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Qt3c2713f9 Nosplash 56D6be0 ARTICLE https://doi.org/10.1038/s42003-020-01181-z OPEN Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt Amber N. Habowski 1, Jessica L. Flesher 2, Jennifer M. Bates3, Chia-Feng Tsai 4, Kendall Martin4, Rui Zhao5, Anand K. Ganesan2,6, Robert A. Edwards 7, Tujin Shi 4, H. Steven Wiley5, Yongsheng Shi1, ✉ Klemens J. Hertel1 & Marian L. Waterman 1 1234567890():,; Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and poly- adenylation dominate changes in the transcriptome as stem cells differentiate into pro- genitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt. 1 Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697, USA. 2 Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA. 3 Institute for Immunology, University of California Irvine, Irvine, CA 92697, USA. 4 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA. 5 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA. 6 Department of Dermatology, University of California Irvine, Irvine, CA 92697, USA. 7 Department of Pathology and ✉ Laboratory Medicine, University of California Irvine, Irvine, CA 92697, USA. email: [email protected] COMMUNICATIONS BIOLOGY | (2020) 3:453 | https://doi.org/10.1038/s42003-020-01181-z | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01181-z he intestinal crypt is a good model for studying how stem Here, we developed a new cell sorting protocol for purification Tcells support a rapidly renewing tissue. Crypts are invagi- and comparative analysis of colon stem cells, their immediate nating structures of single-layer epithelium in which stem daughters (SecPro, AbsPro), and their differentiated cell types, cells reside in a supportive niche at the base where they produce including tuft cells, EEC, and enterocytes (Ent). The protocol can daughter cells (progenitors). Progenitors move up the crypt to be used with non-transgenic mice of any strain and importantly, differentiate and replace mature cells that are dying at the when coupled to bulk RNA sequencing and mass spectrometry- mucosal surface—a process with an average lifespan of only based global proteome profiling, can provide a deeper analysis of 4–5 days1. Constant replacement maintains homeostasis and cellular transcriptomes and proteomes. Using this protocol, we proper absorption of water and nutrients, but the fast timescale of found that while the transcriptome and proteome of each cell birth-to-death places great demand on both stem and daughter type are generally correlated, deeper analyses of the bulk RNA- cells. Stem cells are by necessity non-quiescent and rapidly seq data reveal that loss of stemness and lineage commitment are dividing, and progenitor cells exhibit rapid loss of stemness and accompanied by a greater change in mRNA splicing and poly- commitment to differentiation. Multiple studies have shown how adenylation than in gene expression, a pattern that largely absorptive and secretory cell typescanrespondtowoundingbyde- resolves as progenitor cells mature. Sequencing analysis also differentiation and repopulation of the stem cell compartment2–5. enabled higher resolution of signal transduction systems (Wnt Although this de-differentiation process occurs promptly, it is and Myc signaling), environmental sensing pathways, and pat- unknown if these reverse changes in cell state and gain of stemness terns of lineage distinction, including prostaglandins and Fgf occuronasimilarrapidtimescaleaslossofstemness. signaling pathways. These patterns were seen at both the RNA Quantitative imaging and lineage-tracing tools have shown that and protein level and are likely key to understanding the pro- newly produced progenitor cells lose stemness as they move from cesses of homeostasis, namely: (i) loss of stemness, (ii) lineage the stem cell niche into a compartment called the transit ampli- commitment, and (iii) signaling connections between mature cell fying zone (TAZ)6. The progenitor’s first round of cell division types. We relate how these findings are relevant to the earliest and commitment to either an absorptive (AbsPro) or secretory events that happen during loss of stemness and we highlight ways (SecPro) lineage happens nearly simultaneously with entrance in which mature cells might de-differentiate to re-acquire the into this zone. These changes occur within minutes-to-hours of state of stemness. each other, suggesting that loss of stemness and choice of cell lineage are connected and directed by processes that occur on this timescale. Results Several signal transduction systems are important for the early Flow sorting purification of colon crypt cell populations.To changes in cell state. A decrease in Wnt signaling and activation create a high-resolution profile of colon crypt stem cells and their of the unfolded protein response (UPR) correlates with loss of daughter cells, we developed a new flow sorting protocol using stemness7. In addition, Notch signaling balances commitment to freshly dissected, wild-type C57BL/6 N mouse colons and anti- either the absorptive or secretory lineage through lateral inhibi- bodies to validated intestinal cell surface markers including Cd44 tion signaling4. There has been a longstanding expectation that (Fig. 1a, b, Supplementary Fig. 1). Upon discovery that Cd44 is stem cells are defined by a unique transcriptome and that loss of highly sensitive to TrypLE, and other commonly used proteases16 stemness and lineage commitment are similarly defined by (Supplementary Fig. 2a), we developed a dissociation protocol unique signatures. However, although signaling systems are that uses only ethylenediaminetetraacetic acid (EDTA) and capable of altering transcription, it is not known how much of the mechanical force. This change resulted in a 10-fold increase in rapid changes in cell state are due to the turning ON/OFF of detectable Cd44 antigen surface expression and therefore higher signal-targeted gene programs versus more immediate processes resolution for cell sorting (Fig. 1c, Supplementary Fig. 2b). Using of co- and post-transcriptional processing, such as alternative additional commonly used cell surface markers, six cryptal mRNA splicing and alternative polyadenylation (APA)8–10. Each populations could be isolated. A previously validated intestinal of these processes can quickly modify the nascent transcriptome stem cell signature of Cd44-high, Cd24-low, and cKit-negative and its attendant proteome by altering the coding sequences of was used to identify and isolate an abundant fraction of stem cells mRNAs, the localization or interactions of mRNA and proteins, (Fig. 1c). This cell population directly overlapped with Lgr5- or by changing protein abundance through alterations in mRNA EGFP+ cells from Lgr5-EGFP-IRES-creERT2 mice, confirming stability and/or protein translation rates11–14. their stem cell identity24 (Supplementary Fig. 3a). To study how transcription and post-transcription processes In addition to the stem cell population, five additional Epcam- contribute to stemness and differentiation, it is necessary to positive populations were collected (Fig. 1c) and replicate separate stem cells, daughter cells, and their differentiated biological samples of the six populations were processed for bulk progeny. Multiple cell sorting protocols have been optimized to RNA-seq (Supplementary Data #1). These populations represent isolate stem cells, but each lack resolution of these three cell cellular subtypes from two distinct cryptal lineages (secretory and types15,16. For example, the transgenic stem cell lineage marker absorptive), each revealing a trajectory of differentiation from Lgr5-EGFP enables purification of GFP-bright stem cells, but a stem to mature populations (Fig. 1d). Principal Component mosaic expression pattern of the transgene in the intestine analysis (PCA) of mRNA and protein from the sorted cells has made it difficult to confidently separate daughter cells showed that these cryptal populations were distinct and replicates from GFP-negative stem cells and differentiated cells17,18. tightly clustered (Fig. 1e, f). Known markers for colon crypt cells Single-cell RNA-sequencing captures the diversity when ana- were used to identify cell types (Supplementary Fig. 4), which lyzing mixed cell populations and has been useful for defining clearly demonstrated the presence of stem cells, two distinct intestinal lineage trajectories and diversity of mature cells (for populations of progenitor cells (absorptive and
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