Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions

Abstract

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

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Figure 1: The circular chromosomal conformation capture (4C) assay of intra- and interchromosomal interactions.
Figure 2: Epigenetic control of intra- and interchromosomal networks.
Figure 3: Imprinted domains are functionally connected in trans via DMRs.
Figure 4: Epigenetically controlled networks of neonatal liver are formed during in vitro maturation of embryonic stem cells.

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Acknowledgements

We thank A. Mattsson for expert technical assistance and M. Merkenschlager and P. Fraser for helpful advice. We are also grateful for the assistance of the Wallenberg microarray group at the Royal Technical University at Stockholm. This work was supported by the Swedish Science Research Council (V.R., R.O.), the Swedish Cancer Research Foundation (C.F., R.O.), the Swedish Pediatric Cancer Foundation (B.C.F, R.O.), HEROIC (European Union integrated project) and the Lundberg Foundation (R.O.).

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Z.Z. generated the 4C library and designed and performed the 4C microarray experiments. G.T. performed the 3C validation, analysis of microarray data and quantitative RT-PCR expression analysis. M.S. performed the 3D DNA FISH analysis. A.G. designed the 3D DNA FISH analysis and analysis of microarray data. P.M. designed the 4C approach and assisted in the analysis. S.W. assisted in the generation of microarrays. C.K. performed the DNA blot analysis. M.L. provided the ES cells and derived embryoid bodies and phenotyped the preparations used in this study. K.S.S. performed the statistical analysis. U.S. assisted in the characterization of the ES and derived embryoid bodies. V.P. generated the mutant 142* mouse strain used in this study. V.T. assisted in characterization of PCR primers and conditions. S.K. assisted in characterization of PCR primers and conditions. R.O. designed and supervised the entire 4C project and assisted in the analysis of microarray data.

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The method described in the manuscript is the subject of a patent application. This has not influenced the results and discussion of this report.

Supplementary information

Supplementary Fig. 1

3C validation of randomly picked sequences of the 4C library. (PDF 355 kb)

Supplementary Fig. 2

DNA FISH analysis of chromosomal interactions. (PDF 260 kb)

Supplementary Fig. 3

Markers of undifferentiated and differentiated embryonic stem (ES) cells. (PDF 59 kb)

Supplementary Table 1

The 4C library. (PDF 111 kb)

Supplementary Table 2

Primers for 3C and expression analysis. (PDF 83 kb)

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Zhao, Z., Tavoosidana, G., Sjölinder, M. et al. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38, 1341–1347 (2006). https://doi.org/10.1038/ng1891

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