Ascl2 Reinforces Intestinal Stem Cell Identity
2015; Elsevier BV; Volume: 16; Issue: 2 Linguagem: Inglês
10.1016/j.stem.2015.01.014
ISSN1934-5909
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoAscl2 is a Wnt-responsive master transcription factor that controls the Lgr5+ intestinal stem cell gene expression program. Now in Cell Stem Cell, Schuijers et al., 2015Schuijers J. Junker J.P. Mokry M. Hatzis P. Koo B.K. Sasselli V. van der Flier L.G. Cuppen E. van Oudenaarden A. Clevers H. Cell Stem Cell. 2015; 16 (this issue): 158-170Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar report an Ascl2 positive feedback loop, tuned by previous Wnt pathway activity, that perpetuates intestinal stem cell identity in response to Wnt/R-spondin stimulation. Ascl2 is a Wnt-responsive master transcription factor that controls the Lgr5+ intestinal stem cell gene expression program. Now in Cell Stem Cell, Schuijers et al., 2015Schuijers J. Junker J.P. Mokry M. Hatzis P. Koo B.K. Sasselli V. van der Flier L.G. Cuppen E. van Oudenaarden A. Clevers H. Cell Stem Cell. 2015; 16 (this issue): 158-170Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar report an Ascl2 positive feedback loop, tuned by previous Wnt pathway activity, that perpetuates intestinal stem cell identity in response to Wnt/R-spondin stimulation. The intestinal epithelium undergoes rapid and continuous stem cell-dependent turnover, a property that naturally lends itself to be studied by stem cell biologists. The small intestine is compartmentalized into distinct zones of proliferation (crypts) and differentiation (villi). Rapidly cycling Lgr5+ intestinal stem cells (ISCs) reside in the crypt base, where they support homeostatic epithelial turnover. Lgr5+ ISCs give rise to transit-amplifying (TA) cells with limited self-renewal potential, whose progeny differentiate as they migrate upward to populate the post-mitotic villus compartment (Clevers, 2013Clevers H. Cell. 2013; 154: 274-284Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Wnt signaling is a major driver of crypt proliferation. Wnt ligands bind cell surface Frizzled receptors, inducing the stabilization and nuclear translocation of cytoplasmic β-catenin and transactivation of Wnt target genes in cooperation with Tcf transcription factors. Maintenance of the crypt compartment requires Wnt signals, as inhibiting Wnt signaling via genetic deletion of β-catenin or Tcf4, overexpression of the Wnt antagonist Dkk1, or pharmacologic inhibition induces crypt loss. Conversely, the R-spondin (Rspo) family of secreted proteins binds receptors of the Lgr4–6 and Znrf3/Rnf43 families to dramatically augment Wnt signaling. Rspo treatment results in crypt hyperplasia and intestinal overgrowth, similar to the effects of Apc deletion, β-catenin gain-of-function mutations, or combined Znrf3/Rnf43 deletion (Clevers, 2013Clevers H. Cell. 2013; 154: 274-284Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Accordingly, Rspo treatment also expands the number of Lgr5+ cells in vivo (Yan et al., 2012Yan K.S. Chia L.A. Li X. Ootani A. Su J. Lee J.Y. Su N. Luo Y. Heilshorn S.C. Amieva M.R. et al.Proc. Natl. Acad. Sci. USA. 2012; 109: 466-471Crossref PubMed Scopus (596) Google Scholar). Now in Cell Stem Cell, Hans Clevers and colleagues (Schuijers et al., 2015Schuijers J. Junker J.P. Mokry M. Hatzis P. Koo B.K. Sasselli V. van der Flier L.G. Cuppen E. van Oudenaarden A. Clevers H. Cell Stem Cell. 2015; 16 (this issue): 158-170Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) provide insights into how Ascl2, a Wnt target gene and master regulator of the ISC transcriptional program, controls stem cell identity. Prior studies identified Ascl2 while investigating the transcriptional signature of Lgr5+ ISCs compared to their immediate non-ISC descendants. This analysis revealed enrichment of a subset of genes, including Lgr5, Troy, and Ascl2, whose expression is restricted to the crypt base (van der Flier et al., 2009van der Flier L.G. van Gijn M.E. Hatzis P. Kujala P. Haegebarth A. Stange D.E. Begthel H. van den Born M. Guryev V. Oving I. et al.Cell. 2009; 136: 903-912Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Interestingly, Ascl2 encodes a basic helix-loop-helix transcription factor that binds to the Lgr5 promoter and is itself a Wnt target gene. Ascl2 has been proposed to be a master regulatory gene for Lgr5+ ISCs, since transgenic overexpression in mice induces hyperplastic and ectopic crypts in the villus compartment, while its deletion results in loss of Lgr5+ ISCs (van der Flier et al., 2009van der Flier L.G. van Gijn M.E. Hatzis P. Kujala P. Haegebarth A. Stange D.E. Begthel H. van den Born M. Guryev V. Oving I. et al.Cell. 2009; 136: 903-912Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Schuijers et al., 2015Schuijers J. Junker J.P. Mokry M. Hatzis P. Koo B.K. Sasselli V. van der Flier L.G. Cuppen E. van Oudenaarden A. Clevers H. Cell Stem Cell. 2015; 16 (this issue): 158-170Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar provide mechanistic insights revealing how Ascl2 is regulated and how it specifies Lgr5+ ISC identity. The authors performed ChIP-seq analyses, revealing frequent co-occupancy of ISC identity genes by the trio of Ascl2, β-catenin, and Tcf4 ("tripartite occupancy"). Importantly, Ascl2 was found to act in an auto-regulatory positive feedback fashion; it binds its own regulatory elements and can functionally synergize with β-catenin to increase transcriptional output in luciferase assays that report Ascl2-dependent transcription. In small intestine epithelial organoid cultures, overexpression of Ascl2 increased cell proliferation and the number of Lgr5+ cells, whereas Ascl2 deletion reduced, but did not eliminate, the Lgr5+ population. Impressively, Ascl2 overexpression rescued the deficient growth and ISC gene signature of Tcf4 null organoids, vividly illustrating the functional potency of Ascl2 in conferring ISC identity. Provocatively, the authors observed an apparent memory component in the Ascl2 response to Wnt/Rspo signals. They initially treated organoid cultures with or without Rspo conditioned medium (CM), eliciting induction or loss of Lgr5 and Ascl2 transcripts, respectively, as determined by single-molecule-resolution in situ hybridization analysis. After this treatment, the organoids were subsequently incubated with varying concentrations of Rspo CM. Interestingly, the cellular response to this latter Wnt/Rspo treatment appears to be contextual. Cellular expression of Ascl2 was found to either be high ("on") or very low ("off"), correlating directly with whether a cell expressed Lgr5 or not. In fact, the dose of Rspo CM required to switch Ascl2 expression on or off differed depending on whether or not the organoids were previously treated with Rspo CM. This is consistent with hysteresis, in which a biological system responds not only to a current condition but also to a past condition, and where different stimulus-response curves are seen depending on whether the system began in the "on" or "off" state. Indeed, prior Rspo exposure allowed lower concentrations of Rspo CM to induce high-level "on"-state Ascl2 expression, thus stabilizing this state. Taken together, these findings of auto-regulation, positive feedback, and hysteresis indicate a bistable on/off signaling circuit (Figure 1). Transient niche Wnt/Rspo signals lead to Ascl2 auto-activation via positive feedback. Hysteresis implies that the Lgr5+ ISC phenotype is characterized by inertia where it is more difficult to convert an Lgr5+ ISC from a stem cell state to a non-stem cell state, or vice versa, than to maintain the status quo. Ascl2 expression may thus create a self-perpetuating state within the ISC to reinforce stem cell identity. Moreover, as Schuijers et al. allude, the memory component of this auto-regulatory loop may serve to protect the intestinal epithelium from relatively minor fluctuations of Wnt signals in the local niche compartment. The findings of Schuijers et al. have broad implications for plasticity within the gut. Interconversion of TA cells, or even more differentiated cells, into ISCs may conceivably occur if the alterations in Wnt/Rspo signal are appropriately strong in amplitude. This type of positive feedback control reinforces stem cell identity in cells expressing Ascl2, but also may endow the intestinal epithelium with plasticity to mobilize cells from the lineage-committed pool to revert back (i.e., de-differentiate) to ISCs in response to appropriate niche signals. Emerging evidence shows that committed secretory progenitors have the potential to revert back to Lgr5-expressing cells (Buczacki et al., 2013Buczacki S.J. Zecchini H.I. Nicholson A.M. Russell R. Vermeulen L. Kemp R. Winton D.J. Nature. 2013; 495: 65-69Crossref PubMed Scopus (555) Google Scholar, van Es et al., 2012van Es J.H. Sato T. van de Wetering M. Lyubimova A. Nee A.N. Gregorieff A. Sasaki N. Zeinstra L. van den Born M. Korving J. et al.Nat. Cell Biol. 2012; 14: 1099-1104Crossref PubMed Scopus (542) Google Scholar). This bistable model also may be compatible with de-differentiation of terminally differentiated cells as well, as seen recently with Troy+ chief cells in the stomach (Stange et al., 2013Stange D.E. Koo B.K. Huch M. Sibbel G. Basak O. Lyubimova A. Kujala P. Bartfeld S. Koster J. Geahlen J.H. et al.Cell. 2013; 155: 357-368Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar) and Clara cells in the airway epithelium (Tata et al., 2013Tata P.R. Mou H. Pardo-Saganta A. Zhao R. Prabhu M. Law B.M. Vinarsky V. Cho J.L. Breton S. Sahay A. et al.Nature. 2013; 503: 218-223Crossref PubMed Scopus (449) Google Scholar). ISCs and lineage-committed cells of the intestine do exhibit surprisingly similar patterns of chromatin accessibility in intergenic regulatory regions (Kim et al., 2014Kim T.H. Li F. Ferreiro-Neira I. Ho L.L. Luyten A. Nalapareddy K. Long H. Verzi M. Shivdasani R.A. Nature. 2014; 506: 511-515Crossref PubMed Scopus (173) Google Scholar), thus potentially facilitating such plasticity through binding of fate-determining/lineage-specification factors such as Ascl2. Future studies will address the applications of these findings, including through in vitro or in vivo use of Ascl2 expression and other fate determinant factors to promote ISC identity, to expand the pool of existing stem cells, and/or to direct them into alternative fates. Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal CryptsSchuijers et al.Cell Stem CellJanuary 22, 2015In BriefThe Wnt signaling pathway controls stem cell identity in the intestinal epithelium, but it has remained unclear how the continuous Wnt gradient is translated into discrete cell type specification. Schuijers et al. show that the transcription factor Ascl2 forms a bimodal switch that interprets Wnt levels and specifies stem cells. Full-Text PDF Open Archive
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