We are interested in understanding how the epigenome is established during human development and stem cell differentiation, and how epigenetic information changes over the life course of a person.
To research these topics, we use different types of stem cell (primarily human pluripotent stem cells), stem cell-based embryo models (blastoids and gastruloids), and donated human embryos, in combination with a variety of molecular and genetic approaches to investigate their epigenomes.
This research is important because establishing our epigenomes correctly during development is vital for establishing a healthy pregnancy, and has long lasting consequences on our health. We therefore need to know more about how it happens and why it sometimes goes wrong. Our work also provides new avenues for improving the epigenetic stability of human pluripotent stem cells, and our ability to drive their specialisation towards useful cell types, which are essential requirements to fulfill their promise in regenerative medicine.
TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronized cells and does not require drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq that improves sample quality and includes multiplexing of up to six samples which dramatically improves throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, and instead replication forks accelerate across the first ∼1 Mb of travel irrespective of local transcriptional activity. We propose that this is a consequence of fewer replication forks being active later in S-phase when these distal regions replicate and there being less competition for replication factors.
Implantation of a human embryo into the endometrium is a crucial event in gestation, as it marks the initiation of a pregnancy and is prone to high failure rates. We have limited understanding of these stages because of the inaccessibility of implanting embryos and the lack of suitable model systems. Here, we establish an in vitro model that recapitulates the luminal, glandular, and stromal compartments of the superficial layer of receptive human endometrium. Human embryos and blastoids implant into the endometrial model, achieving post-implantation hallmarks including advanced trophoblast structures that underlie early events in placental development. Single-cell RNA sequencing of the embryo-endometrial interface at day 14 uncovers predicted molecular interactions between conceptus and endometrium. Disrupting signaling interactions between extravillous trophoblast and endometrial stromal cells caused defects in trophoblast outgrowth, demonstrating the importance of crosstalk processes to sustain embryogenesis. This platform opens the opportunity to investigate early stages of human embryo implantation.
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.