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, 33 (6), 737-45

Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. Elegans Neuronal Precursors

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Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. Elegans Neuronal Precursors

Sabrina Murgan et al. Dev Cell.

Abstract

Transcription factors of the TCF family are key mediators of the Wnt/β-catenin pathway. TCF usually activates transcription on cis-regulatory elements containing TCF binding sites when the pathway is active and represses transcription when the pathway is inactive. However, some direct targets display an opposite regulation (activated by TCF in the absence of Wnt), but the mechanism behind this atypical regulation remains poorly characterized. Here, we use the cis-regulatory region of an opposite target gene, ttx-3, to dissect the mechanism of this atypical regulation. Using a combination of genetic, molecular, and biochemical experiments, we establish that, in the absence of Wnt pathway activation, TCF activates ttx-3 expression via a Zic binding site by forming a complex with a Zic transcription factor. This mechanism is later reinforced by specific bHLH factors. This study reveals an atypical mode of action for TCF that may apply to other binary decisions mediated by Wnt signaling.

Figures

Figure 1
Figure 1. Activation of ttx-3 expression by POP-1 and repression by SYS-1
(A) Classic target genes are activated by TCF associated with β-catenin when the Wnt pathway is active and repressed by TCF when the Wnt pathway is inactive. Opposite target genes are activated by TCF when the Wnt pathway is inactive and repressed by the TCF:β-catenin complex when the Wnt pathway is active. (B) Expression of the POP-1∷GFP (qIs74) and SYS-1∷VENUS (qIs95) fusion proteins in the NBSMDD/AIY (Ant.) and NBSIAD/SIBV (Post.) neuroblasts identified with the hlh-16p∷his∷mCherry transgene (promoter of the hlh-16 gene driving an histone fused to mCherry, stIs10546) at epidermal enclosure. (C) Effect of pop-1 and sys-1 loss of function mutants on the initiation of ttx-3 expression (ttx-3p∷gfp, otIs173) at epidermal enclosure. As pop-1(q772) and sys-1(q736) homozygote mutants are lethal (at later embryonic stages), we scored the progeny of heterozygote mothers. Around 1/4 of the progeny displays a phenotype as expected from mendelian segregation (n = number of lineages analyzed). Note that in sys-1 mutants the ectopic expression in NBSIAD/SIBV is always weaker than the endogenous expression in NBSMDD/AIY (see picture). This may be due to the fact that sys-1 loss of function is only partial (due to maternal contribution) and/or to the fact that NBSIAD/SIBV has a lower nuclear concentration of POP-1 than NBSMDD/AIY. See also Figure S1. (D) Expression of a POP-1 version lacking the SYS-1 interaction domain (hlh-16p∷ΔNpop-1) but not a full length version (hlh-16p∷fullpop-1) ectopically activates ttx-3 expression (ttx-3p∷gfp, mgIs18) in the NBSIAD/SIBV neuroblast. The hlh-16 promoter which drives expression in NBSMDD/AIY and NBSIAD/SIBV is used as a driver. 6 independent lines were analyzed for hlh-16p∷ΔNpop-1 and 8 for hlh-16p∷fullpop-1 (none: control without any pop-1 transgene). The percentage of embryos showing ectopic expression in each of the hlh-16p∷ΔNpop-1 lines is low but statistically significant (p<0.01, Fisher's exact test; n=50; error bars show standard error of proportion). Note that the ectopic expression in NBSIAD/SIBV is always weaker than the endogenous expression in NBSMDD/AIY (see picture). Scale bar = 2 μm.
Figure 2
Figure 2. Role of the Zic and bHLH binding sites in the generation of the asymmetry
Activity of multimers of Zic or bHLH binding sites placed in front of gfp. On top the structure of the cis-regulatory element responsible for the initiation of ttx-3 expression is shown (“ttx-3 initiator”). The graphs present the percentage of lineages with expression in both NBSMDD/AIY and NBSIAD/SIBV at similar level (black), expression in both NBSMDD/AIY and NBSIAD/SIBV with higher level in NBSMDD/AIY (grey), or expression only in NBSMDD/AIY (white). Expression in the NBSMDD/AIY and NBSIAD/SIBV neuroblasts was scored early (during interphase) or late (when entering mitosis as monitored by chromosome condensation using hlh-16p∷his∷mCherry and by the appearance of the cleavage furrow) (n>30, error bars show standard error of proportion). The intensity of the GFP signal in the NBSMDD/AIY neuroblast in each line is indicated above the graph (+++: strong, ++: medium, +: low). Numbers below graph indicate independent lines. See also Figure S2.
Figure 3
Figure 3. Regulation of the initiation of ttx-3 expression by REF-2, HLH-2, HLH-3 and HLH-16
(A) Left: expression of ttx-3 (ttx-3p∷gfp) in embryos at epidermal enclosure just after division of the NBSMDD/AIY neuroblast (ventral view, anterior is left, scale bar = 10 μm, note that the expression of ttx-3 in the AIN lineage (AINm, an unrelated neuronal lineage that also expresses ttx-3) is affected by loss of ref-2 and hlh-2 function but not by loss of hlh-3 and hlh-16 function). Right: percentage of NBSMDD/AIY lineages that display expression of ttx-3 (ttx-3p∷gfp) at epidermal enclosure (n>100, error bars show standard error of proportion). See also Figure S3. (B) EMSA using in vitro (reticulocyte lysate) produced REF-2 or POP-1 proteins on probes containing the wild type or mutated Zic site from the ttx-3 promoter. See also Figure S4. (C) Coimmunoprecipitation from C. elegans embryos expressing the REF-2∷VENUS (otEx3091) translational fusion (+) or not (-), using an anti-POP-1 antibody or control anti-HA antibody for immunoprecipitation and an anti-VENUS antibody for western blot. (Note that in addition to the REF-2∷VENUS band (62kD) an additional band corresponding to the antibody used for immunoprecipitation (∼50kD) is also detected. The same quantity of anti-POP-1 and anti-HA antibodies were used in the immunoprecipitations, differences of intensity of the antibody band between anti- POP-1 and anti-HA reflects difference of affinity of these antibodies with the western blot antibodies). (D) Left: expression of the translational fusions REF-2∷VENUS (otEx3091), HLH-2∷GFP (nIs47), HLH-3∷YFP (otEx4140), HLH-16∷GFP (otEx4503) in the NBSMDD/AIY (Ant.) and NBSIAD/SIBV (Post.) neuroblasts at epidermal enclosure (scale bar = 2 μm). Right: percentage of lineages expressing the translational fusion HLH-3∷YFP in both NBSMDD/AIY and NBSIAD/SIBV at similar level (black), in both NBSMDD/AIY and NBSIAD/SIBV with higher level in NBSMDD/AIY (grey), or only in NBSMDD/AIY (white). Two independent HLH-3∷YFP lines (#1 otEx4140, #2 otEx4142) were analyzed. Expression in the NBSMDD/AIY and NBSIAD/SIBV neuroblasts was scored early (during interphase) or late (when entering mitosis as monitored by chromosome condensation using hlh-16p∷his∷mCherry and by the appearance of the cleavage furrow). The effects of control and sys-1 RNAi were tested on otEx4140. (n>30, error bars show standard error of proportion).
Figure 4
Figure 4. Action of REF-2, POP-1, SYS-1 on Zic binding sites and model
(A) Analysis of the activity in COS-7 cells of a multimer of Zic binding sites placed in front of a luciferase reporter. The multimer is cotransfected with vectors expressing REF-2, POP-1, SYS-1 or an empty vector (none). (*: p<0.05, **: p<0.01, ns: not significant, Student's t-test; n=12, error bars show standard error of the mean). (B) Activation of the opposite target gene ttx-3 by the TCF transcription factor POP-1. “HLH” represents the 3 bHLH factors HLH-2, HLH-3 and HLH-16. The smaller circles in the NBSIAD/SIBV neuroblast represent lower levels of POP-1 and HLH-3. (C) General model for the activation of opposite target genes by TCF. X = REF-2 in the case of ttx-3.

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