The molecular basis of LST-1 self-renewal activity and its control of stem cell pool size.

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 mis-expressed. 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 self-renewal 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 RNA-binding protein suggests broad new avenues for combinatorial control.

SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in Caenorhabditis elegans.

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.

PGL germ granule assembly protein is a base-specific, single-stranded RNase.

Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode "P-granules" as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, single-stranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds.

A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality.

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance.

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.

Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

Germline stem cells and their regulation in the nematode Caenorhabditis elegans.

C. elegans germline stem cells exist within a stem cell pool that is maintained by a single-celled mesenchymal niche and Notch signaling. Downstream of Notch signaling, a regulatory network governs stem cells and differentiation. Central to that network is the FBF RNA-binding protein, a member of the widely conserved PUF family that functions by either of two broadly conserved mechanisms to repress its target mRNAs. Without FBF, germline stem cells do not proliferate and they do not maintain their naïve, undifferentiated state. Therefore, FBF is a pivotal regulator of germline self-renewal. Validated FBF targets include several key differentiation regulators as well as a major cell cycle regulator. A genomic analysis identifies many other developmental and cell cycle regulators as likely FBF targets and suggests that FBF is a broad-spectrum regulator of the genome with >1,000 targets. A comparison of the FBF target list with similar lists for human PUF proteins, PUM1 and PUM2, reveals ∼200 shared targets. The FBF hub works within a network controlling self-renewal vs. differentiation. This network consists of classical developmental cell fate regulators and classical cell cycle regulators. Recent results have begun to integrate developmental and cell cycle regulation within the network. The molecular dynamics of the network remain a challenge for the future, but models are proposed. We suggest that molecular controls of C. elegans germline stem cells provide an important model for controls of stem cells more broadly.

Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.

Stem cells are essential for tissue generation during the development of multicellular creatures, and for tissue homeostasis in adults. The great therapeutic promise of stem cells makes understanding their regulation a high priority. PUF RNA-binding proteins have a conserved role in promoting self-renewal of germline stem cells. Here we use a genome-wide approach to identify putative target mRNAs for the Caenorhabditis elegans PUF protein known as FBF. We find that putative FBF targets represent approximately 7% of all protein-coding genes in C. elegans, implicating FBF as a broad-spectrum gene regulator. These putative FBF targets are enriched for regulators of meiotic entry and other components of the meiotic program as well as regulators of key developmental pathways. We suggest that these targets may be critical for FBF's role in stem cell maintenance. Comparison of likely FBF target mRNAs with putative PUF target mRNAs from Drosophila and humans reveals 40 shared targets, including several established stem cell regulators. We speculate that shared PUF targets represent part of a broadly used module of stem cell control.

A sensitized genetic screen to identify regulators of Caenorhabditis elegans germline stem cells.

GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all germline stem cells enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic germline stem cell defect is called the "Glp" phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain germline stem cells. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated 9 glp-1 alleles, 2 lst-1 alleles, and 1 allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered 1 locus that interacts genetically with sygl-1 and generated useful mutations for further studies of germline stem cell regulation.

The impact of mitotic versus interphase chromatin architecture on the molecular flow of EGFP by pair correlation analysis.

Here we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism on a scale more widespread than cell type or organism.

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins.

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3' untranslated region (3' UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3'UTR elements in both Erk2 and p38alpha mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38alpha 3' UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation.

Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(-) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.

Genetic analysis of lysosomal trafficking in Caenorhabditis elegans.

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.

The Stromal Niche for Epithelial Stem Cells: A Template for Regeneration and a Brake on Malignancy.

Stromal restraint of cancer growth and progression-emerging as a widespread phenomenon in epithelial cancers such as bladder, pancreas, colon, and prostate-appears rooted in stromal cell niche activity. During normal tissue repair, stromal niche signals, often Hedgehog-induced, promote epithelial stem cell differentiation as well as self-renewal, thus specifying a regenerating epithelial pattern. In the case of cancerous tissue, stromal cell-derived differentiation signals in particular may provide a brake on malignant growth. Understanding and therapeutic harnessing of the role of stroma in cancer restraint may hinge on our knowledge of the signaling programs elaborated by the stromal niche.