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To identify novel Six1-interacting proteins, we previously screened the fly interactome for Sine oculis-binding partners whose orthologues are also expressed in Xenopus embryos. We identified a zinc-finger MYM-containing protein-Zmym2-based on its sequence similarity in a few domains also found in the Drosophila and vertebrate Sine oculis-binding proteins (Sobp). Because recent studies established Zmym2 as a transcriptional repressor that interacts with Six4 during renal development, herein we assessed whether it interacts with Six1, can modify Six1's transcriptional activity, and is involved in cranial neural crest or placode gene expression. Although during early development Zmym2 is expressed in many of the same tissues as Six1 and contains several domains also found in Sobp, we did not detect any interaction by co-immunoprecipitation and did not detect any effect on Six1 + Eya1 transcriptional activity in cultured cells. Nonetheless, increasing the level of Zmym2 in embryos resulted in broader expression domains of neural border, neural tube and neural crest genes, and smaller placode gene domains. These results suggest that although Zmym2 is unlikely to be a bone fide Six1 interacting protein, it appears to indirectly antagonize Six1 function during cranial placode development, promoting neural plate and neural crest gene expression.
FIGURE 1. Sobp and Zmym2 proteins share several domains. (A) Sobp protein structure (adapted from Tavares et al. 2021) highlighting Box2 and Box3 identified in Drosophila Sobp by Kenyon et al. (2005). Sobp contains two zinc fingers (ZF1, ZF2), a proline rich domain (PRD), two SUMO-interacting motifs (SIM), and a functional nuclear localization signal (NLS). (B) Zmym2 protein contains nine ZFs domains identified in human ZMYM2 and aligned by sequence homology (see Figure 2). The areas of sequence homology with Sobp Box2 and Box3, that were used by Neilson et al. (2010) to identify Xenopus laevis Zmym2 as a putative Six1 cofactor, are indicated. Two putative NLS that are conserved with the human protein and a glucocorticoid-like DNA binding domain that overlaps the first ZF domain were identified. Zmym2 also contains a C-terminal DUF3405 domain. For each sequence, the NLS was identified using cNLS mapper (Kosugi et al. 2009) and the DNA-binding and DUF3405 domains using InterPro (Blum et al. 2021). The protein structures were generated using Dog2.0.1 (Ren et al. 2016).
FIGURE 2. Dotplot showing the amino acid conservation of Zmym2 between the Xenopus laevis long and short homoeologs, Xenopus tropicalis, human, and Drosophila Without children (Woc) ortholog. The nine zinc finger (ZF) domains identified in the human protein have been highlighted in different colors. The first three ZFs are highly conserved between all species whereas ZFs four through nine are highly conserved among all species other than X. tropicalis. Areas where the sequence is identical between all species examined are highlighted on the dotplot by a black background with white text. Areas with high sequence homology but in which there is some species-specific variation are identified by bolded sequence text and a boxed outline. The areas of homology to Sine oculis binding protein (Sobp) Box2 and Box3, which were used by Neilson et al. (2010) to identify Zmym2 as a potential Six1 binding partner, are highlighted by the black (Box2) and gray (Box3) box outlines. The sequence alignments were generated using T-Coffee (Notredame et al. 2000) and assembled into Dotplots using ESPript (Robert and Gouet 2014)
FIGURE 3. Zmym2 does not bind to Six1 or alter its transcriptional activity. (A) HEK293T cells were transfected with either Myc-tagged Six1, HA-tagged Zmym2, or both and were tested for co-immunoprecipitation (Co-IP) with anti-Myc tagged magnetic beads. In the Co-IP assay (IP-anti-Myc), when Six1 was transfected alone, it bound to the Myc-tagged beads (left lane: Zmym2-3HA (), Six1-Myc (+)), whereas when Zmym2 was transfected alone, it did not bind, as expected (middle lane: Zmym2-3HA (+), Six1-Myc ()). When both were transfected (right lane: Zmym2-3HA (+), Six1-Myc (+)), Six1 was immunoprecipitated by the beads but Zmym2 was not detected in the sample. The flowthrough lanes contained detectible Myc-tagged Six1 and HA-tagged Zmym2 (right). (B) HEK293T cells were transfected with a Six1 reporter (pGL3-6xMEF-luciferase) and plasmids encoding either Six1 alone, Eya1 alone, Zmym2 alone, or combinations of Six1+Eya1 and/or Zmym2. Unlike Eya1, Zmym2 does not activate the Six1 reporter alone. Zmym2 also does not activate transcription in the presence of either Six1 or Eya1, nor does it enhance or repress the ability of the Six1-Eya1 complex to activate transcription. Control=empty plasmid, ns=not significant. (C) Luciferase activity of the pGL3-6XMEF-luciferase reporter in HEK293T cells transfected with different combinations of plasmids expressing control, wild type Six1, Eya1, or BOR variants of Six1. Data are normalized to Renilla expression with a constitutive promoter. Although the combination of wild type Six1+Eya1 significantly increased transcriptional activity over control (***p<0.001), neither wild type Six1 nor any of the BOR1 Six1 variants significantly increased transcription (ns) in the presence of Zmym4. (D) Luciferase activity of the pGL3-6XMEF-luciferase reporter in HEK293T cells transfected with different combinations of plasmids expressing control, wild type Six1, Eya1, or BOR variants of Six1. As in panel B, the combination of wild type Six1+Eya1 significantly increased transcriptional activity (***p<0.001), and addition of Zmym2 did not significantly alter this activity (ns). When the Six1 activator Eya1 was added to Six1 BOR variants in the presence of Zmym2, there was no significant difference in the level of transcriptional activity compared to control (ns).
FIGURE 4. Increased Zmym2 expands neural border, neural plate (np), and neural crest domains and reduces placode domains. (A) Examples of early NP stage embryos (st 1314) in which three neural border gene expression domains are broader (red lines) on the zmym2 mRNA injected side (right side with pink lineage tracer) compared to uninjected side of the same embryos (black lines). np, neural plate. (B) The percentage of embryos with increased (dark blue), decreased (light blue) or no change (white) in border gene expression when one side was injected with zmym2 mRNA. Numbers above the bars denote number of embryos analyzed. (C) At later neural plate stages (st 1618), ~52% of embryos showed broader sox2 expression in the np on the zmym2 mRNA injected side (red bar) compared to uninjected side (black bar). No embryos showed decreased expression. Number above the bar denotes number of embryos analyzed. (D) At later NP stages, ~77% of embryos showed broader foxd3 expression in the neural crest domain on the zmym2 mRNA injected side (red arrow). No embryos showed decreased expression. Number above the bar denotes number of embryos analyzed. (E) Examples of later NP stage embryos in which three pre-placodal ectoderm gene expression domains are reduced (red arrows) on the zmym2 mRNA injected side. (F) The percentage of embryos with increased (dark blue), decreased (light blue) or no change (white) in pre-placodal ectoderm gene expression when one side was injected with zmym2 mRNA. Numbers above the bars denote number of embryos analyzed. (G) The expression of sox9 in both the otic placode (OP; stage 16) and otic vesicle (OV; stage 32) was reduced on the side injected with zmym2 mRNA (red arrows) compared to the control side (ctrl) of the same embryo. dlx5 expression in the OV also was reduced (red arrow). (H) The percentage of embryos with increased (dark blue), decreased (light blue) or no change (white) in sox9 and dlx5 otic gene expression when one side was injected with zmym2 mRNA. Numbers above the bars denote number of embryos analyzed.
Figure S1. gBlock containing 3′HA tag (red highlight) inserted into the zmym2 ORF at an in-frame StyI restriction site (light blue highlight) and a 3′ XhoI restriction site (yellow highlight). The HA tag was inserted just before the TAA stop codon (green highlight). Successful insertion of the construct and the tag was confirmed by Western blot.
