99�Ⱦò������µ�ַ

Poised enhancers (PEs), co-marked by H3K4me1 and Polycomb-associated H3K27me3, are common in primed human pluripotent stem cells (hPSCs) resembling post-implantation epiblast but scarce in naive hPSCs modeling pre-implantation epiblast. PEs form abundant chromosomal contacts with developmental genes, but the timing of their emergence, their relationship to enhancer poising, and their functional significance remain unclear. We devised high-resolution, PE-targeted Capture Hi-C to map PE contacts during the transition from naive to primed pluripotency. We find that enhancer poising emerges early in the transition, while the contacts show diverse dynamics. PROTAC-induced degradation of Polycomb repressive complex 2 early in the transition, but not inhibition of its H3K27 methyltransferase activity, weakens PE connectivity. Finally, PE contacts persist after developmental activation or ectopic CRISPRa targeting and can mediate long-range gene induction. Together, these findings reveal the temporal and mechanistic principles of PE connectivity and highlight a potential role of PE contacts in establishing developmental gene expression patterns.

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.