GLP-1 Notch—LAG-1 CSL control of the germline stem cell fate is mediated by transcriptional targets lst-1 and sygl-1

Abstract

Stem cell systems are essential for the development and maintenance of polarized tissues. Intercellular signaling pathways control stem cell systems, where niche cells signal stem cells to maintain the stem cell fate/self-renewal and inhibit differentiation. In the C. elegans germline, GLP-1 Notch signaling specifies the stem cell fate, employing the sequence-specific DNA binding protein LAG-1 to implement the transcriptional response. We undertook a comprehensive genome-wide approach to identify transcriptional targets of GLP-1 signaling. We expected primary response target genes to be evident at the intersection of genes identified as directly bound by LAG-1, from ChIP-seq experiments, with genes identified as requiring GLP-1 signaling for RNA accumulation, from RNA-seq analysis. Furthermore, we performed a time-course transcriptomics analysis following auxin inducible degradation of LAG-1 to distinguish between genes whose RNA level was a primary or secondary response of GLP-1 signaling. Surprisingly, only lst-1 and sygl-1, the two known target genes of GLP-1 in the germline, fulfilled these criteria, indicating that these two genes are the primary response targets of GLP-1 Notch and may be the sole germline GLP-1 signaling protein-coding transcriptional targets for mediating the stem cell fate. In addition, three secondary response genes were identified based on their timing following loss of LAG-1, their lack of a LAG-1 ChIP-seq peak and that their glp-1 dependent mRNA accumulation could be explained by a requirement for lst-1 and sygl-1 activity. Moreover, our analysis also suggests that the function of the primary response genes lst-1 and sygl-1 can account for the glp-1 dependent peak protein accumulation of FBF-2, which promotes the stem cell fate and, in part, for the spatial restriction of elevated LAG-1 accumulation to the stem cell region.

Recent Advances in the Genetic, Anatomical, and Environmental Regulation of the C. elegans Germ Line Progenitor Zone

“…A widely observed phenomenon in this system is redundancy, making synthetic screens of particular promise going forward [ 50 , 52 , 71 , 77 ]. Furthermore, while connections within these networks are well-supported, quantitative experiments have recently revealed a substantial role for GLP-1-independent regulation of network members [ 68 ]. Other inputs that influence the size of the PZ are signals from the Sh1 pair of somatic gonad sheath cells.…”

“…The Notch pathway acts through LAG-1 CSL DNA binding protein as a transcriptional effector [ 68 , 83 ], and activates the target genes sygl-1 and lst-1 [ 31 , 68 , 84 ]. Exhaustive genetic screening for germ line proliferation defects and sterility had failed to turn up the link between Notch signaling and FBF-mediated stem cell fate.…”

“…Two linking genes— lst-1 and sygl-1 —were recently identified by an elegant candidate screen for genes transcriptionally regulated by Notch signaling and post-transcriptionally by FBF binding that function redundantly to maintain GSC self-renewal [ 52 ]. They were later validated as quite possibly the only direct targets of Notch signaling that mediate germ stem cell fate by genome-wide approaches, including the direct binding of promoters by LAG-1 and RNA-seq for GLP-1-responsive transcripts [ 68 ].…”

“…Following on from that question, several secondary targets of Notch signaling in germ cells have been identified, without known roles in the germ line [ 68 ]; any activity in the stem cell self-renewal program is yet to be discovered. Perhaps more crucially, a downstream target of Notch activation that transcriptionally regulates other genes that are indirect targets of Notch (like lip-1 and fbf-2 ) has yet to be described.…”

“…GSCs are nestled in the distalmost plexus of DTC cellular processes [ 26 , 27 ]. They are characterized by nuclear LAG-1 protein [ 68 ], and the active transcription of direct targets of Notch signaling sygl-1 and lst-1 [ 31 , 54 , 68 , 84 , 93 ]. They appear to be isolated from the rest of the germ line by a diffusion barrier in the cytoplasmic core of the germ line rachis [ 9 ], and extend as far proximal as the interface between the DTC and Sh1 [ 10 ].…”