Sox2 enhancer clusters in a living mouse embryonic stem cell.

A new super-resolution method to see 3D genome organization in single cells

Sequencing-based methods like Hi-C and ATAC-seq reveal genome organization as population averages, obscuring cell-to-cell variation. We wanted to see the 3D genome, specifically, the accessible chromatin that drives gene regulation, directly, in individual cells, at nanometer resolution.

The technology: 3D ATAC-PALM. We invented 3D ATAC-PALM by integrating ATAC (Assay for Transposase-Accessible Chromatin) with Tn5-mediated insertion of photoactivatable fluorophores, imaged by lattice light-sheet PALM microscopy. This provides nanoscale, whole-nucleus maps of the accessible genome in single cells (Xie & Dong et al., Nature Methods 2020). Paired with single-molecule residence-time imaging of transcription factors, 3D ATAC-PALM enables simultaneous measurement of chromatin structure and protein binding dynamics in the same cell.

What we discovered. Using 3D ATAC-PALM with chemical and genetic perturbations, we found that:

  • CTCF decompacts accessible chromatin (Xie & Dong et al., Nature Methods 2020).

  • Cohesin prevents spatial mixing of accessible chromatin domains by counterbalancing affinity-driven interactions mediated by BRD2, effectively compartmentalizing the accessible genome (Xie & Dong et al., Nature Genetics 2022).

  • Cohesin loss reshapes gene co-expression patterns in single cells rather than changing population-average expression levels (Dong et al., Nature Genetics 2024), a finding with direct implications for cohesinopathies such as Cornelia de Lange syndrome and for understanding how 3D topology encodes cell-type-specific transcriptional programs.

This last discovery, that genome topology regulates which genes are co-expressed together, set the stage for our next technology, cycleHCR.

Collaborative applications. 3D ATAC-PALM has been used in collaboration to study phase-separation-driven genome reorganization by YAP (Cai et al., Nat. Cell Biol. 2019) and DNA-initiated epigenetic cascades in C9orf72 repeat expansion disease (Liu et al., Neuron 2023).

Related Publications

1.   Liu, Z.@, Legant, WR., Chen, B., Li, L., Grimm, JB., Lavis, LD., Betzig, E. and Tjian, R. (2014). 3D imaging of Sox2 enhancer clusters in embryonic stem cells. eLife, 3:e04236.

2.  Xie, L.*, Dong, P.*, Qi, Y., Marzio, M.D., Chen, X., Banala, S., Legant, W.R., English, B., Hansen, A., Schulmann, A., Lavis, L.D., Betzig, E., Chang, H.Y., Zhang, B., Tjian, R.@, Liu, Z.@ (2020). 3D ATAC-PALM: super-resolution imaging of the accessible genome. Nature Methods, 17(4):430-436.

3.   Xie, L.*, Dong, P.*, Qi, Y., Hsieh, T.S., English, B., Jung, S., Chen, X., Marzio, M.D., Chen, X., Casellas, R., Chang, H.Y., Zhang, B.@, Tjian, R.@, Liu, Z.@ (2022) BRD2 Compartmentalizes the Accessible Genome. Nature Genetics, 54(4):481-491.

4. Dong, P.@, Zhang, S., Gandin, V., Xie, L., Wang, L., Lemire, A.L., Li, W., Otsuna, H., Kawase, T., Lander, A.D., Chang. H.Y., Liu, Z.@ (2024) Cohesin prevents cross-domain gene co-activation. Nature Genetics,56(8):1654-1664.

Collaborations:

1.  Cai, D., Feliciano, D., Dong, P., Flores, E., Gruebele, M., Porat-Shliom, N., Sukenik, S., Liu, Z., Lippincott-Schwartz., J. (2019). Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression. Nat. Cell biol., 21(12):1578-1589.

2.   Liu, Y., Huang, Z., Liu, H., Ji, Z., Arora, A., Cai, D., Wang, H., Liu, M., Simko, E.A., Zhang, Y., Periz, G., Liu, Z., Wang, J. (2023) DNA-initiated epigenetic cascades driven by C9orf72 hexanucleotide repeat. Neuron, 111(8):1205-21.