Central questions in regenerative biology include how stem cells are maintained and how they transition from self-renewal to differentiation. Germline stem cells (GSCs) in Caeno-rhabditis elegans provide a tractable in vivo model to address these questions. In this system, Notch signaling and PUF RNA binding proteins, FBF-1 and FBF-2 (collectively FBF), maintain a pool of GSCs in a naïve state. An open question has been how Notch signaling modulates FBF activity to promote stem cell self-renewal. Here we report that two Notch targets, SYGL-1 and LST-1, link niche signaling to FBF. We find that SYGL-1 and LST-1 proteins are cytoplasmic and normally restricted to the GSC pool region. Increasing the distribution of SYGL-1 expands the pool correspondingly, and vast overexpression of either SYGL-1 or LST-1 generates a germline tumor. Thus, SYGL-1 and LST-1 are each sufficient to drive “stemness” and their spatial restriction prevents tumor formation. Importantly, SYGL-1 and LST-1 can only drive tumor formation when FBF is present. Moreover, both proteins interact physically with FBF, and both are required to repress a signature FBF mRNA target. Together, our results support a model in which SYGL-1 and LST-1 form a repressive complex with FBF that is crucial for stem cell maintenance. We further propose that progression from a naïve stem cell state to a state primed for differentiation relies on loss of SYGL-1 and LST-1, which in turn relieves FBF target RNAs from repression. Broadly, our results provide new insights into the link between niche signaling and a downstream RNA regulatory network and how this circuitry governs the balance between self-renewal and differentiation.

A stem cell’s immediate microenvironment creates an essential “niche” to maintain stem cell self-renewal. Many niches and their intercellular signaling pathways are known, but for the most part, the key downstream targets of niche signaling remain elusive. Here, we report the discovery of two GLP-1/Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic Glp), that function redundantly to maintain germ-line stem cells (GSCs) in the nematode Caenorhabditis elegans. Whereas lst-1 and sygl-1 single mutants appear normal, lst-1 sygl-1 double mutants are phenotypically indistinguishable from glp-1/Notch mutants. Multiple lines of evidence demonstrate that GLP-1/Notch signaling activates lst-1 and sygl-1 expression in GSCs within the niche. Therefore, these two genes fully account for the role of GLP-1/Notch signaling in GSC maintenance. Importantly, lst-1 and sygl-1 are not required for GLP-1/Notch signaling per se. We conclude that lst-1 and sygl-1 forge a critical link between Notch signaling and GSC maintenance.

Highlights d Notch-dependent transcription assayed in its native context is ''bursty'' d Notch-dependent transcriptional bursts are graded across the stem cell pool d Burst duration is the key determinant of Notch-dependent transcriptional probability d Notch NICD strength affects both burst duration and intensity

PUF RNA-binding proteins have diverse roles in animal development, with a broadly conserved role in stem cells. Two paradigmatic PUF proteins, FBF-1 and FBF-2, promote both self-renewal and differentiation in the C. elegans germline. The LST-1 protein is a pivotal regulator of self-renewal and is oncogenic when misexpressed. Here, we demonstrate that LST-1 self-renewal activity resides within a predicted disordered region that harbors two KXXL motifs. We find that the KXXL motifs mediate the binding of LST-1 to FBF, and that point mutations of these motifs abrogate LST-1 selfrenewal activity. The LST-1-FBF partnership is therefore crucial to stem cell maintenance and is a key element in the FBF regulatory network. A distinct region within LST-1 determines its spatial expression and size of the GSC pool. Most importantly, the molecular understanding of how an IDR-rich protein works in an essential partnership with a conserved stem cell regulator and RNAbinding protein suggests broad new avenues for combinatorial control.

Stem cell regulation relies on extrinsic signaling from a niche plus intrinsic factors that respond and drive self-renewal within stem cells. A priori, loss of niche signaling and loss of the intrinsic self-renewal factors might be expected to have equivalent stem cell defects. Yet this simple prediction has not been borne out for most stem cells, including Caenorhabditis elegans germline stem cells (GSCs). The central regulators of C. elegans GSCs include extrinsically acting GLP-1/Notch signaling from the niche; intrinsically acting RNA-binding proteins in the PUF family, termed FBF-1 and FBF-2 (collectively FBF); and intrinsically acting PUF partner proteins that are direct Notch targets. Abrogation of either GLP-1/Notch signaling or its targets yields an earlier and more severe GSC defect than loss of FBF-1 and FBF-2, suggesting that additional intrinsic regulators must exist. Here, we report that those missing regulators are two additional PUF proteins, PUF-3 and PUF-11. Remarkably, an fbf-1 fbf-2; puf-3 puf-11 quadruple null mutant has a GSC defect virtually identical to that of a glp-1/Notch null mutant. PUF-3 and PUF-11 both affect GSC maintenance, both are expressed in GSCs, and epistasis experiments place them at the same position as FBF within the network. Therefore, action of PUF-3 and PUF-11 explains the milder GSC defect in fbf-1 fbf-2 mutants. We conclude that a "PUF hub," comprising four PUF proteins and two PUF partners, constitutes the intrinsic self-renewal node of the C. elegans GSC RNA regulatory network. Discovery of this hub underscores the significance of PUF RNA-binding proteins as key regulators of stem cell maintenance.

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of C. elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1–PUF and SYGL-1–PUF partnerships and their molecular activities in their natural context – nematode stem cells. We confirm LST-1–PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the in vivo functional significance of the LST-1–PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs in vivo. Comparison of PUF-LST-1 and Pumilio–Nanos reveals fundamental molecular differences, making PUF–LST-1 a distinct paradigm for PUF partnerships.

21 22 23 IMPACT STATEMENT 24 A key stem cell regulator partners with a broadly conserved PUF RNA-binding protein to drive self-25 renewal and maintain a stem cell pool.26 27 2 ABSTRACT 28Stem cell self-renewal is essential to development and tissue repair. The C. elegans LST-1 protein is a 29 pivotal regulator of self-renewal and oncogenic when misexpessed. Here we define regions within the 30 LST-1 protein that provide molecular insights into both its function and regulation. LST-1 self-renewal 31 activity resides within a predicted disordered region that harbors two KXXL motifs. These KXXL motifs 32 mediate LST-1 binding to FBF, a broadly conserved Pumilio/PUF RNA-binding protein that represses 33 differentiation. Point mutations of the KXXL motifs abrogate LST-1 self-renewal activity. Therefore, FBF 34 binding is essential to LST-1 function. A second distinct region regulates LST-1 spatial expression and 35 primarily affects LST-1 protein turnover. Upon loss of this regulatory region, LST-1 protein distribution 36 expands and drives formation of a larger than normal GSC pool. Thus, LST-1 promotes self-renewal as a 37 key FBF partner, and its spatial regulation helps determine size of the GSC pool.

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.