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Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication

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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Figure 1: Replication timing of genes in mouse B cell lines.
Figure 2: Replication timing and transcriptional activity of genes in mouse fibroblast cell lines.
Figure 3: Delayed separation of Rag and TdT chromatids in mature T cells.
Figure 4: Delayed chromatid separation of heterochromatin-associated CD8α and λ5 genes in mature B cells.
Figure 5: Late replication of heterochromatin-embedded λ5 and hCD2 transgenes.

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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for critical reading of the manuscript, D. Graf, M.-L. Caparros, K. Takacs and S. Smale for help and discussion of unpublished results, and G. Reed, R. Newton and I. Devonish for photographic and secretarial assistance.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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Correspondence to Véronique Azuara or Amanda G. Fisher.

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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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