Embryonic stem cells (ESCs) possess the capacity for self-renewal and multilineage differentiation, along with high genome stability. Maintaining high-level ribosome biogenesis is essential for ESCs to sustain their identity, and this process begins with RNA polymerase I-mediated transcription of ribosomal RNA (rRNA) genes.

In ESCs, rDNA loci exhibit a hyperactive chromatin state, characterized by low DNA methylation and reduced levels of repressive histone modifications such as H3K27me3, to ensure efficient rRNA transcription and ribosome biogenesis. However, how ESCs establish and maintain this hyperactive nucleolar chromatin state to preserve stem cell identity remains incompletely understood.

In a study published in PLOS Biology, researchers from the Kunming Institute of Zoology of the Chinese Academy of Sciences (CAS) and the Shanghai Institute of Materia Medica of CAS revealed that the transcription factor Tcf15 maintains an open nucleolar chromatin state to safeguard ribosome biogenesis and genome stability in mouse ESCs.

Researchers found that Tcf15, identified through CRISPR/Cas9 screening as a regulator of genomic stability, localizes to the nucleolus and specifically binds 18S and 28S coding regions of rRNA genes. Combining TurboID proximity labeling with mass spectrometry, they identified two key interacting proteins, Tet2 and Rbbp5. Co-immunoprecipitation confirmed that Tcf15 binds each independently. Tcf15 knockdown significantly reduced the binding of both factors to rDNA regions.

Moreover, researchers showed that Tcf15 maintains rDNA chromatin openness through two parallel pathways. The Tcf15-Rbbp5 axis promotes H3K4me3 deposition to counteract PRC2-mediated H3K27me3 modification, while the Tcf15-Tet2 axis sustains rDNA hypomethylation. Upon Tcf15 depletion, H3K27me3 and DNA methylation levels at rDNA regions increase significantly, and H3K4me3 levels decrease.

These two pathways are functionally non-redundant. The Tcf15-Rbbp5 axis directly drives rRNA transcription and ribosome biogenesis. Knockdown of Tcf15 or Rbbp5 reduces 47S precursor rRNA and mature rRNA levels by approximately 50%, with significant decreases in ribosomal subunits and polysomes. The Tcf15-Tet2 axis shows no apparent effect on rRNA transcription, suggesting that it may participate in non-canonical nucleolar functions.

Further analysis showed that impaired ribosome biogenesis through the Tcf15-Rbbp5 axis, while not affecting embryoid body formation rate, reduces translational levels of pluripotency genes (Pou5f1, Klf4) and diminishes embryoid body size. Tcf15 knockdown triggers severe genomic instability, evidenced by the accumulation of DNA damage marker gammaH2AX, increased double-strand breaks, elevated micronuclei, and higher aneuploidy rates.

Integrated RNA-seq and Ribo-seq analyses revealed that Tcf15 depletion does not substantially alter global gene transcription profile or cause a decline in protein synthesis, but downregulates translation efficiency of 819 genes, enriched in RNA splicing, DNA replication, and DNA repair pathways. This impairs DNA replication fork recovery, replication stress signaling, and double-strand break damage signal transduction. Rbbp5 knockdown recapitulates DNA damage phenotype, confirming the central role of the Tcf15-Rbbp5 axis in maintaining genome stability.

This study establishes the first complete molecular pathway linking nucleolar chromatin state to ribosome biogenesis, selective translational control, and genome stability. It reveals a new mechanism by which ESCs maintain the open rDNA chromatin state, and deepens the understanding of why ESCs maintain a high-level ribosome pool.