The architecture of interphase chromosomes and gene positioning are altered by changes in DNA methylation and histone acetylation

Wheat nuclei have a remarkably well defined interphase organisation, and we have made use of this to determine the relationship between interphase chromosome organisation, the positioning of specific transgenes and induced changes in DNA methylation and histone acetylation, using in situ hybridisation and confocal 3D imaging. After germinating seeds either in the presence of 5-Azacytidine (5-AC), which leads to DNA hypomethylation, or trichostatin A (TSA), which results in histone hyperacetylation, the architecture of the interphase chromosome arms changes significantly even though the overall Rabl configuration is maintained. This suggests that specific chromosome segments are remodelled by these treatments but that there is a strong link of both centromeres and telomeres to the nuclear envelope. In lines carrying multiple transgene integrations at widely separated sites, we show that the multiple transgenes, which are usually colocalised during interphase, are dispersed after 5-AC or TSA treatment and that there is an increase in transgene activity. This suggests that the colocalisation/dispersion of the transgenes may be a function of specific interphase chromosome organisation and that these lines containing multiple transgene copies may all be partially transcriptionally repressed.

[1]  N. Dillon,et al.  Transcription Factor Dosage Affects Changes in Higher Order Chromatin Structure Associated with Activation of a Heterochromatic Gene , 2000, Cell.

[2]  C. Allis,et al.  Acetylation and chromosomal functions. , 2000, Current opinion in cell biology.

[3]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Yoshida,et al.  Reversible arrest of proliferation of rat 3Y1 fibroblasts in both the G1 and G2 phases by trichostatin A. , 1988, Experimental cell research.

[5]  P. Christou,et al.  Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase. , 2000, The Plant journal : for cell and molecular biology.

[6]  Tony Kouzarides,et al.  Acetylation: a regulatory modification to rival phosphorylation? , 2000, The EMBO journal.

[7]  A. Wolffe,et al.  Review: chromatin structural features and targets that regulate transcription. , 2000, Journal of structural biology.

[8]  A. Bird,et al.  MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin , 1997, Cell.

[9]  B. Wells.  Low temperature box and tissue handling device for embedding biological tissue for immunostaining in electron microscopy , 1985 .

[10]  J. Jeddeloh,et al.  Maintenance of genomic methylation requires a SWI2/SNF2-like protein , 1999, Nature Genetics.

[11]  Mark Groudine,et al.  A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin , 1999, Cell.

[12]  P. Cook.  A chromomeric model for nuclear and chromosome structure. , 1995, Journal of cell science.

[13]  E. Viégas-Péquignot,et al.  Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. , 1993, Cytogenetics and cell genetics.

[14]  E. Ábrahám,et al.  Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. , 1997, The Plant journal : for cell and molecular biology.

[15]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[16]  G. Felsenfeld,et al.  Chromatin Unfolds , 1996, Cell.

[17]  A. Bird,et al.  Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. , 1992, Nucleic acids research.

[18]  Norbert O. E. Vischer,et al.  Object-image: an interactive image analysis program using structured point collection , 1994 .

[19]  D. Jackson,et al.  Active RNA polymerases are localized within discrete transcription "factories' in human nuclei. , 1996, Journal of cell science.

[20]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[21]  P. Cook.  The organization of replication and transcription. , 1999, Science.

[22]  M. Bennett,et al.  Parental Genome Separation in Reconstructions of Somatic and Premeiotic Metaphases of Hordeum Vulgare × H. Bulbosum , 1992 .

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[24]  S. Gasser,et al.  Nuclear compartments and gene regulation. , 1999, Current opinion in genetics & development.

[25]  Andrew S. Belmont,et al.  Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator , 2001, Nature Cell Biology.

[26]  A. Fisher,et al.  Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. , 1999, Molecular cell.

[27]  A. Wolffe,et al.  Epigenetics: regulation through repression. , 1999, Science.

[28]  J. S. Heslop-Harrison,et al.  rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat x rye hybrids , 1995, Theoretical and Applied Genetics.

[29]  M. Gotta,et al.  Nuclear organization and silencing: trafficking of Sir proteins. , 1998, Novartis Foundation symposium.

[30]  D. Jackson,et al.  Active RNA polymerase I is fixed within the nucleus of HeLa cells. , 1990, The EMBO journal.

[31]  M. Bevan,et al.  GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. , 1987, The EMBO journal.

[32]  D. Jackson,et al.  Visualization of focal sites of transcription within human nuclei. , 1993, The EMBO journal.

[33]  Monika Tsai-Pflugfelder,et al.  The dynamics of yeast telomeres and silencing proteins through the cell cycle. , 2000, Journal of structural biology.

[34]  P. Loidl.  Histone acetylation: facts and questions , 1994, Chromosoma.

[35]  The Arabidopsis Genome Initiative.  Analysis of the genome sequence of the flowering plant Arabidopsis thaliana , 2000, Nature.

[36]  D. Agard,et al.  Perturbation of Nuclear Architecture by Long-Distance Chromosome Interactions , 1996, Cell.

[37]  P. Shaw,et al.  Transcription Sites Are Not Correlated with Chromosome Territories in Wheat Nuclei , 1998, The Journal of cell biology.

[38]  J. S. Heslop-Harrison,et al.  5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine. , 1999, Journal of cell science.

[39]  Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs , 1989, Cell.

[40]  A. Mirsky,et al.  ACETYLATION AND METHYLATION OF HISTONES AND THEIR POSSIBLE ROLE IN THE REGULATION OF RNA SYNTHESIS. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Peter Teague,et al.  Differences in the Localization and Morphology of Chromosomes in the Human Nucleus , 1999, The Journal of cell biology.

[42]  W. F. Thompson,et al.  Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes. , 1997, The Plant journal : for cell and molecular biology.

[43]  A. Travers.  An Engine for Nucleosome Remodeling , 1999, Cell.

[44]  J. Workman,et al.  Alteration of nucleosome structure as a mechanism of transcriptional regulation. , 1998, Annual review of biochemistry.

[45]  A. Bird,et al.  DNA methylation and chromatin modification. , 1999, Current opinion in genetics & development.

[46]  P. Schulze-Lefert,et al.  A contiguous 60 kb genomic stretch from barley reveals molecular evidence for gene islands in a monocot genome. , 1998, Nucleic acids research.