Abstract
Temporal control of DNA replication has been implicated in epigenetic regulation of gene expression on the basis of observations that certain tissue-specific genes replicate earlier in expressing than non-expressing cells. Here, we show evidence that several leukocyte-specific genes replicate early in lymphocytes regardless of their transcription and also in fibroblasts, where these genes are never normally expressed. Instead, the heritable silencing of some genes (Rag-1, TdT, CD8α and λ5) and their spatial recruitment to heterochromatin domains within the nucleus of lymphocytes resulted in a markedly delayed resolution of sister chromatids into doublet signals discernable by 3D fluorescence in situ hybridization (FISH). Integration of transgenes within heterochromatin (in cis) did, however, confer late replication and this was reversed after variegated transgene expression. These findings emphasise that chromosomal location is important for defining the replication timing of genes and show that retarded sister-chromatid resolution is a novel feature of inactive chromatin.
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Acknowledgements
The authors thank S. M. Gartler, R. S. Hansen and T. K. Canfield for advice and support with the PCR-based replication timing assay. We also thank A. Pombo, F. Uhlmann, M. Gomez, N. Brockdorff and L. Aragon for critical reading of the manuscript, D. Graf, M.-L. Caparros, K. Takacs and S. Smale for help and discussion of unpublished results, and G. Reed, R. Newton and I. Devonish for photographic and secretarial assistance. This work was supported by the Medical Research Council, UK., the Biotechnology and Biological Sciences Research Council (R.R.E.W.) and the Wellcome Trust (K.E.B).
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Supplementary information
FigS1 The replication timing of TdT and Rag-1 genes is unaltered following transient or permanent (heritable) gene silencing in developing T cells.
Fig S2 Models linking DNA synthesis and transcriptional memory. (PDF 510 kb)
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Azuara, V., Brown, K., Williams, R. et al. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nat Cell Biol 5, 668–674 (2003). https://doi.org/10.1038/ncb1006
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DOI: https://doi.org/10.1038/ncb1006
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