299797a0. [DOI] [PubMed] [Google Scholar]
  14. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Laimins L. A., Tsichlis P., Khoury G. Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus. Nucleic Acids Res. 1984 Aug 24;12(16):6427–6442. doi: 10.1093/nar/12.16.6427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Moscovici C., Moscovici M. G., Jimenez H., Lai M. M., Hayman M. J., Vogt P. K. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell. 1977 May;11(1):95–103. doi: 10.1016/0092-8674(77)90320-8. [DOI] [PubMed] [Google Scholar]
  18. Mura C. V., Setterfield G., Neelin J. M. Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes. Can J Biochem. 1982 Mar;60(3):215–223. doi: 10.1139/o82-026. [DOI] [PubMed] [Google Scholar]
  19. Mura C. V., Stollar B. D. Fluorescence-activated sorting of isolated nuclei. Heterogeneity of histone H5 immunofluorescence in chicken erythrocyte nuclei. Exp Cell Res. 1981 Sep;135(1):31–37. doi: 10.1016/0014-4827(81)90296-2. [DOI] [PubMed] [Google Scholar]
  20. Mura C. V., Stollar B. D. Interactions of H1 and H5 histones with polynucleotides of B- and Z-DNA conformations. Biochemistry. 1984 Dec 4;23(25):6147–6152. doi: 10.1021/bi00320a039. [DOI] [PubMed] [Google Scholar]
  21. Mura C. V., Stollar B. D. Serological detection of homologies of H1o with H5 and H1 histones. J Biol Chem. 1981 Oct 10;256(19):9767–9769. [PubMed] [Google Scholar]
  22. Norton P. A., Coffin J. M. Characterization of Rous sarcoma virus sequences essential for viral gene expression. J Virol. 1987 Apr;61(4):1171–1179. doi: 10.1128/jvi.61.4.1171-1179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Richards R. I., Heguy A., Karin M. Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids. Cell. 1984 May;37(1):263–272. doi: 10.1016/0092-8674(84)90322-2. [DOI] [PubMed] [Google Scholar]
  24. Savatier P., Bagnis C., Thoraval P., Poncet D., Belakebi M., Mallet F., Legras C., Cosset F. L., Thomas J. L., Chebloune Y. Generation of a helper cell line for packaging avian leukosis virus-based vectors. J Virol. 1989 Feb;63(2):513–522. doi: 10.1128/jvi.63.2.513-522.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  26. Sun J. M., Ali Z., Lurz R., Ruiz-Carrillo A. Replacement of histone H1 by H5 in vivo does not change the nucleosome repeat length of chromatin but increases its stability. EMBO J. 1990 May;9(5):1651–1658. doi: 10.1002/j.1460-2075.1990.tb08285.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sun J. M., Wiaderkiewicz R., Ruiz-Carrillo A. Histone H5 in the control of DNA synthesis and cell proliferation. Science. 1989 Jul 7;245(4913):68–71. doi: 10.1126/science.2740916. [DOI] [PubMed] [Google Scholar]
  28. Sung M. T., Freedlender E. F. Sites of in vivo phosphorylation of histone H5. Biochemistry. 1978 May 16;17(10):1884–1890. doi: 10.1021/bi00603a013. [DOI] [PubMed] [Google Scholar]
  29. Sung M. T., Harford J., Bundman M., Vidalakas G. Metabolism of histones in avian erythroid cells. Biochemistry. 1977 Jan 25;16(2):279–285. doi: 10.1021/bi00621a019. [DOI] [PubMed] [Google Scholar]
  30. Wagner T. E., Hartford J. B., Serra M., Vandegrift V., Sung M. T. Phosphorylation and dephosphorylation of histone (V (H5): controlled condensation of avian erythrocyte chromatin. Appendix: Phosphorylation and dephosphorylation of histone H5. II. Circular dichroic studies. Biochemistry. 1977 Jan 25;16(2):286–290. doi: 10.1021/bi00621a020. [DOI] [PubMed] [Google Scholar]
  31. Wu R. S., Panusz H. T., Hatch C. L., Bonner W. M. Histones and their modifications. CRC Crit Rev Biochem. 1986;20(2):201–263. doi: 10.3109/10409238609083735. [DOI] [PubMed] [Google Scholar]
  32. Yu S. F., von Rüden T., Kantoff P. W., Garber C., Seiberg M., Rüther U., Anderson W. F., Wagner E. F., Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3194–3198. doi: 10.1073/pnas.83.10.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES