Embryonic stem cells (ESCs) possess the capacity for self-renewal and multilineage differentiation, along with high genome stability, making them valuable both as seed cells for regenerative medicine and as models for developmental biology. Maintaining high-level ribosome biogenesis (RiBi) 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, ribosomal DNA (rDNA) loci exhibit a hyperactive chromatin state, characterized by low DNA methylation and reduced levels of repressive histone modifications such as H3K9me2/3 and 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 has remained incompletely understood.

In a study published in PLOS Biology, a team led by Prof. Ping Zheng from the Kunming Institute of Zoology (KIZ) of the Chinese Academy of Sciences, along with collaborators from the Shanghai Institute of Materia Medica, revealed that the transcription factor Tcf15 maintains an open nucleolar chromatin state to safeguard ribosome biogenesis and genome stability in mouse ESCs.

The researchers found that Tcf15, identified through CRISPR/Cas9 screening as a potential regulator of genomic stability, localizes to the nucleolus and specifically binds the 18S and 28S coding regions of rRNA genes. Using TurboID proximity labeling combined with mass spectrometry, the team 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.

The 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 increased significantly, while H3K4me3 levels decreased.

The two pathways are functionally non-redundant. The Tcf15-Rbbp5 axis directly drives rRNA transcription and ribosome biogenesis. Knockdown of Tcf15 or Rbbp5 reduced 47S precursor rRNA and mature rRNA levels by approximately 50%, with significant decreases in ribosomal subunits and polysomes. The Tcf15-Tet2 axis showed no apparent effect on rRNA transcription, suggesting 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, reduced the translational levels of pluripotency genes (Pou5f1, Klf4) and diminished embryoid body size. Tcf15 knockdown triggered severe genomic instability, evidenced by accumulation of the 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 did not substantially alter the global gene transcription profile or cause a comprehensive decline in protein synthesis. Instead, it selectively reprogrammed the translational landscape: translation efficiency of 819 genes was significantly downregulated, enriched in RNA splicing, DNA replication, and DNA repair pathways. Of these, 181 were high-expression proteins in ESCs, including Mcm2/3/6, Parp1, Fen1, Khdc3, Klf4, and Pou5f1. This translational defect directly impaired DNA replication fork recovery, replication stress signaling, and double-strand break damage signal transduction. Rbbp5 knockdown alone recapitulated the 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, revealing a new mechanism by which ESCs maintain the open rDNA chromatin state and deepening the understanding of why ESCs maintain a high-level ribosome pool.

Figure: In wild-type mouse embryonic stem cells, Tcf15 recruits Tet2 or Rbbp5 to activate rDNA chromatin. The Tcf15‑Rbbp5 and Tcf15‑Tet2 complexes exert distinct biological functions. Along the Tcf15‑Rbbp5 axis, Tcf15 facilitates rRNA transcription and ribosome biogenesis, as well as the selective translation of genes involved in DNA replication and DNA damage repair, thereby safeguarding genome stability. By contrast, the Tcf15‑Tet2 axis is not required for rRNA transcription and ribosome biogenesis but may participate in the organization of genome architecture.

Contact:

DONG YuPing, WANG Lin

Kunming Institute of Zoology, Chinese Academy of Sciences,

Kunming, Yunnan 650201, China

E-mail: wanglin2015@mail.kiz.ac.cn