Active chromatin is not simply open: it forms compact domains that cohesin keeps from mixing

The human genome is about two meters long, yet it is folded inside a cell nucleus only about 10 micrometers in diameter. In cells, DNA is wrapped around histone proteins to form nucleosomes [1], which are further organized into chromatin [2]. For decades, chromatin has often been described in textbooks in two simple forms [3]: euchromatin, which is active, open, and accessible (Figure A), and heterochromatin, which is more compact and repressed.

This simple textbook view has long been widely accepted. However, we felt that it did not fully explain how active euchromatin regions are organized in living cells [4]. If euchromatin were simply open and loose, neighboring active regions might easily interact and mix with each other (Figure A). How, then, are neighboring regions kept properly separated for gene regulation?

This question motivated our study. We wanted to understand the physical nature of euchromatin in living human cells, not only from Hi-C genomic maps but also at the level of individual nucleosomes. For this purpose, we first focused on the histone H3 variant H3.3 and fused it to HaloTag (H3.3-Halo) to label euchromatin in human cells. We then combined single-nucleosome imaging and tracking in living cells [5] with super-resolution 3D-structured illumination microscopy (3D-SIM) [6]. Single-nucleosome imaging allowed us to follow the movement of individual nucleosomes, while 3D-SIM enabled us to visualize nano-scale euchromatin structures.

When we observed euchromatin in this way, we found that the textbook view was too simple. Euchromatin formed condensed domains in living human cells (Figure B) [7]. This domain organization appeared to help prevent neighboring domains from mixing, acting as physical insulation.

A key molecule in this story is cohesin. The cohesin complex is a ring-shaped protein complex widely known for forming chromatin loops and organizing the genome [6, 8]. Does cohesin also control the physical behavior of euchromatic domains?

To address this, we rapidly depleted cohesin in living cells and examined how the properties of euchromatin changed. The result was not what such an “open versus closed” model would predict. Cohesin depletion increased the mobility of nucleosomes within euchromatic domains, making the domains more fluid-like (Figure C) [7]. However, this increase in fluidity occurred without altering the overall compaction of euchromatin. Neighboring condensed domains appeared to begin mixing locally.

This was an important point for us. Cohesin constrained condensed euchromatic domains and prevented their local mixing. When cohesin was lost, the domains became more fluid and locally mixed, while their overall compaction remained.

We then asked what the biological relevance of this local mixing was. Euchromatic domains are environments for gene regulation. We focused on transcription bursts of several gene pairs on chromosome 1 and found that the proximal co-burst frequency of these gene pairs significantly increased after cohesin depletion. This suggests that local domain mixing after cohesin depletion weakened transcriptional insulation (Figure C)[7].

Our study therefore revises the view of euchromatin: Active chromatin is not simply open. It can form condensed, dynamic domains whose local mixing is actively controlled. Cohesin constrains euchromatic domains and helps maintain their integrity, providing a physical basis for transcriptional insulation [9] in living cells (Figure B).

This work also suggests a broader message: chromatin should be understood not only as genomic sequence or contact maps, but also as a dynamic physical material in living cells. By watching individual nucleosomes in living cells, we can directly examine how chromatin fluctuates, becomes constrained, and reorganizes.

Finally, because cohesin dysfunction is linked to developmental disorders and cancer [10,11], understanding this physical role of cohesin may also provide new insight into how genome regulation fails in disease.

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