Accurate chromosome segregation and ribosomal gene expression silencing are essential for maintaining genome integrity, and disruptions in these processes are key for oncogenesis and cancer progression. Here, we demonstrate a novel role for the transcriptional co-repressor SKI in regulating rDNA and pericentromeric heterochromatin (PCH) silencing in human cells. We found that SKI localizes to the rDNA promoter on acrocentric chromosomes and is crucial for maintaining H3K9 trimethylation (H3K9me3) and repressing 45S rRNA gene expression. SKI is also associated with BSR and HSATII satellites within PCH, where is necessary for H3K9 methylation and recruitment of SUV39H1 and HP1α, key players for heterochromatin silencing and centromere function. Consequently, SKI deficiency disrupted centromere integrity and resulted in aberrant chromosome segregation, micronuclei formation, and chromosome instability. The identification of SKI as a key participant in the epigenetic-mediated silencing of pericentromeric and ribosomal DNA provides a fundamental insight, paving the way for new research into the intricate relationship between transcriptional regulation and genome instability during cancer progression, and opening novel opportunities for therapeutic intervention.

Replication stress (RS) is a primary driver of genomic instability in cancer, yet the contribution of transcription-coupled repair (TC-NER) to this process remains unclear. Here, we investigate how the TC-NER factor ERCC6 (CSB) shapes mutational landscapes under RS. We found that ERCC6 deficiency biases early damage signaling toward a 53BP1-mediated response, ultimately leading to senescence. Conversely, ERCC6-proficient cells prioritize survival and proliferative recovery but at the expense of distinct genomic alterations. Whole-exome sequencing reveals that ERCC6 proficiency is associated with the retention of stress-induced mutations specifically within coding regions of transcriptionally active loci, whereas ERCC6-deficient cells accumulate variants primarily in intergenic regions. These findings suggest that while ERCC6 safeguards transcriptional continuity during RS, its activity is associated with a biased retention of stress-induced mutations within coding regions in the surviving cell population. These findings reveal a previously unrecognized link between transcription-coupled repair and mutation distribution in human cells, linking TC-NER to context-dependent somatic evolution and tumor heterogeneity.

Replication stress (RS) is a primary driver of genomic instability in cancer, yet the contribution of transcription-coupled repair to this process remains poorly understood. Here, we investigate how the TC-NER factor ERCC6 (CSB) shapes mutational landscapes under RS. We demonstrate that ERCC6 deficiency impairs replication restart and biases early damage signaling toward a 53BP1-mediated response, ultimately leading to senescence. Conversely, ERCC6-proficient cells prioritize survival and proliferative recovery but at the cost of distinct genomic alterations. Whole-exome sequencing reveals that ERCC6 drives the retention of stress-induced mutations specifically within coding regions of transcriptionally active loci, whereas ERCC6-deficient cells accumulate variants primarily in intergenic regions. These findings uncover a survival-mutagenesis trade-off: ERCC6 safeguards transcriptional continuity during replication stress but promotes mutational burdens in functional genomes. This mechanism parallels bacterial adaptive mutagenesis, identifying ERCC6 as a context-dependent driver of somatic evolution and tumor heterogeneity.

Replication and transcription share the DNA template and must be coordinated to preserve genome integrity. Although temporally organized across the cell cycle, essential transcriptional programs—encoding replication machinery, canonical histones, and DNA repair factors—operate concurrently with DNA synthesis during S phase. Transcription–replication conflicts (TRCs) therefore constitute a recurrent endogenous source of replication stress (RS), particularly under hypertranscriptional or chromatin-constrained conditions. A frequent outcome of TRCs is the formation of R-loops–RNA:DNA hybrids that can stall or collapse replication forks, leading to DNA damage. This review summarizes current evidence supporting a broader involvement of ERCC6/CSB at the transcription–replication interface beyond its established role in transcription-coupled repair. We discuss how ERCC6 participates in RNA polymerase II processing, chromatin remodeling, R-loop metabolism, replication fork protection, and repair pathway engagement following RS, operating through both its ATPase domain and intrinsically disordered regions. Conversely, in homologous recombination-deficient contexts, ERCC6 may favor mutagenic restart mechanisms, including break-induced replication; and recent evidence indicates that ERCC6 status influences cellular fate and the genomic distribution of stress-induced mutations, linking transcription-coupled repair with transcription-dependent mutagenesis. These observations support a model in which ERCC6 coordinates transcription-associated repair with RS responses, shaping genome maintenance and mutational outcomes, with implications for cancer and therapeutic strategies.