dGLYAT is required for cell polarity disruption-induced cell invasion
It is well known that disrupting cell polarity by depleting scrib along the anterior/posterior (A/P) compartment boundary of the wing discs leads to JNK-dependent invasive cell migration [16], in which cells migrate away from the A/P boundary with up-regulated expression of matrix metalloprotease 1 (MMP1) that is able to degrade the basement membrane [17]. Using this well-established in vivo cell invasion model, we examined the role of dGLYAT in cell polarity disruption-induced cell invasion. Compared with ptc>GFP control (Figure 1a-a’’), knockdown of scrib induced extensive cell migration and MMP1 up-regulation (Figure 1b-b” and quantified in Figure 1f-g). Both phenotypes were significantly suppressed in heterozygous dGLYATc02982 mutants (Figure 1c-c”), which has a piggyBac insertion into the second exon that deletes the critical Gcn5-related N-acetyltransferases (GNAT) domain [14]. To corroborate this result, we depleted dGLYAT by RNAi-mediated knockdown, and confirmed that expression of a dGLYAT-IR dramatically inhibited scrib depletion-induced cell migration and MMP1 elevation, compared with LacZ expression as a negative control (Figure 1d-d”, e-e”).
To further confirm the effect of dGLYAT on cell invasion, we checked other markers of epithelial-mesenchymal transition (EMT), a critical process in malignant transformation [18]. During the process of EMT, expressions of cell-cell junction proteins, such as E-cadherin, are switched off in epithelial cells [19]. Consistently, loss-of-scrib resulted in reduced expression of E-cadherin (Figure 2a-a’ and 2b-b’), which was suppressed by expression of dGLYAT-IR, but not LacZ (Figure 2c-c’ and 2d-d’). Taken together, these data suggest that dGLYAT is required for cell polarity disruption-induced EMT-like cell migration.
dGLYAT is necessary for Egr-JNK pathway-triggered invasive cell migration
Scrib depletion triggers Eiger-JNK pathway-mediated invasive cell migration [15]. To investigate whether dGLYAT is involved in Egr-JNK pathway-activated cell migration, we used ptc-Gal4 to drive ectopic Egr expression. Compared with the control (Figure 3a-a”), ectopic expression of Egr driven by ptc-Gal4 resulted in invasive cell migration, accompanied by up-regulated MMP1 expression (Figure 3b-b”). Both phenotypes were considerably suppressed in heterozygous dGLYAT mutants, or by expressing dGLYAT-IR, but not LacZ (Figure 3c-e” and quantified in Figure 3f-g). As MMP1 also serves as a reporter of JNK signaling, these results suggest that dGLYAT is necessary for Egr-JNK pathway-triggered invasive cell migration.
dGLYAT is required for cell polarity disruption-induced JNK pathway activation
As loss-of-cell polarity-induced cell invasion depends on JNK signaling, and depletion of dGLYAT impedes ptc>scrib-IR-induced cell invasion and MMP1 expression, a transcriptional target of JNK signaling, we proposed that dGLYAT might be required for cell polarity disruption-triggered JNK pathway activation. To test this possibility, we examined the expression of two well-recognized JNK pathway reporters, puc-LacZ and TRE-RFP [20]. In agreement with previous reports, knockdown of scrib along the A/P compartment boundary triggered JNK pathway activation, as indicated by elevated expression of puc-LacZ (Figure 4a-b) and TRE-RFP (Figure 4d-e), both of which were significantly suppressed by knockdown of dGLYAT (Figure 4c, f). Collectively, these results suggest that dGLYAT is required for loss-of-cell polarity-induced JNK pathway activation. dGLYAT regulates Gadd45 transcription
According to the Flybase (http://flybase.org/reports/FBgn0054010), dGLYAT encodes a Gcn5-related N-acetyltransferases (GNAT) family member that catalyzes the transfer of an acetyl moiety from Coenzyme A (Ac-CoA) to diverse substrates [21]. GNATs are evolutionarily conserved regulators that acetylate key amine of small molecules and proteins involved in numerous cellular processes. Some members of GNAT superfamily are involved in histone acetylation [22], which affects gene transcription [23]. Based on these information, we proposed that dGLYAT might be required for the transcriptional activation of JNK pathway positive regulator(s). In this scenario, loss of dGLYAT would compromise JNK pathway activation via reduced expression of the positive regulator(s). To find the potential regulator(s), we conducted the whole genome mRNA-seq assays, and analyzed differentially expressed genes (DEGs) between control and dGLYAT-depleted groups. We found that 13 genes were up-regulated and 29 genes down-regulated upon dGLYAT knockdown (Figure 5a, log2FC≥1.0 or log2FC≤ −1.0, adjusted P value<0.001 and Additional file 1: Table S1). We paid specific attention to the 29 down-regulated genes, whose expression profiles are presented (Figure 5b). Intriguingly, among the 29 down-regulated genes, Gadd45 has been previously reported as a positive regulator of JNK pathway in apoptosis and egg asymmetric development [24, 25]. We performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay to validate the mRNA-seq results, and confirmed that Gadd45 mRNA level was significantly reduced 2 hours and 6 hours after heat shock-induced dGLYAT knockdown (Figure 5c). The relative short time interval (2 hours) between dGLYAT knockdown and Gadd45 mRNA reduction implys dGLYAT may directly affect Gad45 transcription.
Loss of Gadd45 suppresses scrib depletion-induced JNK activation and cell invasion
Given that loss of dGLYAT decreased Gadd45 mRNA expression and suppressed JNK-dependent cell invasion, we hypothesized that dGLYAT regulates JNK-dependent cell invasion through Gadd45. In agreement with this assumption, ptc>scrib-IR-induced invasive cell migration was significantly suppressed by knockdown of Gadd45 with two independent RNAi lines (Figure 6a-d). Furthermore, scrib depletion-triggered JNK pathway activation, revealed by up-regulated MMP1 and puc-LacZ expression, were significantly impeded by loss of Gadd45 (Figure 6e-l, quantified in Figure 6m-n). Collectively, these results suggest that Gadd45 is required for loss-of-cell polarity-induced JNK-dependent cell invasion.