Abstract
Eukaryotic genomes are highly organized within the cell nucleus. Genome organization not only implies the preferential positioning of genetic elements in the interphase nucleus but also the topographic distribution of biological processes. We have investigated the relationship between spatial organization and genome function in single cells. Myc, c-Met, Igf2r, Asb4, and Zac1 genes have the same radial distribution, but they are not positioned in close proximity with respect to each other. Three-dimensional mapping of their transcription sites uncovered a gene-specific pattern of relative positioning with respect to the nucleolus. We found that the Zac1 gene transcription preferentially occurs juxtaposed to the nucleolus, and that its mRNA accumulates at this site of transcription. Nucleoli isolation followed by qRT-PCR provided evidence for a physical interaction between Zac1 mRNA and the nucleolus. Actinomycin-D treatment induced disassembly of the nucleolus, loss of the RNA-FISH signal, and dramatic increase of the ZAC protein level. However, inhibition of RNA polymerase II had no effect over the Zac1 FISH signal and the protein expression. Induction of cell cycle arrest, which involves participation of the ZAC protein, provoked mRNA release from its retention site and protein synthesis. Our data demonstrate that Zac1 mRNA preferentially accumulates in close proximity to nucleoli within the cell nucleus. In addition, our results suggest a functional link between such spatial distribution and protein expression.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M (2005) Nucleolar proteome dynamics. Nature 433:77–83
Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585
Bridger JM, Boyle S, Kill IR, Bickmore WA (2000) Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts. Curr Biol 10:149–152
Cook PR (1999) The organization of replication and transcription. Science 284:1790–1795
Dekker J (2008) Gene regulation in the third dimension. Science 319:1793–1794
Dostie J, Zhan Y, Dekker J (2007) Chromosome conformation capture carbon copy technology. Curr Protoc Mol Biol Chapter 21: Unit 21.14.
Dundr M, Misteli T, Olson MO (2000) The dynamics of postmitotic reassembly of the nucleolus. J Cell Biol 150:433–446
Erickson JM, Rushford CL, Dorney DJ, Wilson GN, Schmickel RD (1981) Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments. Gene 16:1–9
Henderson AS, Eicher EM, Yu MT, Atwood KC (1974) The chromosomal location of ribosomal DNA in the mouse. Chromosoma 49:155–160
Huang SM, Schonthal AH, Stallcup MR (2001) Enhancement of p53-dependent gene activation by the transcriptional coactivator Zac1. Oncogene 20:2134–2143
Kalmarova M, Smirnov E, Masata M, Koberna K, Ligasova A, Popov A, Raska I (2007) Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli. J Struct Biol 160:49–56
Kumaran RI, Thakar R, Spector DL (2008) Chromatin dynamics and gene positioning. Cell 132:929–934
Loreni F, Thomas G, Amaldi F (2000) Transcription inhibitors stimulate translation of 5' TOP mRNAs through activation of S6 kinase and the mTOR/FRAP signalling pathway. Eur J Biochem 267:6594–6601
Manuelidis L, Borden J (1988) Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction. Chromosoma 96:397–410
Misteli T (2007) Beyond the sequence: cellular organization of genome function. Cell 128:787–800
Okuwaki M (2008) The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein. J Biochem 143:441–448
Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL, Bolland DJ, Corcoran AE, Fraser P (2007) Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. PLoS Biol 5:e192
Parada LA, McQueen PG, Munson PJ, Misteli T (2002) Conservation of relative chromosome positioning in normal and cancer cells. Curr Biol 12:1692–1697
Parada LA, McQueen PG, Misteli T (2004a) Tissue-specific spatial organization of genomes. Genome Biol 5:R44
Parada LA, Sotiriou S, Misteli T (2004b) Spatial genome organization. Exp Cell Res 296:64–70
Pederson T (1998) The plurifunctional nucleolus. Nucleic Acids Res 26:3871–3876
Piras G, El Kharroubi A, Kozlov S, Escalante-Alcalde D, Hernandez L, Copeland NG, Gilbert DJ, Jenkins NA, Stewart CL (2000) Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines. Mol Cell Biol 20:3308–3315
Politz JC, Tuft RA, Prasanth KV, Baudendistel N, Fogarty KE, Lifshitz LM, Langowski J, Spector DL, Pederson T (2006) Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm. Mol Biol Cell 17:1239–1249
Prasanth KV, Prasanth SG, Xuan Z, Hearn S, Freier SM, Bennett CF, Zhang MQ, Spector DL (2005) Regulating gene expression through RNA nuclear retention. Cell 123:249–263
Romanova L, Korobova F, Noniashvilli E, Dyban A, Zatsepina O (2006) High resolution mapping of ribosomal DNA in early mouse embryos by fluorescence in situ hybridization. Biol Reprod 74:807–815
Rozenfeld-Granot G, Krishnamurthy J, Kannan K, Toren A, Amariglio N, Givol D, Rechavi G (2002) A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1. Oncogene 21:1469–1476
Shimizu N, Kawamoto JK, Utani K (2007) Regulation of c-myc through intranuclear localization of its RNA subspecies. Biochem Biophys Res Commun 359:806–810
Smith RJ, Arnaud P, Konfortova G, Dean WL, Beechey CV, Kelsey G (2002) The mouse Zac1 locus: basis for imprinting and comparison with human ZAC. Gene 292:101–112
Valleley EM, Cordery SF, Bonthron DT (2007) Tissue-specific imprinting of the ZAC/PLAGL1 tumour suppressor gene results from variable utilization of monoallelic and biallelic promoters. Hum Mol Genet 16(8):972–981
Visintin R, Amon A (2000) The nucleolus: the magician's hat for cell cycle tricks. Curr Opin Cell Biol 12:752
Wiblin AE, Cui W, Clark AJ, Bickmore WA (2005) Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells. J Cell Sci 118:3861–3868
Wsierska-Gadek J, Horky M (2003) How the nucleolar sequestration of p53 protein or its interplayers contributes to its (re)-activation. Ann N Y Acad Sci 1010:266–272
Acknowledgements
We would like to thank Dr. Tom Misteli and Dr. Paul Edwards for critical reading of the manuscript. This work was supported by funding from Fondo de Investigación Sanitaria (ISCiii), Grant #05/1117 to LAP; Department of Industry and Department of Health of the Basque Government and CIBERHED. FR is a CIBERHED postdoctoral fellow associated to CIC bioGUNE. This research was supported in part by the Intramural Research Program of the NIH, Center of Information Technology.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by T. Misteli
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary File 1
(DOC 67 kb)
Supplementary Fig. 1
Schematic of the Zac1 gene structure and CpG methylation status of the differential methylated region (DMR). Location of exons/introns in the Zac1 gene and position of the primers used in the different PCR reactions are depicted according to the nucleotide sequences deposited in Ensembl Mouse Exon View for ENSMUST00000068371; http://www.ensembl.org (a). Methyl-specific PCR (MSP) assay was performed to detect differentially methylated CpGs of the Zac1 gene promoter in the MEF cell line (G7) and neurons (Neu) from mouse embryos. The digital image is automatically generated by the 2100-Bioanalyzer (Agilent Technologies). MSP assay showed that the CpGs of the promoter region exist in both methylated (M) and unmethylated (U) forms (b). Methyl-specific PCR (MSP) assay was performed to test the completion of the bisulfate-mediated C>T conversion. The results from two experiments show that the MSP primers only amplified unmethylated DNA (c) (AI 520 kb)
Supplementary Fig. 2
NORs mapping in the normal embryonic mouse fibroblast G7 cell line. Sequential FISH analysis with of pA and pB rDNA probes (arrows) followed by multicolor spectral karyotyping showing the localization of ribosomal genes only on chromosomes 12, 15, and 18, but not on chromosome 10 (AI 1696 kb)
Supplementary Fig. 3
Zac1 locus and its primary transcripts co-localize in the interphase nucleus. RNA FISH with probes for exon X (Fragment Zac1-5; red, arrows) and intron X–XI (Fragment Zac1-4, green, arrows) on G7 cells counterstained with DAPI. Arrows indicate RNA signals (a). Sequential RNA/DNA FISH analysis with the Zac1-5 fragment (red) and the BAC probe (green). Scale bars = 4 µm (b) (AI 1053 kb)
Supplementary Fig. 4
Zac1 gene transcription after Actinomycin D withdrawal. Zac1 gene RNA FISH (green, arrows) with a probe for exon X (Zac1-5 fragment) combined with nucleolus immunostaining with an antibody against NPM (red) after Actinomycin D removal from the culture dish. Counterstaining with DAPI (blue), scale bar = 2 μm (AI 647 kb)
Supplementary Fig. 5
3D reconstruction of a fibroblast nucleus with high level of Zac1 gene transcription. RNA FISH was performed with the Zac1-5 fragment labeled with digoxigenin-12-dUTP and revealed with anti-digoxigenin conjugated to FITC (green). The nucleus was counterstained with DAPI (blue) (AVI 6213 kb)
Rights and permissions
About this article
Cite this article
Royo, F., Paz, N., Espinosa, L. et al. Spatial link between nucleoli and expression of the Zac1 gene. Chromosoma 118, 711–722 (2009). https://doi.org/10.1007/s00412-009-0229-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00412-009-0229-1