Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2- mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain.

lysine-specific demethylase, repressing target genes [12]. The SLUG domain has also been 50 reported to contribute to repression [13] and members of the Snail family that lack a SNAG domain 51 repress through an alternative domain known as the CtBP domain [14]. 52 In Scratch1, the repressor domain has also been attributed to the amino-terminus of the 53 protein, but deletion of the SNAG domain in human Scratch1 does not reduce its ability to repress 54 E-box-driven transcription [4]. Further, the Scratch family lacks an obvious alternative module 55 such as the CtBP domain. Thus, the repressor domain in the Scratch family remains undefined. As 56 it is generally accepted that the Snail and Scratch families originated through gene duplication, 57 with the signature domains of each family arising through divergence of an ancestral gene [15], 58 understanding the functional contribution of each module could provide us with further insight 59 about the molecular and functional evolution of this superfamily. 60 In this work, we have investigated the biological role of different domains in Scratch2 (Scrt2) 61 through deletion and point mutations in the chicken orthologue. Our data suggest that Scrt2 62 requires SNAG for its repression activity. Also, we identified another conserved motif-HINGE-     factor for the assay. Control conditions were the same, except that the tested construct (pMES or 132 pCIG) did not contain cScrt2 or its variants. following the kit manufacturer's instructions (Dual luciferase assay reporter system, Promega).

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Statistical analysis was performed using one-way ANOVA. The level of significance adopted was 140 p <0.05.

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The zinc-finger domain of Scrt2 determines subcellular localization 144 The main activity reported for members of the Snail superfamily is transcriptional 145 repression. This is due to the joint effect of the zinc-finger domain, mediating nuclear translocation  In most members of the Snail superfamily, transcriptional repressor activity requires the 183 conserved amino-terminus SNAG domain (Fig. 2) [13]. Accordingly, removing the SNAG domain 184 (cScrt2ΔSNAG) decreased Scrt2-mediated transcriptional repression significantly (Fig. 1Q), 185 without affecting its nuclear localization (Fig. 1O). transcriptional repression similar to the native form cScrt2WT, suggesting that SCRATCH is 202 required neither for nuclear localization nor for repressor activity (Fig. 1Q).

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To further investigate the role of the SCRATCH domain, we analyzed its evolution in the 205 context of Scrt2 proteins through sequence alignment (Fig. 2). We observed that the full

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To test this hypothesis, we generated a series of single mutants at residues 77 and 78 to 216 simulate the changes in residue charges prior and after phosphorylation. We replaced the original 217 amino acids either with the neutral residues closest in structure to tyrosine or serine, or with acidic 218 residues. Thus, Y77 was replaced with phenylalanine (cScrt2-Y77F) or glutamate (cScrt-Y77E) 219 and S78 with alanine (cScrt-S78A) or aspartate (cScrt2-S78D). All four single mutations impaired 220 Scrt2-mediated transcriptional repression (Fig. 3M). localization, as all constructs were found in the nucleus (Fig. 3B, E, H and K). Moreover, these 234 mutations did not change protein expression levels (data not shown).

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As our homology analysis suggested a co-evolution of the HINGE and SCRATCH domains, 236 we hypothesized that the two domains act together, which would mean that removing the 237 SCRATCH domain in the background of Y77 or S78 single mutants should further decrease Scrt-2 238 repressor activity. Contrary to our hypothesis, removal of the SCRATCH domain restored the 239 repressor activity of cScrt2-Y77F and cScrt-S78A (Fig. 4).

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Statistical significance was calculated using one-way ANOVA multiple comparisons. *p<0.0001; 245 **p=0.02; #p=0.0001; @p=0.0009. 248 Considering that single mutations of either Y77 or S78 decreased Scrt2 repressor activity 249 and that invertebrates lack the entire HINGE domain (Fig. 2), we next tested the effect of   one of the importin-binding basic residues identified in Snail1 (Fig. S4), indicating that 333 conservation of five of the basic residues is sufficient for nuclear localization. Also, the zinc-finger 334 domain is sufficient to direct protein-DNA interaction at E-box motifs (Fig. S5).

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Thus, we confirm that Scrt2, with a general structure similar to the Snail family members, 336 relies on SNAG for transcriptional repression and the zinc-finger domain for nuclear translocation 337 and DNA-binding. We also show that Scrt2 has additional domains that modulate transcriptional 338 repression. Together, our data extends current knowledge on the modular structure of Snail 339 superfamily members and provides support for the hypothesis that modularity in this superfamily 340 arose from duplication and divergence from a common ancestral protein.