Retrospective analysis of enhancer activity and transcriptome history

nature biotechnology Article https://doi.org/10.1038/s41587-023-01683-1 Retrospective analysis of enhancer activity and transcriptome history Received: 1 July 2021 Accepted: 20 January 2023 Published online: xx xx xxxx Ruben Boers 1,5, Joachim Boers1,5, Beatrice Tan 1,5, Marieke E. van Leeuwen Evelyne Wassenaar1, Erlantz Gonzalez Sanchez1, Esther Sleddens1, Yasha Tenhagen 1, Eskeatnaf Mulugeta 2, Joop Laven3, Menno Creyghton1, Willy Baarends1, Wilfred F. J. van IJcken 4 & Joost Gribnau 1 , 1 Check for updates Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. In this study, we used an inducible DCM–RNA polymerase subunit b fusion protein which labels active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. This DCM–RNA polymerase fusion protein enables transcribed genes and active enhancers to be tagged and then examined at later stages of development or differentiation. We apply this DCM-time machine (DCM-TM) technology to study intestinal homeostasis, revealing rapid and coordinated activation of enhancers and nearby genes during enterocyte differentiation. We provide new insights in absorptive–secretory lineage decision-making in intestinal stem cell (ISC) differentiation and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation-associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes. Embryonic development and cell differentiation are intricate processes directed by crosstalk between cells that affect cell fate decisions and the establishment of cell-type-specific gene expression programs1–3. Lineage tracing studies have been crucial to understand these processes. Initial studies applied light microscopy to follow cleavage divisions, and, more recently, barcoding, cre-lox and other genetic systems have been used to mark precursors or progenitors for readout at later stages of development or differentiation4. The present advance of single-cell RNA sequencing (scRNA-seq) technologies provides a wealth of expression data that can be used to predict developmental trajectories in silico and can be linked to genetic lineage-tracing techniques to rebuild lineage trees5–7. Application of these tracing technologies to study the epithelium of the small intestine provided critical insights in homeostasis and regeneration. Turnover of this epithelium happens within 7 days and starts with division of the intestinal stem cell (ISC) located at the bottom of the intestinal crypt8. ISCs give rise to progenitors that divide moving up the intestinal crypt, meanwhile committing to the absorptive or secretory lineage. Absorptive progenitors mature into enterocytes, whereas secretory progenitors give rise to Paneth, tuft, enteroendocrine and goblet cells. ISCs are flanked by Paneth cells that provide Wnt, Notch and epidermal growth factor (EGF) signals required for self-renewal. Loss of ISC–Paneth cell contact facilitates cell differentiation, aided by bone morphogenetic protein (BMP) signaling that further supports maturation of differentiated cell types. Notch signaling also plays a crucial role in lineage commitment remaining high in absorptive progenitors and is downregulated in secretory progenitors. Lineage-tracing and scRNA-seq experiments have been instrumental in identification and characterization of the crypt-based columnar cell as the ISC9 but also showed that several 1 Department of Developmental Biology, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands. 2Department of Cell Biology, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands. 3Department of Obstetrics and Gynaecology, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands. 4Erasmus Center for Biomics, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands. 5 These authors contributed equally: Ruben Boers, Joachim Boers, Beatrice Tan. e-mail: Nature Biotechnology Article other cell types, including enteroendocrine, Paneth and immature enterocytes, provide a reservoir of cells that can replenish the ISC niche in injury-induced regeneration10–12. Although these examples highlight the successful application of lineage-tracing and scRNA-seq technologies to build relationships between cellular trajectories, they cannot keep track of cell state changes following this trajectory and provide limited depth and temporal information with respect to gene expression changes13. To facilitate whole-genome cell state tracing, we developed a system to tag transcribed genes with DCM methylation labels to be examined at later stages of development or differentiation. We made use of a fusion between DCM and RNA polymerase 2 subunit b to DCM-label gene bodies of transcribed genes. DCM methylation of CmeC(A/T)GG penta-nucleotides is a bacterial form of cytosine methylation detected at only very low levels in most mammalian cell types but is maintained when introduced on transgenes in somatic cells without affecting transgene expression14. Our study demonstrates that DCM-time machine (DCM-TM) marks both active genes as well as enhancers and confirms that DCM methylation is propagated to daughter cells with limited effect on gene expression. Thus, DCM-TM provides a powerful technology to trace genome-wide gene transcription and enhancer activity back in time without relying on in silico assumptions. We applied DCM-TM to study homeostasis in the small intestine, generating gene and enhancer activity maps that trace the ISC state to the enterocyte state. We found that gene and enhancer activity changes during enterocyte differentiation are not mediated by heterochromatin changes, and we show that the H2A variant H2A.Z is preloaded at ISC enhancers that become activated in the enterocyte. Application of DCM-TM also indicated that commitment of progenitors to the absorptive lineage is a one-way event that does not involve a temporarily dynamic absorptive–secretory intermediate state. Results DCM–POLR2B labels active genes To develop a gene activity tagging system, we fused the bacterial methyltransferase DCM to the N-terminal end of mouse RNA polymerase 2 subunit b (Polr2b; Fig. 1a) and introduced this DCM–Polr2b fusion gene into the Col1a1 locus in an embryonic stem cell (ESC) line harboring the m2rtTA trans-activator expressed from the Rosa26 locus (Supplementary Fig. 1a,b)15. Addition of doxycycline (dox) leads to expression of the fusion protein at levels lower than endogenous POLR2B, and fusion RNA and protein expression is depleted 24 hours after removal of dox (Supplementary Fig. 1c–e). To detect DCM methylation, we developed methylated DNA sequencing (MeD-seq), a technology based on LpnPI-mediated digestion of CpG an (...truncated)