PBRM-1/PBAF-regulated genes in a multipotent progenitor in Caenorhabditis elegans

Abstract

The Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic cells of the adult reproductive system. The 2 SGPs originate in the mesodermal layer and are born through a division that produces one SGP and one head mesodermal cell (hmc). One hmc terminally differentiates, and the other dies by programmed cell death. The polybromo-associated BAF (PBAF) chromatin remodeling complex promotes the multipotent SGP fate. The complete loss of PBAF causes lethality, so we used a combination of Cre/lox recombination and GFP nanobody-directed protein degradation to eliminate PBRM-1, the signature subunit of the PBAF complex, from 83 mesodermal cells, including SGPs, body muscles, and the hmc. We used RNA sequencing to identify genes acting downstream of PBAF in these cells and identified 1,955 transcripts that were significantly differentially expressed between pbrm-1(-) and pbrm-1(+) in the mesoderm of L1 larvae. We found that genes involved in muscle cell function were overrepresented; most of these genes had lower expression in the absence of PBRM-1, suggesting that PBAF promotes muscle differentiation. Among the differentially expressed genes were 125 that are normally expressed at higher levels in SGP vs hmc and positively regulated by pbrm-1 and 53 that are normally expressed at higher levels in hmc vs SGP and are negatively regulated by pbrm-1; these are candidate regulators of the SGP/hmc fate decision. We validated one candidate gene using a fluorescent reporter; the hsp-12.3 reporter was derepressed in SGPs in pbrm-1 mutants, suggesting that hsp-12.3 expression is normally repressed by pbrm-1 in SGPs.

Keywords: C. elegans; pbrm-1; PBAF; SGP; SWI/SNF chromatin remodeling; differential gene expression; hmc; multipotent progenitor; muscle.

Fig. 1.

Generation of a GFP-tagged loxP-flanked pbrm-1 allele. a) The engineered pbrm-1 locus contains GFP-coding sequences (stippled) immediately downstream of the last pbrm-1 coding exon, followed by a 3× FLAG tag (striped). One loxP site remains between GFP and 3× FLAG following removal of the self-excising cassette. An additional loxP site was inserted upstream of the start of pbrm-1b. Cre-mediated recombination removes the last 6 exons of pbrm-1 and all GFP-encoding exons; the deleted locus is expected to produce a strong loss of function because it removes all common pbrm-1 exons. b) PBRM-1::GFP is found in the nucleus of many and perhaps all cells across development, from embryogenesis through adulthood. Images shown are embryos, L1 larvae, and L4 larvae.

Fig. 2.

pbrm-1 conditional knockout in SGPs. a–c) A Cre readout reporter shows where hnd-1 promoter driven Cre recombinase is active. The readout reporter is driven by a ubiquitous promoter and switches from mCherry to GFP expression upon recombination between loxP sites. b and c) hnd-1p::Cre promotes recombination in SGPs, body wall muscles (arrow heads), and M mesoblast daughters; DIC image b) and GFP fluorescence c). d–g) A combination of Cre/lox recombination and GFP nanobody-directed protein degradation was used to eliminate PBRM-1::GFP from SGPs. Driver constructs are indicated above images: no driver d) hnd-1p::Cre e), hnd-1p::GFP-nanobody::ZIF-1::Cre f), hnd-1p::ZIF-1 alone g). DIC image (left), GFP fluorescence (middle), and inverted monochrome fluorescent image (right). The gonad primordium (dashed line) and SGPs are indicated; the asterisks mark germ cells. All fluorescent images are 1 s exposures with identical adjustments. PBRM-1::GFP fluorescence in SGPs is reduced by hnd-1p::Cre recombination e) and made invisible by the addition of ZIF-1–mediated degradation f); hnd-1p::ZIF-1 does not affect PBRM-1::GFP.

Fig. 3.

Genes regulated by pbrm-1 in mesodermal tissues. a) Volcano plot showing the distribution of DEGs between pbrm-1(TS-KO) and pbrm-1(control). b) Venn diagram indicating the proportion of pbrm-1-regulated genes that are also expressed in SGPs or embryonic muscles. The number of genes in each circle is indicated; 558 genes are expressed in both SGPs and muscles. c) GO biological process categories are statistically overrepresented among the SGP-expressed pbrm-1-regulated genes. Fold enrichment is plotted.

Fig. 4.

Genes regulated by pbrm-1 in SGPs. Expression of reporters in wild-type (WT) and pbrm-1(ok843) mutants. Paired images are DIC (left) and GFP fluorescence (right). Arrows point to SGPs; arrowheads point to hmc; the gonad primordium is outlined (dashed line). Scale bars are indicated. hsp-12.3::GFP expression in SGP a) and hmc b); fluorescent exposures are 500 ms. a) hsp-12.3::GFP is expressed very faintly in wild type SGPs (top), and this expression increases in pbrm-1(ok843) mutants (bottom). b) hsp-12.3::GFP is expressed in hmc (top); this expression is unchanged in pbrm-1(ok843) mutants (bottom). c) txdc-12.1::GFP is expressed in WT SGPs (top), and this expression is unchanged in pbrm-1(ok843) mutants (bottom); fluorescent exposures are 50 ms. Paired WT and pbrm-1 mutant images were taken with identical exposures and adjustments for comparison. d) Percentage of SGPs with dim (white), distinct (light grey), or bright (dark grey) GFP in SGPs. Unpaired Student's t-tests were used to compare expression in WT and pbrm-1 mutants; *P ≤ 0.05.