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.
SUMMARY The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.
Highlights d ATAC-Me simultaneously measures chromatin accessibility and DNA methylation (DNAme) d Enhancer accessibility and DNAme change independently during cell fate transitions d Loss of DNAme is delayed in nascent open chromatin regions d Transcriptional changes track with new enhancer accessibility despite prolonged DNAme
Topoisomerase II alleviates DNA entanglements that are generated during mitotic DNA replication, transcription, and sister chromatid separation. In contrast to mitosis, meiosis has two rounds of chromosome segregation following one round of DNA replication. In meiosis II, sister chromatids segregate from each other, similar to mitosis. Meiosis I, on the other hand, segregates homologs, which requires pairing, synapsis, and recombination. The exact role that topoisomerase II plays during meiosis is unknown. In a screen reexamining Caenorhabditis elegans legacy mutants isolated 30 years ago, we identified a novel allele of the gene encoding topoisomerase II, top-2(it7). In this study, we demonstrate that top-2(it7) males produce dead embryos, even when fertilizing wild-type oocytes. Characterization of early embryonic events indicates that fertilization is successful and sperm components are transmitted to the embryo. However, sperm chromatin is not detected in these fertilized embryos. Examination of top-2(it7) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during anaphase I of meiosis, resulting in anucleate sperm. top-2(it7) chromosome-segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not dependent on SPO-11-induced double-strand DNA breaks. Finally, we show that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7) mutants.
Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions, such as assay for transposase-accessible chromatin using sequencing (ATAC-seq), with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay and workflow, enabled high-resolution testing of ~50 million unique DNA fragments tiling ~101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer or both. Although few MPRA studies have explored silencing activity, we demonstrate silencers occur at similar frequencies to activators, and they represent a distinct functional group enriched for unique TF motifs and repressive histone modifications. We further show that Tn5 cut-site frequencies are retained in the ATAC-STARR plasmid library compared to standard ATAC-seq, enabling TF occupancy to be ascertained from ATAC-STARR data. With this approach, we found that activators and silencers cluster by distinct TF footprint combinations and these groups of activity represent different gene regulatory networks of immune cell function. Altogether, these data highlight the multi-layered capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.
Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln, and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in C. elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamate residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.
Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the Olinked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid β-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD
25Genetic and environmental manipulations, such as dietary restriction (DR), can improve both 26 health span and lifespan in a wide range of organisms, including humans. Changes in nutrient 27 intake trigger often overlapping metabolic pathways that can generate distinct or even opposite 28 outputs depending on several factors, such as when DR occurs in the lifecycle of the organism or 29 the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the 30 diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not 31 yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis 32 elegans is a DR model that is associated with lengthened lifespan and reproductive potential 33 (Angelo and Van Gilst 2009). As the metabolic pathways regulating ARD have not yet been 34 explored in depth, we performed a candidate-based genetic screen analyzing select nutrient-35 sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three 36 phases of ARD (initiation, maintenance, and recovery), we find that ARD initiation is regulated 37 by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) 38 pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in 39 our screen, we found that ARD recovery was modulated by energy sensing, stress response, 40 insulin-like signaling, and the TOR pathway. We also discovered that fatty acid β-oxidation 41 regulates ARD initiation through a pathway involving the O-GlcNAc cycling enzyme, 42 acting with the nuclear hormone receptor NHR-49. Consistent with these findings, our analysis 43 revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings 44 thus identify novel conserved genetic pathways required for ARD entry and recovery and 45 identify new genetic interactions that provide insight into the role of OGT and OGA. 46 47 an intense and controversial area of research (Kennedy et al. 2007; Mair and Dillin 2008). While 63 different forms of DR can trigger divergent pro-longevity pathways with distinct outcomes 64 (Greer and Brunet 2009), they often share common features including involvement of 65 overlapping nutrient-sensing factors and changes to metabolism, particularly fat and 66 carbohydrate stores (Houtkooper et al. 2010; Lapierre and Hansen 2012; Finkel 2015; Solon-Biet 67 et al. 2015). 68 DR-dependent pro-longevity pathways are highly conserved, suggesting research in 69 model organisms, such as the nematode Caenorhabditis elegans, can inform dietary 70 5 interventions and therapeutic targets relevant for improving human health span. For any 71organism, there is a delicate balance between nutrient acquisition, development, aging, and 72 reproduction to ensure the health and propagation of the species. C. elegans is an exceptional 73 model that has been used extensively to examine the intersection of longevity and these 74 biological processes because it has a...
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