A new study has uncovered how an exceptionally scarce protein can orchestrate the assembly of large‑scale gene-silencing structures inside cells, and what happens when that process breaks down.
The findings, published today in Molecular Cell, identify a self-clustering mechanism in the Polycomb protein CBX2 that is essential for initiating the formation of gene-repressive condensates and guiding stem cells toward their proper fates.
Polycomb complexes are essential for establishing and maintaining cell identity, yet the physical principles behind their repression have remained elusive. The challenge is that some of these molecules are typically present at extremely low levels, making them hard to quantify using classical methods.
Using ultra-sensitive single‑molecule imaging and genetic engineering, the researchers, led by the University of North Carolina at Charlotte, count these molecules one-by-one, discovering that condensates in mouse embryonic stem cells (mESCs) form around an unexpectedly small core: three molecules of the protein CBX2.
"This is remarkable. CBX2 is one of the least abundant Polycomb proteins in stem cells, yet it can dictate the assembly of these large regulatory structures and act as a seed for the entire condensate," said UNC Charlotte Associate Professor and Irwin Belk Distinguished Scholar of Biology, Xiaojun Ren. "CBX2 is doing far more with far less than anyone expected."
A low-abundance protein with an outsized influence to shape the epigenome
The team found that these tiny CBX2 clusters recruit two Polycomb repressive complexes 1 and 2, creating multicomponent repressive hubs that shape the epigenome. Despite its scarcity, CBX2 proved essential for organizing and positioning its hallmark histone modification H3K27me3, impacting gene repression. As cells differentiate, the condensates change: neural progenitor cells (NPCs) contain roughly 15 CBX2 molecules per condensate, five times as many as in mESCs, highlighting that cell fate reprograms repressive condensates.
Genome-wide CUT&Tag and CUT&RUN analyses revealed that CBX2 binds preferentially at PRC2 nucleation sites, where H3K27me3 deposition begins. When CBX2 was removed, both PRC2 and H3K27me3 redistributed across the genome, weakening repression at key developmental genes.
A separation-of-function mutant reveals CBX2's true nature
To test whether CBX's ability to self-cluster is required for its function, the researchers engineered a CRISPR-edited variant, CBX2^PSM, that cannot self-interact but still binds to chromatin normally.
The results were striking:
CBX2^PSM failed to form condensates in live cells.
It required over 100-fold higher concentrations to condense in vitro.
Despite normal chromatin binding, H3K27me3 became mislocalized, accumulating in DNA-dense regions.
Genome-wide profiling showed a loss of H3K27me3 at Polycomb domains and gains at atypical sites.
These changes closely mirrored the effects of a full CBX2 knockout, demonstrating that self-clustering, and not chromatin binding alone, is essential for organizing Polycomb domains.
Clustering is required for cells to choose their fate
The consequences extended beyond chromatin structure. When the team induced differentiation, they found:
Embryoid bodies derived from CBX2^PSM cells showed severely impaired outgrowth.
CBX2^PSM cells failed to efficiently generate neural progenitor cells.
The number of Nestin-positive cells was significantly reduced compared to wild-type controls.
"Without CBX2's ability to self-cluster, the cells simply cannot execute their developmental programs," said Ren. "Just as a raindrop needs a tiny speck of dust to form in a humid sky, these three molecules act as the 'seeds' that pull the cell's Polycomb proteins together into a functional droplet."
A new model for how Polycomb condensates form
The findings challenge the long-standing assumption that Polycomb condensates form through classical liquid-liquid phase separation in mESCs. With only approximately three molecules per condensate in the mESCs, CBX2 cannot form a traditional interaction network.
Instead, the team proposes a nucleation and bridging induced phase separation (NBiPS) model:
CBX2 binds to nucleation sites, forming small CBX2-chromatin structures.
This structure recruits CBX7 and CBX7-PRC1, which bridge chromatin fibers.
PRC2 is then stabilized at these sites, promoting H3K27me3 deposition.
The condensate grows into a functional Polycomb domain.
This model unifies previous in vitro and in vivo observations and explains how low-abundance proteins can initiate large-scale chromatin organization.
Implications for development and disease
Polycomb dysfunction is linked to developmental disorders and cancers. By revealing how condensates form and how their composition is regulated, the study provides a new framework for understanding how gene-silencing programs are established and maintained.
