Biology subjects

Friman, E. T.

Publications and source records attributed to Friman, E. T..

13 recordsLinked to original sources

bioRxiv2026

Loop extrusion dynamics and cooperative activation independently regulate enhancer-driven transcription

How enhancers transmit their activation signals to target promoters is a fundamental unanswered question. Gene activation by distal enhancers is affected by the three-dimensional organisation of chromatin driven by cohesin-mediated loop extrusion, but how the dynamic loop extrusion cycle shapes transcriptional outcomes is not fully understood. In addition, genes are typically regulated by multiple enhancers and cases of cooperativity between enhancers have been observed that depend on the relative position of enhancers and gene. The reason for such context-dependent cooperativity is unknown, and whether it is linked to loop extrusion has not been explored. Here, we use synthetic gene activation to show that NIPBL depletion, which decreases loop extrusion rate, reduces long-range activation in a dose-dependent manner. In contrast, WAPL depletion, which reduces cohesin turnover, can both increase and decrease distal gene activation depending on the presence of adjacent CTCF binding sites. These effects on gene activation correlate with the impact on chromatin contacts. Depletion of the co-activator BRD4 also affects long-range activation, but not by changing chromatin contacts. Synthetic activation from two locations results in super-additive responses, which can be predicted by an independently determined response model. These predictions hold for dual activation from proximal and/or distal sites, and with perturbed NIPBL, WAPL, or BRD4. Distal activation therefore appears to be equivalent in nature to proximal activation, and context-dependent cooperativity can arise simply from different level of activation inputs operating on a non-linear response function.

molecular biology↗

bioRxiv2026

Benchmarking tissue- and cell type-of-origin deconvolution in cell-free transcriptomics

Plasma cell-free RNA (cfRNA) reflects tissue- and cell-type-specific activity across pathological states and is a promising biomarker for organ injury and disease. Computational deconvolution methods are widely used to infer organ and cell-type contributions to cfRNA profiles. However, most were originally developed for single-tissue bulk transcriptomes and their performance in body-wide cfRNA settings, where any tissue or cell type can contribute, remains poorly characterised. Here, we present a systematic benchmarking of tissue- and cell type-of-origin deconvolution for plasma cfRNA that considers both methodological and reference-related sources of variability under realistic cfRNA simulation settings. We evaluated seven commonly used deconvolution methods across distinct algorithmic classes and multi-organ reference configurations derived from bulk and single-cell atlases. We assessed performance using simulation frameworks that model multi-organ mixtures, technical noise, and transcript degradation. We further examined deconvolution methods across multiple previously published clinical cfRNA cohorts spanning diverse disease contexts. Across both tissue- and cell-type-level analyses, deconvolution performance was strongly influenced by both method choice and reference parameters. Tissue-of-origin inference was comparatively robust across simulated and clinical datasets, recovering disease-associated organ signals and concordance with biochemical markers. In contrast, cell type-of-origin inference showed greater variability and reduced consistency across analytical settings, leading to divergent interpretations in both simulations and published clinical cfRNA cohorts. Together, these findings demonstrate that methodological and reference-related variability are major sources of uncertainty in cfRNA deconvolution, with tissue-level inference being more robust than cell-type-level inference. Our benchmarking framework provides guidance for reference selection and comparative interpretation in cfRNA deconvolution.

bioinformatics↗

bioRxiv2026

Identifying severe COVID-19 risk variants modulating enhancer reporter activity in lung cells

Common genetic variants contribute to risk for complex human diseases. However, despite thousands of associations, variants modulating disease risk and their functional impact remain largely unknown. This includes SARS-CoV-2 infection, where outcomes range from asymptomatic to fatal. Most host risk variants associated with COVID-19 disease, identified through genome wide association studies, are located in the non-coding genome and may function by altering gene expression in disease-relevant cells and tissues. To address this at scale, we tested >4800 severe COVID-19-associated variants to determine the impact of individual variants and variant combinations on regulatory activity using Self-Transcribing Active Regulatory Region sequencing, a massively-parallel reporter assay, in a lung epithelial cell line (A549). We identify 166 variants within active sequences, of which 29 modulate activity allele-specifically. Evaluating variant combinations, we observe both additive and non-additive effects on regulatory activity. We employ state-of-the-art deep learning models to interpret allele-specific variant effects on regulatory activity and endogenous genomic features. Our work provides a set of prioritised severe COVID-19-associated variants that modulate regulatory activity in lung epithelial cells, candidate transcription factors, and candidate target genes with potential to be disease modifying.

molecular biology↗

bioRxiv2025

Disease-associated genetic variants can cause mutations in tissue-specific protein isoforms

Genetic variants can cause protein-coding mutations that result in disease. Variants are typically interpreted using the reference transcript for a gene. However, most human multi-exon genes encode alternative isoforms. Here, we show that coding exons in alternative isoforms harbour more population variants than exons of reference isoforms, consistent with their reduced evolutionary constraint, and that these variants are more likely to cause nonsynonymous coding mutations. Common and rare disease-associated variants mapping to alternative transcripts can lead to amino acid substitutions predicted to be structurally damaging in the corresponding protein isoform. The alternative transcripts to which disease-associated variants map demonstrate high tissue-specific expression, with many unannotated in reference human genomes, revealed only by long-read RNA-sequencing. As an example, we report an unannotated alternative transcript of the inflammasome regulator DPP9 that is lung epithelium-specific and which harbours a common genetic variant associated with severe COVID-19 and lung fibrosis. The variant causes a p.Leu8Pro missense mutation in an alternative first exon, predicted to disrupt the encoded alpha helix. These findings highlight the importance of considering alternative isoforms, their tissue-specific expression, and full-length transcripts in variant interpretation, with implications for uncovering underappreciated mechanisms of both common and rare disease.

genomics↗

bioRxiv2023

DNA-binding factor footprints and enhancer RNAs identify functional non-coding genetic variants

Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, these highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritisation of variants is commonly based on functional genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyse six markers of active regulatory elements for their ability to identify functional variants. We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA as markers for functional variants. This signature provides high precision, trading-off low recall, thus substantially reducing candidate variant sets to prioritise variants for functional validation. We present this as a framework called FINDER - Functional SNV IdeNtification using DNase footprints and Enhancer RNA, and demonstrate its utility to prioritise variants using leukocyte count trait and analyse variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritising variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches.

genomics↗

bioRxiv2023

Acetylation of H3K115 at the nucleosome dyad is associated with fragile nucleosomes at active regulatory sites.

Acetylation of lysine residues in the tail domain of histone H3 is well characterized, but lysine residues in the histone globular domain are also acetylated. Histone modifications in globular domain have regulatory potential because of their impact on nucleosome stability but remain poorly characterized. In this study we report the genome-wide distribution of acetylated H3 lysine 115 (H3K115ac), a residue on the lateral surface at the nucleosome dyad, using chromatin immunoprecipitation. In mouse embryonic stem cells, we find that detectable H3K115ac is enriched at the transcription start site of active CpG island promoters, but also at polycomb repressed promoters prior to their subsequent activation during differentiation. By contrast, at enhancers H3K115ac enrichment is dynamic, changing in line with gene activation and chromatin accessibility during differentiation. Most strikingly, we show that H3K115ac is detected as enriched on "fragile" nucleosomes within nucleosome depleted regions at promoters, and active enhancers where it coincides with transcription factor binding, and at CTCF bound sites. These unique features suggest that H3K115ac correlates with, and could contribute, to nucleosome destabilization and that it might be a valuable marker for identifying functionally important regulatory elements in mammalian genomes.

genomics↗

bioRxiv2023

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and physical proximity

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation by Capture-C, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent but are influenced by enhancer-bound protein complexes whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

genomics↗

bioRxiv2023

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops

Polycomb-repressive complex 1 (PRC1) has a strong influence on 3D genome organization, mediating local chromatin compaction as well as localized and chromosome-wide clustering of target loci. Several subunits of PRC1 have been shown to have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexandiol, which disrupts liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation combined with deep sequencing analyses, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci - at megabase and tens of megabase length scales - can be reversibly disrupted by the addition and subsequent removal of 1,6-hexandiol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to the reduction of PRC1 binding following 1,6-hexandiol treatment as the addition of 2,5-hexandiol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules, characteristic of liquid-liquid phase separation, have a role in polycomb-mediated genome organization.

molecular biology↗

bioRxiv2022

Ultra-long-range interactions between active regulatory elements

Contacts between enhancers and promoters are thought to relate to their ability to activate transcription. Investigating mechanisms that drive such chromatin interactions is therefore important for understanding gene regulation. Here, we have determined contact frequencies between millions of pairs of cis-regulatory elements from chromosome conformation capture datasets and analysed a collection of hundreds of DNA-binding factors for binding at regions of enriched contacts. This analysis revealed enriched contacts at sites bound by many factors associated with active transcription. We show that active regulatory elements, independent of cohesin and polycomb, interact with each other across distances of 10s of megabases in vertebrate and invertebrate genomes and that interactions correlate and change with activity. However, these ultra-long-range interactions are not dependent on RNA polymerase II transcription or several transcription cofactors. We propose that long-range interactions between cis-regulatory elements are driven by three distinct mechanisms - cohesin-mediated loop extrusion, polycomb contacts, and association between active regions.

genomics↗

bioRxiv2019

Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

genomics↗

bioRxiv2018

Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells

The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-{beta} stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

molecular biology↗

bioRxiv2018

Mitotic chromosome binding predicts transcription factor properties in interphase

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.

genomics↗

bioRxiv2018

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

developmental biology↗