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Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5[superscript +] intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (nonISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoidinitiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how dietmodulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors., Howard Hughes Medical Institute, Ellison Medical Foundation (Aging Grant), United States. Department of Defense (PRCRP Career Development Award CA120198), National Institutes of Health (U.S.) (Grants R01 CA103866, AI47389, K08 CA198002, R00 AG045144, R00 AG041765, and DK043351), National Institutes of Health (U.S.) (Cancer Center Support Core Grant P30-CA14051), Kathy and Curt Marble Cancer Research Fund, American Federation for Aging Research, V Foundation for Cancer Research (Scholar Grant), Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (Post-doctoral Fellowship), Massachusetts General Hospital (Fellowship T32DK007191), Damon Runyon Cancer Research Foundation (Robert Black Fellowship)

The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in embryonic stem cell (ESC) self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed., National Science Foundation (U.S.) (Graduate Research Fellowship), Whitehead Institute for Biomedical Research (Jerome and Florence Brill Graduate Student Fellowship), Croucher Foundation (Research Fellowship), Virginia and Daniel K. Ludwig Graduate Fellowship, National Institutes of Health (U.S.) ((NIH) Kirschstein National Research Service Award (1 F32 GM099153-01A1)), National Institutes of Health (U.S.) (NIH grant HD 045022), National Institutes of Health (U.S.) (NIH grant R37CA084198)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, 2014., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student-submitted PDF version of thesis., Includes bibliographical references (pages 197-200)., This work is concerned with mRNA processing in mammalian cells and proceeds in two parts. In the first part, I introduce a computational framework for inferring the abundances of mRNA isoforms using high-throughput RNA sequencing data. This framework was applied to study the targets of the ubiquitous splicing factor hnRNP H in human cells. In the second part, I describe an experimental study of the Musashi (hnRNP-like) family of RNA-binding proteins in stem cells and cancer cells, which incorporates computational analyses that rely heavily on the framework developed in part one. In sum, this work provides a computational framework of general use in global analyses of RNA processing and its protein regulators, as well as functional insights into a family of poorly understood RNA-binding proteins. Several related analyses and techniques developed as part of the thesis are described in Appendix A-C. Appendix A describes a study of activity-dependent gene expression and mRNA processing in the mouse olfactory bulb. It uses computational techniques developed in part one of the thesis. Appendix B describes a technique for quantitative visualization of alternative splicing from RNA sequencing data and its integration into a genome browser. Appendix C describes a method for clonal analysis of neural stem cell growth and differentiation in culture using live imaging and `microdot' plates, developed as part of the work presented in part one of the thesis., by Yarden Katz., Ph. D.

Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system., National Institutes of Health (U.S.) (Grant HD 045022), National Institutes of Health (U.S.) (Grant R37CA084198)

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV cross-linking–immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression., National Science Foundation (U.S.), National Institutes of Health (U.S.)

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types., National Institutes of Health (U.S.) (Grant RO1-CA084198), National Institutes of Health (U.S.) (Grant U01-CA184897), National Institutes of Health (U.S.) (Grant R01-GM085319), National Science Foundation (U.S.). Graduate Research Fellowship (Grant 1122374)