Biology subjects

Di Tommaso, E.

Publications and source records attributed to Di Tommaso, E..

3 recordsLinked to original sources

bioRxiv2026

Replication stress at centromeres biases the segregation of DNA damage

Replication-associated errors can cause DNA damage to accumulate on the newly synthesized strand over time. In specific cases such as stem cells, retention of the immortal strand used as template preserves one daughter cell into pluripotency while correlating with terminal differentiation of the damage one. In somatic cells, DNA damage distribution after mitosis remains unclear. Here, we uncovered a mechanism of non-random segregation of the DNA damage marker gH2AX occurring during a single cell division cycle. Replication stress using hydroxyurea (HU) upon release into S phase in RPE-1, BJ, hCEC D29 and fibroblasts showed reproducible Non-Random Segregation (NRS) of gH2AX in the ensuing G1, a phenotype not observed in any of the cancer cell lines analyzed. Notably, removal of R-loops led to a reduction of cells with NRS, whether RNaseH1 was over-expressed globally or exclusively targeted to centromeres, indicating that centromeric DNA-RNA hybrids contribute to NRS of the damage. In line with our previous evidence of centromeric chromatin disruption leading to R-loops, rapid removal of the histone H3 variant CENP-A causes damage and NRS, although to a lower extent than HU alone. This implies that additional mechanisms contribute to centromeric R-loops and NRS of damage in the daughter cells upon mitotic exit. Mechanistically, chemical inhibition of the catalytic activity of Rad51 led to a significant drop in NRS without a change in the total amount of damaged cells, implying involvement of the Homologous Recombination (HR) pathway to accumulation of gH2AX to only one chromatid. In turn, this affects the spindle-kinetochore with a measurable length asymmetry, inducing mechanical and/or epigenetic signals that affect the orientation of the sister chromatids on the metaphase plate to bias segregation. Altogether, we found replication-induced asymmetric segregation of DNA damage during mitosis that is influenced by centromeric R-loops, Rad51 activity and spindle dynamics, with implications on cell fate, chromosome and genome stability in the daughter cells.

cell biology↗

bioRxiv2026

The diploid reference genome of a human embryonic stem cell line

Advances in DNA sequencing and assembly technologies are spurring a shift from haploid reference genomes to sample-specific diploid assemblies. Here, we generated the first telomere-to-telomere (T2T) diploid reference for the widely used human embryonic stem cell (hESC) line, H9 (WAe009-A). This haplotype-resolved assembly is highly accurate with comprehensive annotation of genes, segmental duplications, methylation, and chromatin conformation. Pangenomic and phased-locus inference point to H9s mixed ancestry with a predominant European component. H9-specific genomic features include near-perfect telomeres [~]1.65-fold longer than other T2T assemblies, consistent with telomerase activity during pluripotency; chromosome 17 inversions that can predispose offspring to neurological syndromes; and expansions of ncRNA clusters, with overall genomic stability maintained despite extensive culturing. Mapping multi-omic datasets to the genome, we demonstrate the power of this resource for allele-specific, high-precision transcriptomic, genetic, and epigenetic analyses, with far-reaching implications for human development and disease.

genomics↗

bioRxiv2023

The complete human diploid reference genome of RPE-1 identifies the phased epigenetic landscapes from multi-omics data

Comparative analysis of recent human genome assemblies highlights profound sequence divergence that peaks within polymorphic loci such as centromeres. This raises the question about the adequacy of relying on human reference genomes to accurately analyze sequencing data derived from experimental cell lines. Here, we generated the complete diploid genome assembly for the human retinal epithelial cells (RPE-1), a widely used non-cancer laboratory cell line with a stable karyotype, to use as matched reference for multi-omics sequencing data analysis. Our RPE1v1.0 assembly presents completely phased haplotypes and chromosome-level scaffolds that span centromeres with ultra-high base accuracy (>QV60). We mapped the haplotype-specific genomic variation specific to this cell line including t(Xq;10q), a stable 73.18 Mb duplication of chromosome 10 translocated onto the microdeleted chromosome X telomere t(Xq;10q). Polymorphisms between haplotypes of the same genome reveals genetic and epigenetic variation for all chromosomes, especially at centromeres. The RPE-1 assembly as matched reference genome improves mapping quality of multi-omics reads originating from RPE-1 cells with drastic reduction in alignments mismatches compared to using the most complete human reference to date (CHM13). Leveraging the accuracy achieved using a matched reference, we were able to identify the kinetochore sites at base pair resolution and show unprecedented variation between haplotypes. This work showcases the use of matched reference genomes for multi-omics analyses and serves as the foundation for a call to comprehensively assemble experimentally relevant cell lines for widespread application. HighlightsO_LIWe generated the complete phased genome assembly of one of the most widely used non-cancer cell lines (RPE-1) with a stable diploid karyotype C_LIO_LIWe used this genome as a matched reference to analyze sequencing data from RPE-1 C_LIO_LIMapping to the RPE1v1.0 genome improves alignment quality, faithful assignment of reads to each haplotype, and epigenome peak calling accuracy uncovering inter-haplotype variation C_LIO_LIUse of the matched reference genome enables epigenetic precision in identifying for the first time the kinetochore site at base pair resolution for each haplotype C_LIO_LIThe RPE-1 genome represents a new telomere-to-telomere (T2T) human diploid reference for the scientific community that will advance genetic and epigenetic research across fields using this cell line C_LI

genomics↗