98% of the DNA in our body is non-coding, i.e. does not carry the information needed to build proteins. Non-coding has sometimes been equated with ‘non-functional’, or called ‘junk’ in the past; today we know that this is far from the truth. Scattered throughout non-coding DNA is a plethora of so-called regulatory elements, including enhancers, silencers and insulators. These regulatory elements function like molecular switches to control which genes are active (and thus produce proteins) in which cells. This process of gene expression control is vital to allow cells – which all contain the same genes – to specialise to carry out different tasks, and to help them respond to changes.
Enhancers are a type of regulatory element that control gene expression over long distances. They contact their target genes via chromosomal interactions, often bridging large distances in the genome, with the intervening DNA ‘looping out’. To understand how enhancers work, we study them in the context of the three-dimensional organisation of the genome. Our aim is to find regulatory elements and to understand which genes they control. We also aim to uncover the molecular mechanisms by which regulatory elements find their target genes in the three-dimensional space of the cell nucleus, and to understand how altering the function of regulatory elements can lead to developmental malformations and disease. We study these questions in pluripotent stem cells – cells that have the potential to create all cell types in the adult body. We use a combination of molecular, genetic, biochemical and imaging approaches to study pluripotent stem cells in their ‘ground state’, and when they start to form new cell types – a process called cell lineage specification.
Through high-resolution mapping and experimental perturbation of the spatial genome architecture, we aim to reveal gene regulatory principles that underpin cell states and cell fate transitions. This may ultimately pave the way for us to experimentally engineer 3D genome folding to achieve predictable outcomes on gene expression and cell fate choice, with potential implications for gene therapy and regenerative medicine.
Although human pluripotent stem cells (hPSCs) can generate all tissues of the body, hPSCs in vitro frequently exhibit differentiation biases or failure that pose substantial challenges for disease modeling and regenerative medicine. The origins of these biases remain incompletely understood and extend beyond reprogramming artifacts. Here we show that loss of default neural differentiation capacity and failure to form brain organoids are linked to erosion of bivalent chromatin marks at developmental gene loci, independent of DNA methylation, driving acquisition of a posterior epiblast-like state and premature developmental gene expression. We develop a chemical chromatin restoration (CHR) approach that rescues this differentiation bias by reinstating transcriptional programs and chromatin landscapes characteristic of the competent anterior epiblast-like state, restoring broad differentiation potential. These findings establish locus-specific patterns of repressive and activating histone post-translational modifications as a tractable and experimentally targetable determinant of hPSC fate competency, and offer an effective route to rescue differentiation-compromised hPSC lines for applications in disease modeling and regenerative medicine.
Innate lymphoid cells (ILCs) are rare tissue-resident lymphocytes that functionally mirror cells of CD4 T helper lineage but lack antigen receptors. Type 3 ILCs (ILC3s) are enriched at barrier sites, regulating inflammation and promoting tissue integrity. Here we profile the promoter-anchored chromosomal contacts of primary human ILC3s using low-input, high-resolution targeted chromosome conformation capture and compare them with those in CD4 T cells. We use these data to link Crohn's disease genome-wide association study variants with target genes, implicating both known and unanticipated candidates, including CLN3, a causal gene for Batten disease. We show that Cln3 overexpression in a mouse ILC3-like cell line alters stimulation-induced transcriptional programs and cytokine secretion. Extending our approach to five additional immune genome-wide association study traits reveals enrichment for regulators of ILC3 activation. Our work develops methods, maps long-range gene regulation in ILC3s, and prioritizes immune disease risk genes with roles in this clinically relevant immune cell type.
Chinese hamster ovary (CHO) cells are the leading mammalian system for recombinant therapeutic protein production. However, optimizing transgene expression remains challenging due to the limited understanding of the regulatory mechanisms controlling gene expression in CHO cells. Towards overcoming this barrier, here we provide a systematic characterization of cis-regulatory elements in CHO cells. Using genome-wide STARR-seq, a high-throughput method for quantifying enhancer strength, we identified regions with enhancer activity in the CHO cell genome. By integrating these data with ATAC-seq and histone modification profiles, we were able to characterize the chromatin state of these regions. Our analysis revealed thousands of newly identified enhancer sequences. The most active sequences could drive transgene expression at levels similar to or higher than strong viral enhancers. Notably, half of the regions found to have enhancer activity were within inaccessible chromatin in their native context. We observed that accessible enhancers were primarily near to transcriptional start sites and associated with ubiquitously-expressed genes, whereas inaccessible enhancers were predominantly intergenic and associated with tissue-specific genes. Additionally, through a deep-learning-based approach ETS and YY1 transcription factor (TF) binding motifs were identified as key determinants of enhancer identity and strength. Disrupting YY1 binding motifs led to reduced enhancer activity, thereby highlighting the importance of YY1 as a transcriptional activator in CHO cells. Our study demonstrates the first comprehensive map of functionally-validated enhancers in CHO cells and generates new insights into gene regulation and the role of TFs in determining enhancer strength. This study helps to lay the foundation for strategic engineering of CHO cell transcriptional networks to achieve enhanced biopharmaceutical production.