A conserved role for Snail as a potentiator of active transcription

Abstract

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of "Snail-activated" genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.

Keywords: Drosophila embryogenesis; Snail; Twist; activation; repression; spatiotemporal gene expression; transcription factor.

Figure 1.

Identification of Snail direct target genes. (A) Schematic outline of the ChIP and expression profiling experiment, highlighting the stages and genotypes used. (B) Overlap of Twist- and Snail-bound regions using 300-bp windows centered on the Snail peak summit. (C) ChIP signals (log₂ mean immunoprecipitation/mock signal) showing Snail (red) and Twist (blue) occupancy on two known Snail-repressed enhancers (green bar). The gene model is shown below; thick lines indicate exons, thin lines introns, and arrows indicate direction of transcription for the gene of interest (black) and surrounding genes (gray). The chromosome arm and genome coordinates are indicated along the dashed line. (D) ChIP signals (log₂ mean immunoprecipitation/mock signal) showing Snail and Twist occupancy on known active mesodermal enhancers (green bar). The tin A enhancer is not active in the mesoderm (brown bar). The gene model is indicated as in C. (E) Differentially expressed genes in two snail alleles at both time points. Central histogram: Two-hundred-fifty-five genes are up-regulated (red), and 223 genes down-regulated (green) in one or more conditions. Up-regulated genes: Twenty-three genes are associated with Snail-only CRMs, 20 with Snail-Twist cobound CRMs, and 12 with both types. Down-regulated genes: 21 are associated with Snail-only CRMs, 20 are associated with Snail–Twist cobound, and 10 are associated with both types. Heat maps show expression changes of cobound genes in sna^V2 and Df(sna) at stage 5 and stage 7, respectively. The names of known Snail- or Twist-regulated genes are underlined. *(vepD) ventrally-expressed-protein-D (FBgn0053200).

Figure 2.

Snail positively regulates tin B-374 enhancer activity both in vitro and in vivo. Luciferase assays in Kc cells on the wntD-lacZ enhancer (A) and the tin B-374 enhancer (B). The X-axis indicates the amount of DNA transfected (ng), and the Y-axis is the average fold luciferase activity across replicates (n = 3), normalized to Renilla. (A) Dorsal-mediated enhancer activation is repressed upon cotransfection of Snail (Students two-tailed t-test, P < 0.05). (B) Snail augments the activating effect of Twist on the tin B-374 enhancer approximately twofold (Students two-tailed t-test, P < 0.01). (C) In vivo activity of the tin B-374 enhancer (lacZ; green) and expression of the endogenous tinman (tin) gene (red). Expression of sna (blue) marks the mesoderm (arrow), while derepression of sim (blue) in the mesoderm identifies sna mutant embryos. All embryos are orientated with anterior to the left and dorsal up. Images show a single confocal plane. Expression of tin in the anterior domain (arrowhead) is independent of sna.

Figure 3.

Snail-positive regulation of Mef2 I-D[L] requires the Snail motif. (A) Luciferase assay in Kc cells. The X-axis indicates the amount of DNA transfected (ng), and the Y-axis is the average fold of luciferase across replicates (n = 3), normalized to Renilla. Snail significantly potentiates Twist-mediated Mef2 I-D[L] enhancer activation (dark-gray bars) (Students two-tailed t-test, P < 0.01). Disruption of two putative Snail motifs, shown in B, abrogates Snail's effect (light-gray bars). (B) Two mutated Snail motifs, Sna1 and Sna3, are highlighted in the sequence of the Mef2 I-D[L] enhancer (asterisks and red letters indicate the base exchanges). The Sna2 motif overlaps an essential Twist motif and was therefore not mutated. (C) In vivo activity of the Mef2 I-D[L] enhancer. (Top panel) In wild-type embryos, the Mef2 I-D[L] enhancer initiates reporter gene expression (lacZ; green) in mesoderm at stage 5, similar to the endogenous Mef2 gene (red). Enhancer activity and Mef2 expression are ablated in sna¹⁸ mutant embryos but maintained in sna^V2 mutant embryos. (Bottom panels) Mutation of the Snail motifs 1 and 3 (Mef2 I-D[L] ΔSna1,3) drastically reduces lacZ expression. Expression of sna (blue) marks the mesoderm, while derepression of sim (blue) in the mesoderm was used to distinguish sna mutant embryos from wild-type embryos. LacZ expression in the head fold is caused by the eve minimal promoter in the reporter vector.

Figure 4.

Snail-activated and -repressed enhancers contain subtle differences in their Snail and Twist motifs. (A) De novo motif discovery in cobound activated and repressed CRMs. Position weight matrices (PWMs) are shown as sequence logos, with known motifs for Snail (Jaspar MA0086.1), Twist (FlyReg), Zelda (SOLEXA_5), and Dorsal (FlyReg). An alternative Snail and a Twist-like motif are found in both sets, while only repressed CRMs are enriched for a Dorsal-like motif. (*) Alternative Snail motif used for analysis shown in B and C. (B) Distribution of PWM match scores (P-value < 1 × 10⁻³) for the alternative CAGGTA motif across the four classes of Snail-bound CRMs showing all PWM matches (Patser scores, left box plot) or the cumulated match scores (right box plot), summing up putative high-affinity and low-affinity sites. In both cases, no significant differences in the number of motifs were observed. (C) The base-pair distance between Twist and CAGGTA Snail motifs is greater in activated compared with repressed cobound CRMs. The Y-axis shows the mean enrichment of Snail–Twist distances over random expectations (smoothed using a 10-bp window), with 95% confidence intervals (dotted lines). Red asterisks indicate where the signal deviates from random (confidence interval remains less than one for greater than five consecutive values). (Top panel) In activated cobound CRMs, Twist motifs are preferentially enriched at a distance of 50–65 bp. (Bottom panel) In repressed cobound CRMs, Twist motifs cluster around Snail motifs at a distance of 10–20 and 40–50 bp. No enrichment of Twist motifs around CAGGTA Snail motifs is seen in Snail-only CRMs, as expected.

Figure 5.

Differentially enriched motifs predict Snail's regulatory output. (A, left column) Significantly enriched motifs in activated compared with repressed cobound CRMs. The right column shows enrichment of the same motifs compared with the genome. Log₂ fold enrichment values (hypergeometric P-value ≤ 0.025). (B) Work flow of machine learning approach (SVM) to discriminate between activated and repressed CRMs and the in silico mutagenesis to pinpoint the most important motifs for experimental testing. (C) Receiver–operator characteristic [ROC] curves showing SVM performance for activated and repressed CRMs. Area under the curve (AUC) is indicated. (D) The most important motifs used by the SVM to discriminate between activated and repressed CRMs (selected discriminative features). (E) In silico mutatgenesis predicts that the Tll (Tll_MA0459-1 [AAAAGTCAAM]) and ME6 (VATTWGCAT) motifs are the most important for activated cobound CRMs, affecting 50% and 33.3% of the confidently predicted activated peaks, respectively; 8.3% of CRMs depend on the Eyg motif. See Supplemental Table S9 for motif information.

Figure 6.

The Tll motif is essential for the Snail-activated CycE_401 enhancer. (A) An intronic region of the Cyclin E gene is cobound by Twist and Snail (blue and red ChIP signal [log₂ mean immunoprecipitation/mock], respectively). Tll-like, Twist, and Snail motifs are indicated above the ChIP signal; bold red letters mark mutated nucleotides, including a base mutated based on an earlier version of predictions (asterisk). The gene model is shown below; thick lines indicate exons, thin lines indicate introns, and arrows indicate direction of transcription. (B) In vivo activity of the CycE_401 enhancer (genome coordinates are in the Supplemental Material). In situ hybridization of the reporter lacZ gene (green), endogenous CyclinE gene (red), and sna or sim (blue) in wild-type and sna mutant embryos, as indicated. (Top panel) The CycE_401 enhancer drives lacZ expression in a striped pattern in mesoderm (white arrows), partially recapitulating Cyclin E expression. Enhancer activity is dramatically reduced in sna¹⁸ mutant embryos (amorph, second row) compared with sna^V2 (hypomorph). (Bottom panel) Mutation of the Tll-like motif reduces enhancer activity in mesoderm and ectoderm. All embryos are stage 7, with anterior shown to the left, and are single confocal planes. LacZ expression in the head fold is caused by the eve minimal promoter used in the reporter vector.