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. 2014 Mar 31;28(6):697-710.
doi: 10.1016/j.devcel.2014.01.028. Epub 2014 Mar 6.

DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation

Moritz Horn  1 , Christoph Geisen  2 , Lukas Cermak  3 , Ben Becker  2 , Shuhei Nakamura  2 , Corinna Klein  2 , Michele Pagano  3 , Adam Antebi  4
Affiliations

DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation

Moritz Horn et al. Dev Cell. .

Abstract

Developmental timing genes catalyze stem cell progression and animal maturation programs across taxa. Caenorhabditis elegans DRE-1/FBXO11 functions in an SCF E3-ubiquitin ligase complex to regulate the transition to adult programs, but its cognate proteolytic substrates are unknown. Here, we identify the conserved transcription factor BLMP-1 as a substrate of the SCF(DRE-1/FBXO11) complex. blmp-1 deletion suppressed dre-1 mutant phenotypes and exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits, including entry into the dauer diapause and longevity. BLMP-1 protein was strikingly elevated upon dre-1 depletion and dysregulated in a stage- and tissue-specific manner. The role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as we observed direct protein interaction and degradation function for worm and human counterparts. Taken together, posttranslational regulation of BLMP-1/BLIMP-1 by DRE-1/FBXO11 coordinates C. elegans developmental timing and other life history traits, suggesting that this two-protein module mediates metazoan maturation processes.

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Figures

Figure 1
Figure 1. blmp-1 Promotes Terminal Differentiation in the Hypodermis
(A) Seam cell lineages from WT worms and various mutants as inferred from inspection. Terminal differentiation is indicated by fusion of the seams (eye-shaped cells) and adult alae formation (three horizontal bars). dre-1 mutants exhibit precocious seam cell terminal differentiation, whereas blmp-1 and the double mutants show incomplete formation of adult alae (three dashed horizontal bars). (B) Representative images of seam cell adherence junctions as visualized by ajm-1::gfp in WT and dre-1 L3 larvae grown on L4440 empty vector control or blmp-1 RNAi, respectively. Arrowheads indicate seam-seam boundaries. (C) blmp-1 and nhr-25 RNAi suppress dre-1 precocious seam cell fusion in L3 larvae. ***p<0.001 (Fisher's exact). Mean+95%CI (n>30). (D) Representative DIC images of young adult WT and blmp-1 mutant worms. Arrowheads indicate seam cell positions, the origin of adult alae synthesis. (E) blmp-1 and dre-1;blmp-1 mutants show poor formation of adult alae. The blmp-1 mutant phenotype is rescued by a blmp-1::gfp transgene. (n>20). Scale bars = 10 μm. See also Table S1.
Figure 2
Figure 2. blmp-1 Impedes Dorsal Migration of the Gonad
(A) Schematic representation of gonadal migration programs of WT worms and various mutants as inferred from inspection. Arrowheads indicate positions of the dtcs. (B) Representative DIC images of WT and dre-1;daf-12 young adult worms grown on L4440 empty vector control or blmp-1 RNAi, respectively, showing one gonadal arm. Arrowheads indicate the position of the dtc. v = vulva. (C) blmp-1 depletion specifically suppresses gonadal migration defects of dre-1;daf-12 and dre-1;lin-29(n546) mutants. ***p<0.001 (ANOVA). Mean+SEM (n=3, 40 gonadal arms each). (D) Representative DIC images of 27 h post-hatching WT and blmp-1 mutant larvae staged by the vulval precursor cells (arrowheads, upper panel). Dtcs migrate precociously in the blmp-1 mutant (arrowheads, lower panel). (E) In blmp-1 mutants, as well as WT and dre-1;daf-12 mutants grown on blmp-1 RNAi dtcs turn dorsally ahead of time. ***<0.001 (ANOVA). Mean±SD. ns = not significant. nd = not determined. Scale bars = 10 μm.
Figure 3
Figure 3. blmp-1 Interacts with Precocious and Retarded Heterochronic Loci
(A) Epistasis and synergy experiments of blmp-1(tm548)/blmp-1i with precocious (hbl-1(ve18), lin-41i, lin-42(n1089), sop-2(bx91)) and retarded (let-7(n2853ts), lin-29(n546), mab-10(e1248)) heterochronic loci (n>20). L4440 empty vector functions as control. Partial seam fusion refers to an incomplete seam syncytium with remaining unfused seam cells. Partial alae formation refers to poorly formed or incomplete adult alae. Temperature-sensitive alleles of sop-2 and let-7 were observed after shifting L1 larvae from 15°C to 23°C or 20°C, respectively. (B) Representative pictures of seam cell adherence junctions as visualized by ajm-1::gfp in hbl-1 and lin-42 L3 larvae grown on L4440 empty vector control or blmp-1 RNAi, respectively. Arrowheads indicate seam-seam boundaries. (C) Representative DIC images of hbl-1 and let-7 mutants showing adult alae formation under control (L4440) and blmp-1 depleted conditions at the early L4 or young adult (yA) stage. Arrowheads indicate positions of the seams. Scale bars = 10 μm. See also Figure S1.
Figure 4
Figure 4. dre-1 and blmp-1 Show Opposite Phenotypes in Dauer Formation and Lifespan
(A) Dauer formation of WT, dre-1(dh99), blmp-1(tm548) and dre-1(dh99);blmp-1(tm548) animals scored on plates lacking cholesterol at 27°C. *p<0.05, ***p<0.001 (ANOVA). Mean+SEM (n≥8, >100 worms each). (B) Dauer formation of WT, daf-2(e1368), daf-7(e1372) and daf-9(dh6) animals scored on regular NGM plates at 25°C under control (L4440) or blmp-1 depleted conditions . In the latter genotypes, blmp-1 depletion led to L3/L4 arrest without dauer alae and SDS sensitivity. ***p<0.001 (ANOVA). Mean+SEM (n=3, 15 worms each). (C) Representative DIC images of daf-2(e1368) mutants grown on L4440 empty vector control or blmp-1 RNAi at 25°C for 48 h. Arrowheads indicate dauer alae. Scale bar = 10 μm. (D) Lifespan analysis: WT animals were grown on RNAi from the L4 stage as indicated. Dashed lines represent an independent repeat of the experiment. First given p value (Mantel-Cox Log Rank method) corresponds to the experiment displayed with solid lines.
Figure 5
Figure 5. DRE-1 Targets BLMP-1 for Proteasomal Degradation
(A) Representative DIC and fluorescence images of BLMP-1::GFP expressing L4 larvae grown on L4440 empty vector control or dre-1 RNAi. (B) BLMP-1::GFP levels in seam and hypodermal cells are significantly increased upon dre-1 and cul-1 depletion. ***p<0.001 (ANOVA). Mean+SEM (n≥3, ≥10 cells of 5 worms each). (C) Representative DIC and fluorescence images of BLMP-1::GFP expressing L4 larvae treated with 100 μM bortezomib or DMSO control. (D) Proteasome inhibition (bortezomib and rpn-8 RNAi) increases BLMP-1::GFP levels in seam and hypodermal cells. **p<0.01, ***p<0.001 (ANOVA). Mean+SEM (n≥3, ≥10 cells of 5 worms each). (E) Western blot analysis of endogenous BLMP-1 protein levels in WT, dre-1 and blmp-1 L4 larvae after various RNAi treatments from egg stage onwards. The asterisk marks a nonspecific band. The arrow points to the BLMP-1 band. (F) Quantification of Western blot analyses. *P<0.05, **P<0.01, ***P<0.001 (ANOVA). Mean +SD (n=5). (G) qPCR analysis of WT L4 larvae treated with the indicated RNAis starting at the egg stage. **p<0.01 (ANOVA). Mean+SEM (n=3). (H) L3 larvae expressing integrated DRE-1::GFP or NHR-25::GFP as control were grown from egg stage onwards on L4440 empty vector control or blmp-1 RNAi expressing bacteria, respectively and utilized for anti-GFP immunoprecipitation (IP). Lysates and IP were analyzed by immunoblotting. Detected sizes: BLMP-1: ≈ 98 kDa, DRE-1::GFP: ≈ 145 kDa and NHR-25::GFP: ≈ 120 kDa se = seam cell. hyp = hypodermal cell. Scale bars = 10 μm. See also Figures S2 and S3.
Figure 6
Figure 6. FBXO11 Directly Interacts with BLIMP-1 and Triggers its Degradation
(A) anti-FLAG IP of human FLAG-HA-tagged (F/H) RFP, -FBXO5, -FBXO10, -FBXO11 and -FBXO1 1(Q491L/Jeff) from HEK293T cells treated with 15 μM MG132. Human His-BLIMP-1 was co-expressed. Detected protein sizes: see panel C. Quantification of RFP/F-box-protein binding intensity to BLIMP-1 was normalized to BLIMP-1 input and FBXO1 1-BLIMP-1 association. ***p<0.001 (ANOVA). Mean+SD (n=5). nd = not detectable. (B) HEK-293T cells were transfected with His-tagged BLIMP-1, FLAG-tagged FBXO11 or -FBXO11(F-boxMUT), and an empty vector as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of BLIMP-1 was performed in the presence of E1 and E2s. Where indicated, ubiquitin was added. Samples were analyzed by immunoblotting (left panel). Immunoblots of whole-cell extracts and immunoprecipitations are shown in the right panel. Detected size SKP1 ≈ 19kDA, other proteins as indicated in panels C and D. (C) Human BLIMP-1 stability was assessed in HEK293T cells transfected with His-BLIMP-1 and -RFP in combination with either F/H-tagged RFP, -FBXO11, -FBXO11 (Δ F-box), -FBXO1 1(Q491L/Jeff), -FBXO5 or –FBXO10. Cells were treated with cycloheximide (CHX) or CHX ± 15μM MG132 for 0, 1, 2 or 4 hrs prior to harvesting and analysis by immunoblotting. Human BLIMP-1 stability was quantified from ≥3 independent experiments and normalized to co-expressed His-RFP. Mean+SD (n≥3). (D) Stability of endogenous BLIMP-1 was analyzed in ARP1 multiple myeloma cells infected with either viruses expressing two different FBXO11 shRNAs or an empty virus (CTRL), and selected for 72 hours. Cells were then treated with CHX for the indicated times and protein extracts were analyzed by immunoblotting. Detected sizes: BLIMP-1 ≈ 90 kDA, FBXO11 ≈ 125 kDA. See also Figure S4.
Figure 7
Figure 7. BLMP-1::GFP Levels are Dynamically Regulated by DRE-1
(A) BLMP-1::GFP levels in the dtcs were assessed in ≥5 worms per grouped DA (developmental age). ***p<0.001 (ANOVA). Mean+SD. (B) Representative DIC images of age-matched L3 larvae with zoom in on BLMP-1::GFP expression in the dtcs. Black box indicates the area enlarged in the fluorescence image. White circle roughly marks the dtc nucleus. Scale bars = 10 μm. (C) BLMP-1::GFP levels in seam and hypodermal cells were assessed in ≥5 worms per grouped DA. *p<0.05 (ANOVA). Mean+SEM. (D and E) Model for dre-1/blmp-1 action in (D) gonadal tissue: BLMP-1 is downregulated in a dre-1-dependent manner in mid-L3 to allow the dorsal gonadal turn. In dre-1 or dre-1/daf-12 mutants BLMP-1 levels persist to later ages, leading to retarded migration defects, whereas the dorsal turn is preceded in the absence of BLMP-1. (E) somatic tissue: Elevated BLMP-1 levels contribute to seam cell terminal differentiation, leading to precocious terminal differentiation in dre-1 mutants and consequentially impaired terminal differentiation in the absence of BLMP-1. Double-slashes indicate the end of data assessment. (F) Summary of gene-ontology/biological processes derived from DAVID analysis (Huang et al., 2009) of differentially regulated genes (q<0.05, fold change >±1.5) in WT vs blmp-1 mutant animals from RNA-Seq datasets (n=3). ReviGO was used for visualization of the resulting GO term classes and its semantic similarity (Supek et al., 2011). Bubble color, size, and line widths respectively indicate significance of enrichment, frequency of GO term in underlying data sets, and degree of similarity. More intense colors indicate a higher significance of enrichment. (G) qPCR analysis of blmp-1-regulated molting and cuticle-related genes in L3 larvae and young adult worms. *P<0.05, **P<0.01, ***P<0.001 (t-test). Mean+SEM (n≥3). See also Figure S5.
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