David Santiago-Algarra

The cell cycle is a highly regulated process that ensures the accurate replication and transmission of genetic information from one generation of cells to the next. It is a fundamental biological process and plays a crucial role in development, growth, and maintenance of all living organisms, and its dysregulation can lead to a number of pathologies such as autoimmune diseases, neurodegenerative diseases, and cancer. In this work we present a novel approach to study the gene expression and chromatin accessibility dynamics during the cell cycle in mouse embryonic stem cells. To achieve this, we combined high-depth single-cell multiome sequencing, biophysical modeling, and advanced deep learning techniques. First, we used DeepCycle, a deep learning tool that assigns a cell cycle phase to every cell based on its spliced and unspliced mRNA levels. We then developed a biophysical model that describes the dynamics of gene-specific mRNA transcription, nuclear export, and degradation rates during the cell cycle. Our model obtains genome-wide estimates of mRNA transcription, nuclear retention, and degradation half-lives of genes exhibiting oscillatory dynamics in mESCs. The key feature of single-cell multiome sequencing is that it simultaneously provides readouts for gene expression as well as chromatin accessibility in the same cells. By applying our approach to these data we showcase first-of-its-kind exploration of chromatin accessibility during the cell cycle at high temporal resolution. We believe that our work will be pivotal in formulating a coherent quantitative description of mRNA metabolism. Highlights Integration of single-cell and single-nucleus multiome profiling with deep learning and biophysical modeling uncovers cell cycle regulation of RNA metabolism Genome-wide estimation of mRNA transcription, nuclear export, and degradation rates during the cell cycle in mESCs Identification of a prominent wave of transcription during early G1 in unperturbed asynchronously proliferating mESCs Characterization of temporal waves of post-transcriptional mRNA regulation controlling both mRNA degradation and nuclear export during the cell cycle Chromatin accessibility dynamics within TF footprints reveal key cell cycle regulators

Gene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response.