Biology subjects

Giunta, S.

Publications and source records attributed to Giunta, S..

9 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 consequences of array size for human centromere performance in mitosis

Long-read sequencing and assembly of human genomes have revealed extreme variation in centromeric alpha-satellite array sizes across chromosomes and individuals. However, assessing the impact of centromeric array size on centromere function remains challenging. In this study, using quantitative FISH and microscopy assays, we established a framework for the systematic functional evaluation of naturally occurring variations in centromeric array size during mitosis. By comparing 8 pairs of homologous chromosomes exhibiting 1.24 - 4.6 -fold variation in centromeric array size, we uncovered size-based differences in function. We found that between a pair of homologous chromosomes, the chromosome harboring the smaller centromeric array is more prone to chromosome missegregation. Centromere size-based differences in chromosome missegregation rates cannot be explained by differential enrichment of molecular factors like centromeric histone CENP-A, CENP-B, or most kinetochore proteins. However, the smaller centromere out of a homologous pair consistently exhibits increased cohesion fatigue, suggesting the involvement of the cohesin complex in size-based differences in centromere cohesion. Consistent with our hypothesis, complete deprotection of the cohesin complex by depletion of shugoshin-1, removes the size-based bias in centromere cohesion. These results suggest that while CENP-A-enriched core centromeres are essential for kinetochore assembly, variation in overall centromeric array size has a significant impact on centromere performance and the fidelity of chromosome segregation during mitosis.

Cell Biology↗

bioRxiv2026

Characterization of Human Ectocentromeric Sites.

Centromeres are composed of DNA repeats within chromosomes primary constriction. CENP-B is the only centromeric protein known to bind a specific motif, the CENP-B box, promoting kinetochore stability. We recently uncovered degenerate CENP-B binding motifs outside centromeres, whose position and orientation defines chromosome specific banding patterns. Here, we leveraged telomere-to-telomere assemblies to map conservation of these ectocentromeric sequences (ECS) across hundreds of haplotypes. We found strong negative selection acting on their occurrence along chromosome arms, implying functional constraints incompatible with stochastic drift. We classified four categories: (i) ECSs that lack CENP-B binding ([~]84%); (ii) ECSs bound by CENP-B ([~]10%); (iii) ECSs near CENP-B-enriched accessible chromatin ([~]6%); (iv) we further identified [~]700 CENP-B binding sites outside centromeres without CENP-B boxes. Integrating chromatin conformation capture (HiC), neocentromeres and meiotic recombination mapping with CENP-B CUT&RUN, methylation and ATAC-seq data, we found heterogenous functionalities driven by distance-dependent enrichment and local contacts of boxes in inverted orientation on the same strand, analogous to ALU repeats affecting topological folding. CENP-B knockdown significantly reduced neighboring gene expression, revealing a moonlighting regulatory role outside centromeres. Our findings characterizes human ectocentromeric sites as evolutionarily constrained and functionally heterogeneous elements along chromosome arms with context-dependent roles in chromatin state. Graphical AbstractEctocentromeric sites exhibit heterogeneous CENP-B occupancy and context-dependent chromatin functions. Ectocentromeric sequences (ECSs) along chromosome arms fall into four categories: (i) CENP-B box motifs alone, lacking protein binding, embedded within repressed or boundary chromatin and contributing to TAD organization. Motifs with opposite orientation (forward and reverse complement) paired on the same strand may further promote self-complementary chromatin contacts analogous to Alu inverted repeats, reshaping topology with long-range looping contacts; (ii/iii) ECSs bound by CENP-B protein, associated with specific open chromatin state downstream of H3K27me3-marked compacted chromatin that modulate local accessibility and gene expression; and (iv) CENP-B binding peaks lacking a canonical box motif, located proximal to transcription start sites and linked to active gene expression. Together, ectocentromeric sites represent functionally heterogeneous elements with context-dependent roles spanning chromosome architecture, chromatin state, and transcription regulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/728588v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1683fb7org.highwire.dtl.DTLVardef@12f1c9dorg.highwire.dtl.DTLVardef@1ffc699org.highwire.dtl.DTLVardef@14790dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

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↗

bioRxiv2025

The complete genome of a songbird

Bird genomes are the smallest among amniotes, but remain challenging to assemble due to their structural complexity. This study presents the first fully phased, diploid, telomere-to-telomere (T2T) reference genome for the zebra finch (Taeniopygia guttata), a model organism for neuroscience and evolutionary genomics. Combining multiple sequencing strategies resulted in closing nearly all gaps, adding [~]90 Mbp of previously missing sequence (7.8%). This includes T2T assemblies for all microchromosomes, including dot chromosomes, and the previously almost entirely missing chr16. The T2T genome is comprehensively annotated for genes, repeats, structural variants, and long-read methylation calls. Complete centromeric structures were assembled and annotated along with kinetochore binding sites. Relative to the previous high-quality reference of the Vertebrate Genomes Project, 2,778 (8.51%) previously unassembled or unannotated genes were identified, of which 9% overlap with segmental duplications. This first complete genome of a songbird, now the new public reference, illuminates avian genome architecture and function.

genomics↗

bioRxiv2025

Diploid genome assembly of human fibroblast cell lines enables clone specific variant calling, improved read mapping and accurate phasing

Human cell lines are fundamental tools in biomedical research and are widely used in disease modeling, drug development, and many other domains. Here, we present chromosome-level, phased diploid genome assemblies of two popular human cell lines: the BJ foreskin fibroblast line and the IMR-90 fetal lung fibroblast line. Our high-quality assemblies, generated using long-read and Hi-C sequencing data, reveal substantial structural variation, including more than 50,000 insertions, deletions, duplications, and inversions compared to the recent T2T-CHM13v2.0 reference. Our assemblies provide detailed maps of genetic variation, enabling more accurate variant calling and the ability to phase reads when using newly generated or historical sequencing data on these cell lines or their derivatives. All assemblies and associated data have been made available as a resource for the research community. We envision that diploid genome assembly will become a cornerstone approach for personalized medicine in the near future.

genomics↗

bioRxiv2025

Heterochromatin boundaries maintain centromere position, size and number

Centromeres are chromosomal loci that ensure proper chromosome segregation by providing a platform for kinetochore assembly and spindle force transduction during cell division. Human centromeres are defined primarily by a unique chromatin domain featuring the histone H3 variant, Centromere Protein A (CENP-A), that marks a single active centromere locus per chromosome. CENP-A chromatin typically occupies a small subregion of low DNA methylation within multi-megabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. However, the mechanisms defining and maintaining precise centromere position and domain size, and the role of the underlying alpha satellite DNA sequence, are poorly characterised. Using an experimentally-induced neocentromere in RPE1 cells, we discovered that the SUV39H1 and H2 methyltransferases tri-methylate H3K9 at neocentromere boundaries to maintain CENP-A domain size independent of DNA methylation or satellite sequences. Furthermore, we found that the CENP-A domain at canonical alpha-satellite-based centromeres is characterized by local depletion of H3K9me3-mediated heterochromatin, coinciding with the DNA methylation dip region. We identified the SETDB1 methyltransferase as key to maintaining H3K9me3 within flanking active higher order alpha satellite arrays while SUV39s and SUZ12 contribute to globally heterochromatinize both alpha satellites and neighbouring repeats. Loss of this heterochromatin boundary results in the progressive expansion of the primary CENP-A domain, erosion of DNA methylation, and the nucleation of new centromeres across alpha satellite sequences. Our study identifies the functional specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and number.

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↗

bioRxiv2020

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S-phase, but not other cell cycle stages, caused accumulation of R-loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R-loop-dependent manner, unfinished replication and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S-phase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/277103v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@4e6dc4org.highwire.dtl.DTLVardef@1089c90org.highwire.dtl.DTLVardef@5ee5c2org.highwire.dtl.DTLVardef@3e300d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗