A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans

Abstract

Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development.

Keywords: P granules; chromatin; development; genome-wide RNAi screen; germline.

Figure 1

let-418 mutants arrest as L1. (A) Primordial germ cells and blast cells are mitotically arrested in let-418 mutants. a–d show differential interference contrast microscopy images of wt (a, b, and d) and let-418 (c) developing larvae. e–h show V lineage and division pattern in wt (e, f, and h) and let-418 (g) revealed by the reporter construct scm::gfp. (3d = days). i–l display mesoblast (M) lineage and division pattern in wt (i, j, and l) and let-418 (k) larvae visualized by the reporter construct hlh-8::gfp. (B) LET-418 is required to survive and recover from starvation. Recovery and survival assays of indicated genotypes. Larvae were allowed to hatch at 25° in the presence or absence of food; starved larvae were returned to food following the indicated number of days. All larvae were maintained at 15°. wt, wild-type.

Figure 2

Suppression of ectopic P granule expression. (A) Perinuclear P granules are observed in somatic cells of let-418 and set-26(RNAi) worms, but are missing in animals that are fed with mes-4 or htz-1 RNAi. Presence of the P granule was revealed by the reporter construct pgl-1::gfp. Bar, 20 µm. (B) pgl-1 mRNA level is decreased in htz-1;let-418 but not in set-26;let-418 animals. mRNA level of pgl-1 was measured by qRT-PCR and represented as fold induction of mRNA expression vs. wt. Total mRNA was isolated from wt and let-418 L1 animals treated with the indicated suppressor RNAi. *** indicates a P value ≤ 0.001 mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; RNAi, RNA interference; wt, wild-type.

Figure 3

set-26 suppression of M cell mitotic arrest. (A) Inactivation of set-26 in let-418 mutant background allows the M cell to divide. However, set-26;let-418 animals show reduced numbers of muscle cell precursors compared to wt. Numbers in brackets correspond to the number of germ cells indicative of the developmental stage. (B) M cell division pattern is revealed by the reporter construct hlh-8::gfp. Developing L1 shows BWM cell precursors (filled arrowheads) and L4 larva show SM precursors (open arrowheads). SM precursor cells are mislocalized in set-26;let-418 animals. BWM, body wall muscle; CC, coelomocytes; eL1, early L1 with four germ cells (Z1–4); SM, sex myoblast; wt, wild-type.

Figure 4

LET-418, NURF-1, and HTZ-1 regulate common target genes. (A) Upregulation of the germline gene expression in let-418 depends on NURF-1 and HTZ-1. mRNA levels of deps-1, pie-1, and pgl-1 were measured by qRT-PCR and represented as fold induction of mRNA expression vs. wt. Total mRNA was isolated from wt and let-418 L1 animals treated with the indicated suppressor RNAi. ama-1 was used to normalize. (B) Upregulation of DAF-16 target expression in let-418 depends on NURF-1 and HTZ-1. mRNA levels of dct-3, W08A12.4, and fbxa-165 were measured by qRT-PCR and represented as fold induction of mRNA expression vs. wt. Total mRNA was isolated from wt and let-418 L1 animals treated with the indicated suppressor RNAi. ama-1 was used to normalize. mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; RNAi, RNA interference; wt, wild-type.

Figure 5

LET-418 and HTZ-1, and LET-418 and NURF-1, share a significant number of common direct targets (P-value < 0.001). P-values were determined by Fisher’s exact test. Gene ontology categories that are highly enriched for LET-418/HTZ-1 (A) and LET-418/NURF-1 (B) common targets are shown. The percentage of enrichment is indicated by the bars.

Figure 6

Model for the action of LET-418 and the suppressors. In the presence of food, larval development is initiated and LET-418, together with a network of other chromatin factors, is responsible for modulating the transcription of germline genes, a subset of DAF-16 targets, and some other prodevelopmental arrest genes. This network of chromatin factors includes activator proteins that might compete for regulation at the promoter of target genes and repressors whose localization could be determined by LET-418. MES-2 was shown recently to interact with LET-418 (Käser-Pébernard et al. 2016).