Biology subjects

Naughton, C.

Publications and source records attributed to Naughton, C..

7 recordsLinked to original sources

bioRxiv2026

Cross-chemistry single-nucleus RNA-seq identifies gene length and CpG-island promoters as determinants of transcriptional noise.

Cell-to-cell transcriptional heterogeneity, or noise, is an intrinsic property of the transcriptome with implications for development, disease progression, and aging. Bulk RNA-seq masks this variability by averaging gene expression across cells, whereas single-cell RNA sequencing (scRNA-seq) resolves it. Nevertheless, separating biological noise from technical variance remains challenging, particularly across platforms with different chemistries. We benchmarked two widely adopted technologies, Evercode WT (SPLiT-seq, Parse Biosciences) and Chromium (10x Genomics), on human lymphoblastoid nuclei. Evercode WT achieved targeted sequencing depth and nuclei number far more reliably, and its random-hexamer priming yielded more intronic reads and non-coding RNA genes; Chromium recovered more cells and detected polyadenylated transcripts and cell-line markers more sensitively. Despite these opposing biases, the platforms showed comparable gene detection and strongly correlated expression profiles. Using datasets from both platforms, we defined a noise metric detrended from mean expression and showed that per-gene estimates were reproducible across chemistries. Noise was lower in G2M than in G1 and was most strongly associated with gene length rather than exonic length. Expression of genes with CpG-island promoters was less variable than that of those without. This study establishes a platform-independent basis for quantifying transcriptional noise and a framework for selecting an appropriate scRNA-seq platform.

genomics↗

bioRxiv2026

CpG islands act as topological sinks for transcription-induced DNA supercoiling

Strong evolutionary selection has maintained CpG-dense islands (CGIs) at the promoters of constitutively expressed genes throughout the vertebrate genome, suggesting an important role in regulating DNA topology. Here, using Twist-seq, a psoralen-based approach for quantitative genome-wide profiling of DNA supercoiling, we reveal distinct topological states across human gene promoters. We show that CGI promoters accumulate elevated levels of negative supercoiling relative to non-CGI promoters and define localised topological domains at highly transcribed genes. Integrating genome-wide analyses with reaction-diffusion modelling and coarse-grained molecular dynamics simulations, we find that this behaviour is encoded by the intrinsic physical properties of CGI DNA. The GC-rich sequence context promotes nucleosome depletion and focuses torsional stress onto embedded AT-rich pockets, driving localised DNA melting and plectoneme-tip bubble formation within promoter-proximal nucleosome-free regions. This provides an energetically favourable pathway for redistributing transcription-induced torsional stress through transient strand separation and writhe, consistent with increased ssDNA formation at CGI promoters observed by ssDNA-seq. We propose that CGIs function as sequence-encoded topological sinks that buffer supercoiling while maintaining a promoter architecture permissive for transcription initiation, thereby preserving promoter integrity and genome stability.

molecular biology↗

bioRxiv2026

CENP-B binds hairpin motifs in chromosome arms influencing gene expression

CENP-B, a centromeric protein known for its role in binding the B box sequence of centromeric DNA, has long been recognized as important, though not essential, for kinetochore attachment and chromosome segregation. Here, we identify an unexpected, non-centromeric role for CENP-B. We demonstrate that CENP-B binds to specific non-centromeric sites along chromosome arms, predominantly at promoters, and depletion of CENP-B leads to dysregulated gene expression. Binding is enriched in G2 phase cells and, importantly, occurs independently of the canonical B box motif. Instead, CENP-B binding in chromosome arms is defined by regions of negatively supercoiled DNA containing repetitive sequences, such as multiple CCAAT boxes, that are prone to forming secondary structures. Consistently, we find that CENP-B binds to hairpin DNA in vitro via its DNA binding domain. The chromosome arm binding pattern is conserved across cell types and is particularly prominent in the promoters of transcriptionally active replication-dependent histone genes. These findings reveal a previously unrecognized centromere-independent binding activity of CENP-B.

cell biology↗

bioRxiv2024

Coordinated control of genome-nuclear lamina interactions by Topoisomerase 2B and Lamin B receptor

Lamina-associated domains (LADs) are megabase-sized genomic regions anchored to the nuclear lamina (NL). Factors controlling the interactions of the genome with the NL have largely remained elusive. Here, we identified DNA topoisomerase 2 beta (TOP2B) as a regulator of these interactions. TOP2B binds predominantly to inter-LAD (iLAD) chromatin and its depletion results in a partial loss of genomic partitioning between LADs and iLADs, suggesting that its activity might protect specific iLADs from interacting with the NL. TOP2B depletion affects LAD interactions with lamin B receptor (LBR) more than with lamins. LBR depletion phenocopies the effects of TOP2B depletion, despite the different positioning of the two proteins in the genome. This suggests a complementary mechanism for organising the genome at the NL. Indeed, co-depletion of TOP2B and LBR causes partial LAD/iLAD inversion, reflecting changes typical of oncogene-induced senescence. We propose that a coordinated axis controlled by TOP2B in iLADs and LBR in LADs maintains the partitioning of the genome between the NL and the nuclear interior. HighlightsO_LILADs and iLADs differ in supercoiling state C_LIO_LITOP2B controls genome partitioning between nuclear lamina and nuclear interior C_LIO_LITOP2B depletion preferentially affects genome interactions with LBR C_LIO_LISimilar impact of TOP2B depletion and LBR depletion on genome-NL interactions C_LIO_LICo-depletion of TOP2B and LBR recapitulates LAD reshaping typical of oncogene-induced senescence. C_LI

genomics↗

bioRxiv2022

Gene structure heterogeneity drives transcription noise within human chromosomes

Classical observations have long suggested there is a link between 3D gene structure and transcription1-4. However, due to the many factors regulating gene expression, and to the challenge of visualizing DNA and chromatin dynamics at the same time in living cells, this hypothesis has been difficult to quantitatively test experimentally. Here we take an orthogonal approach and use computer simulations, based on the known biophysical principles of genome organisation5-7, to simultaneously predict 3D structure and transcriptional output of human chromatin genome wide. We validate our model by quantitative comparison with Hi-C contact maps, FISH, GRO-seq and single-cell RNA-seq data, and provide the 3DGene resource to visualise the panoply of structures adopted by any active human gene in a population of cells. We find transcription strongly correlates with the formation of protein-mediated microphase separated clusters of promoters and enhancers, associated with clouds of chromatin loops, and show that gene noise is a consequence of structural heterogeneity. Our results also indicate that loop extrusion by cohesin does not affect average transcriptional patterns, but instead impacts transcriptional noise. These findings provide a functional role for intranuclear microphase separation, and an evolutionary mechanism for loop extrusion halted at CTCF sites, to modulate transcriptional noise.

molecular biology↗

bioRxiv2021

Centromere formation remodels chromatin fibre structure

Human centromeres appear as constrictions on mitotic chromosomes and form a platform for kinetochore assembly in mitosis. Biophysical experiments led to a suggestion that repetitive DNA at centromeric regions form a compact scaffold necessary for function, but this was revised when neocentromeres were discovered on non-repetitive DNA. To test whether centromeres have a special chromatin structure we have analysed the architecture of a neocentromere. Centromere formation is accompanied by RNA pol II recruitment and active transcription to form a decompacted, negatively supercoiled domain enriched in open chromatin fibres. In contrast, centromerisation causes a spreading of repressive epigenetic marks to surrounding regions, delimited by H3K27me3 polycomb boundaries and divergent genes. This flanking domain is transcriptionally silent and partially remodelled to form compact chromatin, similar to satellite-containing DNA sequences, and exhibits genomic instability. We suggest transcription disrupts chromatin to provide a foundation for kine-tochore formation whilst compact pericentromeric heterochromatin generates mechanical rigidity.

molecular biology↗

bioRxiv2018

Aberrant chromatin resolution in G2/M leads to chromosome instability

Cells coordinate interphase to mitosis transition but recurrent cytogenetic lesions appear at common fragile sites (CFSs) in a tissue-specific manner following replication stress, marking regions of instability in cancer. Despite such a distinct defect no model fully explains their molecular configuration. We show that CFSs are characterised by impaired chromatin folding manifested as disrupted mitotic structures visible using molecular FISH probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Subsequently cytogenetic and molecular lesions arise due to faulty condensin loading at CFSs, through a defect in condensin I mediated compaction and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS formation, concomitantly providing an environment for MIDAS, which, if not resolved, result in chromosome instability.

molecular biology↗