Biology subjects

Zabet, N. R.

Publications and source records attributed to Zabet, N. R..

15 recordsLinked to original sources

bioRxiv2026

A Framework for Quantifying DNA Methylation Heterogeneity and Detecting Co-methylated loci from Native Nanopore Sequencing

DNA methylation is an important epigenetic mechanism involved in gene regulation. Most methods focus on analysing DNA methylation averaged from multiple reads, yet these average methylation profiles obscure heterogeneity between individual DNA molecules and coordinated methylation states across loci. Native Oxford Nanopore Technology (ONT) sequencing directly captures long, native DNA molecules together with their base modifications, allowing methylation to be studied at single-molecule resolution. Here, we present a scalable framework for genome-wide methylation analysis using ONT sequencing at single-molecule resolution, focused on two features that site-level summaries cannot recover. First, it quantifies molecule-to-molecule heterogeneity in DNA methylation by detecting Variable Methylated Domains (VMDs) and Variable Methylated Regions (VMRs). Second, it identifies coordinated methylation, as co-methylated positions (CMPs) and regions (CMRs), from the states observed on the same individual DNA molecules. Using data from human LCL cells, we demonstrate that substantial molecule-level methylation heterogeneity is masked by site-level summaries. Co-methylation analysis reveals coordinated patterns between CpG sites and genomic regions, including shared and sex-specific patterns, uncovering methylation organisation not apparent from average methylation levels. We also show that CMPs can be used to detect TF-pairs that are predicted to have coordinated binding. This framework is integrated within the DMRcaller R/Bioconductor package.

bioinformatics↗

bioRxiv2026

Explainable AI identifies H3K18ac as a new marker of active enhancers

Enhancers are non-coding regions of DNA that regulate gene transcription, yet the mechanisms underlying enhancer activity remain incompletely understood. Despite extensive experimental and computational efforts, we still lack accurate enhancer maps in many human cells, tissues and disease contexts. Here, we developed several Artificial Intelligence (AI) models (Convolutional Neural Networks (CNN), XGBoost, Logistic Regression (LR) and an eXplainable Artificial Intelligence type2 Fuzzy Logic based System (type2-FLS)) to predict enhancers across different human and mouse cell lines. While all models display high accuracy in the cell lines they were trained on, our results confirmed that type2-FLS, and, partially, CNN, LR and XGBoost perform consistently well in cell lines unseen during training, supporting the generalisation of the models. Most importantly, type2-FLS identified H3K18ac as an important enhancer mark along with many novel putative enhancers, which display the same epigenetic signatures as experimentally identified ones. We have validated some of these novel enhancers by both global epigenetic perturbations and directed enhancer epigenetic rewriting (CRISPRi). Interestingly, seven epigenetic marks in humans and five in mouse are sufficient to annotate enhancers without losing accuracy. Overall, we have deciphered the epigenetic code of mammalian enhancers and annotated enhancers in multiple human and mouse cell lines.

genomics↗

bioRxiv2026

HiCPotts: An R/Bioconductor package to identify significant interactions in chromosome conformation capture data and model sources of biases.

MotivationChromosome Conformation Capture methods, including Hi-C, micro-C or Capture-C, are used to map chromatin interactions genome-wide. Most of the existing computational methods do not account for sources of biases (such as DNA accessibility, GC content or TE content) in the data. ResultsWe previously developed ZipHiC, a Bayesian method based on a the hidden Markov random field (HMRF) model and the Approximate Bayesian Computation (ABC), that uses zero-inflated Poisson distribution to model the noise, signal and false signal of the data and showed that this approach was able to detect biases from DNA accessibility, GC content and TE content in both Hi-C and micro-C data. Here, we present HiCPotts, another Bayesian method based on the HMRF model and the ABC that uses a zero-inflated Negative Binomial distribution instead to model the noise and signal of the data. We systematically show that HiCPotts reduces false positives and increases recovery of true interactions compared to ZipHiC, but also compared to other methods such as FastHiC, Juicer and HiCExplorer. Most importantly, we provide an R/Bioconductor package that allows modelling the noise, signal and false signal using various distributions such as the zero-inflated Negative Binomial (ZINB) and the zero-inflated Poisson distribution (ZIP). Availabilityhttps://bioconductor.org/packages/HiCPotts/

bioinformatics↗

bioRxiv2026

Convergent evolution of cluster-wide Hox gene regulation in Bilateria

The anteroposterior collinear expression of Hox genes is a hallmark of animal development that underpins the diversification of body plans1 and life cycles2. However, the origin and drivers of this coordinated expression remain elusive: while vertebrates rely on complex cluster-wide Hox gene regulation3-8, insects define gene-specific, sub-cluster regulatory domains9-11. Here, we discover a new mode of Hox gene regulation in segmented worms (Annelida). By combining chromatin conformation data with histone modifications profiling in Owenia fusiformis, we show that a large distal enhancer forms developmentally regulated, long-range contacts across the Hox cluster, and its activation coincides with the consolidation of a cluster-wide topologically associating domain (TAD), loss of Polycomb-mediated repression, and Hox gene transcription. This chromatin structure also occurs in the annelids Dimorphilus gyrociliatus and Capitella teleta, the latter showing additional subTAD structures that correlate with Hoxs temporal collinearity12. Moreover, related phyla with intact, organised Hox clusters and spatial collinearity, such as nemerteans and chitons, show annelid-like chromatin organisations, whereas phyla with disorganised13 Hox clusters do not. Coordinated Hox gene regulation from a "global control region" is thus ancestral to Lophotrochozoa, indicating that complex regulatory logics based on cluster-wide, long-range chromatin interactions with distal enhancers evolved convergently in vertebrates and spiralians.

developmental biology↗

bioRxiv2024

Modelling medulloblastoma pathogenesis and treatment in human cerebellar organoids.

Faithful genetically engineered in vivo models of medulloblastoma (MB) are currently available only for some molecular subgroups, in keeping with recent studies showing the unique role of human-specific progenitors in the development of G3 and G4 MB subgroups. We generated human cerebellar organoids (CbO) from expanded potential stem cells (EPSC) and characterised their epigenetic and transcriptomic profile compared to the developing human cerebellum. We show the presence of sub compartment-specific cerebellar lineages linked to MB formation, including populations expressing signature genes of putative G3 and G4 MB cells-of-origin. We show that these lineages can be genetically engineered to model MB tumour onset. Moreover, we demonstrate that CbO sustain proliferation and invasion of G3/4 MB cells in a 3D co-culture model (CbO-MB) while preserving their molecular identity, and that treatment of CbO-MB with anti-tumour compounds recapitulate the efficacy of in vivo drug testing in xenograft models.

cancer biology↗

bioRxiv2024

Gene body methylation buffers noise in gene expression in plants

Non-genetic variability in gene expression is an inevitable consequence of stochastic nature of processes driving transcription and translation. Largely thought to be deleterious to cell fitness, it is not uniform across the transcriptome. This implies the existence of (molecular) determinants affecting the degree of gene expression variability, although this remain poorly understood in multicellular systems. In this study, we found a link between gene body methylation and noise in gene expression in Arabidopsis thaliana. More specifically, genes with high levels of noise show low levels of gene body methylation, while genes with lower level of noise in gene expression show higher level of gene body methylation. Most importantly, loss of CpG methylation in gene bodies lead to a significant number of genes displaying higher noise in gene expression. This could be compensated by low but significant gain of non-CpG methylation at promoters of certain genes. Overall, our results show that gene body methylation has a functional role and specifically controls the noise in gene expression for a large number of genes.

molecular biology↗

bioRxiv2024

DNA methylation contributes to plant acclimation to naturally fluctuating light

Plants in the natural environment experience continuous dynamic changes in light intensity. We have limited understanding on how plants adapt to such variable conditions. Here, we exposed Arabidopsis thaliana plants to naturally fluctuating light regimes alongside traditional square light regimes such as those often found in control environment growth chambers. The physiological response was highly consistent across experiments, indicating the involvement of an epigenetic mechanism, leading us to investigated differences in DNA methylation. Our results identified a large number of alterations in DNA methylation patterns between fluctuating light acclimated plants, and square light acclimated plants, demonstrating natural fluctuations in light impacts the plant epigenetic mechanisms. Most importantly, there are more differences in DNA methylation patterns between different light pattern regimes than between different light intensities. These differences in DNA methylation were accompanied by significant changes in gene expression, some of which correlated with altered DNA methylation. Interestingly, several transposable elements which displayed differential methylation were found to be differentially expressed between light regimes. Our data suggests DNA methylation plays a role in acclimation to natural light which may directly regulate gene expression and impact transposable element activation.

genomics↗

bioRxiv2024

Systematic investigation of interindividual variation of DNA methylation in human whole blood

Interindividual genetic variability is well characterised, but we still lack a complete catalogue of loci displaying variable and stable epigenetic patterns. Here, we report a catalogue of stable and variable interindividual DNA methylation in human whole blood by analysing the DNA methylation patterns in 3642 individuals using the IlluminaEPIC array. Our results showed that 41,216 CpGs display stable methylation (SMPs) and 34,972 CpGs display variable methylation levels (VMPs). This catalogue will be a useful resource for interpretation of results when associating epigenetic signals to phenotypes. We observed that SMPs are highly enriched in CpG islands, depleted at CpG shelves and open sea regions of the genome. In addition, we found that the VMPs were under higher genetic control than the SMPs and that trans mQTL pairs are often located in the same TAD or connected by chromatin loops. A subset of these VMPs (784) were classified as putative epialleles and our results demonstrate that these loci located in regulatory regions exhibit a link with gene expression.

genomics↗

bioRxiv2023

Identification of mammalian transcription factors that bind to inaccessible chromatin

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We asked whether TFs preferred to bind to DNA in open or dense chromatin conformation and found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

genomics↗

bioRxiv2022

H4K16ac activates transcription of transposable elements and contribute to their cis regulatory function

Mammalian genomes harbour a large number of transposable elements (TEs) and their remnants. Many epigenetic repression mechanisms are known to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here we demonstrated that the male-specific lethal (MSL) complex mediated acetylation of histone H4 lysine 16 (H4K16ac) activates transcription of long interspersed nuclear elements (LINE1, L1) and long terminal repeats (LTRs). Furthermore, we show that the H4K16ac marked L1 and LTR subfamilies function as enhancers and are enriched with chromatin features associated with active enhancers and looping factors. L1 and LTRs enriched with histone acetylations are bound by chromatin looping factors and these regions loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape of a significant portion of the mammalian genome by maintaining an active chromatin landscape at TEs. One Sentence SummaryH4K16ac activates LINE1 and ERV/LTR transcription and rewires the cis-regulatory landscape of a significant portion of the mammalian genome by increasing the transcriptional activity at TEs.

genomics↗

bioRxiv2021

ZipHiC: a novel Bayesian framework to identify enriched interactions and experimental biases in Hi-C data

Several computational and statistical methods have been developed to analyse data generated through the 3C-based methods, especially the Hi-C. Most of existing methods do not account for dependency in Hi-C data. Here, we present ZipHiC, a novel statistical method to explore Hi-C data focusing on detection of enriched contacts. ZipHiC implements a Bayesian method based on a hidden Markov random field (HMRF) model and the Approximate Bayesian Computation (ABC) to detect interactions in two-dimensional space based on Hi-C contact frequency matrix. ZipHiC uses data on the sources of biases related to contact frequency matrix, allows borrowing information from neighbours using the Potts model and improves computation speed by using the ABC model. In addition to outperforming existing tools on both simulated and real data, our model also provides insights into different sources of biases that affects Hi-C data. We show that some datasets display higher biases from DNA accessibility or Transposable Elements content. Furthermore, approximately half of the detected significant interactions connect promoters with other parts of the genome indicating a functional biological role. Finally, we found that the micro-C datasets display higher biases from DNA accessibility compared to a similar Hi-C experiment, but this can be corrected by ZipHiC.

bioinformatics↗

bioRxiv2021

Characterising sex differences of autosomal DNA methylation in whole blood using the Illumina EPIC array

Sex differences are known to play a role in disease etiology, progression and outcome. Previous studies have revealed autosomal epigenetic differences between males and females in some tissues, including differences in DNA methylation patterns. Here, we report for the first time an analysis of autosomal sex differences in DNAme using the Illumina EPIC array in human whole blood (n=1171). We identified 554 sex-associated differentially methylated CpG sites (saDMPs) with the majority found to be hypermethylated in females (70%). These saDMPs are enriched in CpG islands and CpG shores and located preferentially at 5UTRs, 3UTRs and enhancers. Additionally, we identified 311 significant sex associated differentially methylated regions (saDMRs). Transcription factor binding site enrichment revealed enrichment of transcription factors related to critical developmental processes and sex determination such as SRY and SOX9. Our study reports a reliable catalogue of sex associated CpG sites and elucidates several characteristics of these sites.

genomics↗

bioRxiv2021

The role of insulators and transcription in 3D chromatin organisation of flies

The DNA in many organisms, including humans, is shown to be organised in topologically associating domains (TADs). In Drosophila, several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. Here, we show that depletion of BEAF-32, Cp190, Chro and Dref leads to changes in TAD organisation and chromatin loops. Their depletion predominantly affects TAD borders located in heterochromatin, while TAD borders located in euchromatin are resilient to these mutants. Furthermore, transcriptomic data has revealed hundreds of genes displaying differential expression in these mutants and showed that the majority of differentially expressed genes are located within reorganised TADs. Our work identifies a novel and functional role for architectural proteins at TAD borders in Drosophila and a link between TAD reorganisation and subsequent changes in gene expression.

genomics↗

bioRxiv2019

Dissecting the binding mechanisms of transcription factors to DNA using a statistical thermodynamics framework.

Transcription Factors (TFs) bind to DNA and control activity of target genes. Here, we present ChIPanalyser, a user-friendly, versatile and powerful R/Bioconductor package predicting and modelling the binding of TFs to DNA. ChIPanalyser performs similarly to state-of-the-art tools, but is an explainable model and provides biological insights into binding mechanisms of TFs. We focused on investigating the binding mechanisms of three TFs that are known architectural proteins CTCF, BEAF-32 and su(Hw) in three Drosophila cell lines (BG3, Kc167 and S2). While CTCF preferentially binds only to a subset of high affinity sites located mainly in open chromatin, BEAF-32 binds to most of its high affinity binding sites available in open chromatin. In contrast, su(Hw) binds to both open chromatin and also partially closed chromatin. Most importantly, differences in TF binding profiles between cell lines for these TFs are mainly driven by differences in DNA accessibility and not by differences in TF concentrations between cell lines. Finally, we investigated binding of Hox TFs in Drosophila and found that Ubx binds only in open chromatin, while Abd-B and Dfd are capable to bind in both open and partially closed chromatin. Overall, our results show that TFs display different binding mechanisms and that our model is able to recapitulate this diverse repertoire of mechanisms.

genomics↗

bioRxiv2018

Chromatin architecture reorganisation during neuronal cell differentiation in Drosophila genome

Compartmentalisation of the genome as topologically associating domains (TADs) may have regulatory role in development and cellular functioning, but the, mechanism involved in TAD establishment is still unclear. Here, we present the first high-resolution contact map of Drosophila melanogaster neuronal cells (BG3) and identified different classes of TADs by comparing this to genome organisation in embryonic cells (Kc167). We find new rearrangements during differentiation in neuronal cells reflected as enhanced long-range interactions, which is supported by pronounced enrichment of CTCF at cell type specific borders. Furthermore, we show the presence of strong divergent transcription corroborated with RNA Polymerase II occupancy and increased DNA accessibility at the TAD borders. Interestingly, TAD borders that are specific to neuronal cells are enriched in enhancers controlled by neuronal specific transcription factors. Our results suggest that TADs are dynamic across developmental stages and reflect the interplay between insulators, transcriptional states and enhancer activities.

genomics↗