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.
Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1[Lsh/HELLS]), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro. We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1[Dnmt1]. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.
This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.
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