The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex (SKR-1/2-CUL-1-F-box (SCF)), targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin-derivative/mutant AtTIR1 pair (C. elegans AID version 2 (C.e.AIDv2)) that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID targets, addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expands the utility of the AID system and broadens the number of proteins that can be effectively targeted with it.

High-temporal resolution transcriptomics studies have outlined a number of remarkable features associated with global gene expression patterns in developing C. elegans larva (Hendriks et al., 2014;Kim et al., 2013). These studies indicate that between 10 to 20% of the post-embryonic transcriptome exhibits highly reproducible periodic expression patterns.Periodic transcription occurs in a variety of environmental conditions and is independent of life history, indicating that it is under tight genetic control. Importantly, the transcriptional rhythm follows the cycle of post-embryonic molting, a process that demarcates patterns of stagespecific developmental programs. Under various environmental conditions that modulate overall developmental pace, the timing of transcription onset scales accordingly, such that the phase of transcription onset within the molting cycle is preserved (Hendriks et al., 2014;Kim et al., 2013). Currently, it is not known how these transcriptional rhythms are generated, how they are integrated into the execution of stage-specific cellular programs, and how environmental or internal cues modulate features of these transcriptional patterns to achieve robust progression through developmental programs, even after prolonged developmental arrests.The heterochronic GRN is integrated with global aspects of transcription as each of the miRNAs in this pathway exhibits an oscillatory expression. The most promising candidate gene that integrates the rhythm of C. elegans post-embryonic molting to changes in repetative transcriptional patterns is lin-42. The lin-42 gene encodes the C. elegans ortholog of PERIOD/Per proteins that are an essential component of the circadian clock in mice, Drosophila and humans

The C. elegans transcriptome exhibits reproducible, periodic patterns that are phase-locked with features of the larval molting cycle, but the gene regulatory networks underlying this interdependency are unknown. We show here that repeated transcriptional pulses of the lin-4 temporal patterning miRNA are generated by cooperative binding between the C. elegans orthologs of master circadian regulators Rev-Erb and ROR to elements upstream of the lin-4 gene. Remarkably, the precise timing and length of lin-4 transcriptional pulses are dictated by the phased overlap of NHR-85Rev-Erb and NHR-23ROR temporal expression patterns. We also demonstrate that LIN-42Period functions in a similar capacity to its circadian orthologs to negatively regulate periodic transcription but does so by limiting the duration of NHR-85Rev-Erb/NHR-23ROR cooperative activity at the lin-4 gene.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex (SKR-1/2-CUL-1-F-box (SCF)), targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin-derivative/mutant AtTIR1 pair (C. elegans AID version 2 (C.e.AIDv2)) that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID targets, addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expands the utility of the AID system and broadens the number of proteins that can be effectively targeted with it.

SummaryWhile precise tuning of gene expression levels is critical for most developmental pathways, the mechanisms by which the transcriptional output of dosage-sensitive molecules is established or modulated by the environment remain poorly understood. Here, we provide a mechanistic framework for how the conserved transcription factor BLMP-1/Blimp1 operates as a pioneer factor to decompact chromatin near its target loci hours before transcriptional activation and by doing so, regulates both the duration and amplitude of subsequent target gene transcription. This priming mechanism is genetically separable from the mechanisms that establish the timing of transcriptional induction and functions to canalize aspects of cell-fate specification, animal size regulation, and molting. A key feature of the BLMP-1-dependent transcriptional priming mechanism is that chromatin decompaction is initially established during embryogenesis and maintained throughout larval development by nutrient sensing. This anticipatory mechanism integrates transcriptional output with environmental conditions and is essential for resuming normal temporal patterning after animals exit nutrient-mediated developmental arrests.