Inhibition of Neural Crest Formation by Kctd15 Involves Regulation of Transcription Factor AP-2

Inhibition of neural crest formation by Kctd15 involves regulation of transcription factor AP-2

Valeria E. Zarelli and Igor B. Dawid1

Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Contributed by Igor B. Dawid, January 7, 2013 (sent for review August 23, 2012)

The neural crest develops in vertebrate embryos within a discrete NC formation delimits the NC domain, preventing its lateral ex- domain at the neural plate boundary and eventually gives rise to a pansion into the placodal domain (31). Although Kctd15 inhibits the migrating population of cells that differentiate into a multitude of canonical Wnt pathway, a key signal in NC induction, the level of derivatives. We have shown that the broad-complex, tramtrack and Wnt inhibition seemed insufficient to explain its effectiveness in bric a brac (BTB) domain-containing factor potassium channel tetra- blocking NC formation, and thus we pursued the lead that the re- merization domain containing 15 (Kctd15) inhibits neural crest for- lated protein KCTD1 can interact with AP-2 in human cells (32, 33). mation, and we proposed that its function is to delimit the neural Here, we demonstrate that Kctd15 interacts with AP-2α and is crest domain. Here we report that Kctd15 is a highly effective in- highly effective in repressing the transcriptional activity of three AP- hibitor of transcription factor activating enhancer binding protein 2 2 family members. Kctd15 binds to the activation domain of AP-2α, (AP-2) in zebrafish embryos and in human cells; AP-2 is known to be preventing its function in a reconstituted model system. We propose critical for several steps of neural crest development. Kctd15 inter- that inhibition of AP-2 is a key mechanism in the role of Kctd15 in acts with AP-2α but does not interfere with its nuclear localization regulating NC formation during embryonic development. or binding to cognate sites in the genome. Kctd15 binds specifically Results to the activation domain of AP-2α and efficiently inhibits transcriptional activation by a hybrid protein composed of the regulatory The expression of several genes required during NC formation is kctd15 fi protein Gal4 DNA binding and AP-2α activation domains. Mutation suppressed in mRNA injected zebra sh embryos (31). We Tfap2 of one proline residue in the activation domain to an alanine (P59A) pursued the relationship between AP-2 and Kctd15 as yields a protein that is highly active but largely insensitive to Kctd15. family genes are expressed in the NC and epidermis in the embryo and are required for NC formation (16–21, 28, 30). To compare These results indicate that Kctd15 acts in the embryo at least in part kctd15 tfap2a by specifically binding to the activation domain of AP-2α, thereby the and expression domains we related both to the well-defined NC marker foxD3. The expression domains of kctd15 blocking the function of this critical factor in the neural crest tfap2a foxD3 induction hierarchy. and extended laterally beyond the domain, but the overlap region was different: tfap2a overlapped foxD3 throughout its domain, whereas kctd15 overlapped just barely at the outer neural plate border | Tfap2 | FoxD3 | transcriptional regulation edge (Fig. 1A). This pattern is consistent with our model in that Kctd15 is in a position to inhibit AP-2 function in the region just he neural crest (NC) is a uniquely vertebrate migratory cell lateral to the NC domain. Consistently, overexpression of kctd15 Tpopulation that gives rise to multiple derivatives, including inhibits expression of tfap2a (Fig. S1A), just as it inhibits the ex- craniofacial skeleton, peripheral nervous system, and melano- pression of other transcription factors in this region (31), em- cytes, whereas the neural plate border is a broad competence phasizing the requirement for balanced expression between these domain that contains progenitors for the NC and placodes (1, 2). two factors in the neural plate boundary. When exposed to appropriate stimuli, progenitors acquire NC fate and express multiple factors that activate downstream genes Physical Interaction Between Kctd15 and AP-2α. To test for physical (3–8), and several signaling cascades, including bone morphoge- interaction between Kctd15a and AP-2α we overexpressed hu- netic protein (BMP), wingless-type MMTV integration site family man untagged AP-2α and human Flag-ONE-Strep (FOS)-tagged (Wnt), FGF, retinoic acid, and Notch are vital in NC induction KCTD15 in HEK293T cells. AP-2α bound to KCTD15, whereas (9–12). Deficits in NC development are responsible for many no signal was recovered from empty vector-transfected cells (Fig. birth defects (13–15), broadening the interest in this system. 1B). We tested for confounding effects of the tag on zebrafish An important regulatory component in NC development is Kctd15 behavior and found that FOS-tagged kctd15 mRNA is as transcription factor AP-2, a target of Wnt signaling (16–21). The effective as the untagged form in inhibiting NC formation in AP-2 family contains five members (22), among which AP-2α,-β, zebrafish embryos (Fig. S1B). We tested for presence of an and -γ act in NC formation. After dimerization, AP-2 binds to DNA AP-2α/Kctd15 complex in embryos at the stage of NC formation. at sites with the consensus sequence 5′-GCCNNNGGC-3′, affect- Such a complex could be visualized (Fig. 1C) after increasing ing genes in a broad range of biological processes (22–27). AP-2 can sensitivity by using a high level of embryo extract. be divided into two broad regions: the N-terminal part containing the transactivation domain (AD) and the C-terminal region har- Kctd15 Is a Highly Effective Inhibitor of AP-2 Activity. To test for boring the DNA-binding domain (DBD) that also mediates di- functional interactions between Kctd15 and AP-2 we used a re- merization (22, 24). In zebrafish and Xenopus embryos, AP-2 is porter construct, AP2-Luc, that contains three copies of the AP-2 expressed in the NC anlage and has a role in NC formation (20, 23, consensus binding site controlling the expression of luciferase 28, 29). In zebrafish AP-2α is encoded by tfap2a,andlockjaw and (24, 34, 35) (Fig. 2A). Zebrafish AP-2α strongly activated the montblanc mutations in this gene exhibit defects in NC derivatives (28, 30), but induction of the NC is not abolished owing to partial functional redundancy between AP-2α and AP-2γ (30). In contrast, Author contributions: V.E.Z. and I.B.D. designed research; V.E.Z. performed research; V.E.Z. and I.B.D. knockdown of AP-2α blocks NC development in Xenopus (20, 29). analyzed data; and V.E.Z. and I.B.D. wrote the paper. We have shown that the BTB domain-containing factor Kctd15 The authors declare no conflict of interest. regulates NC specification (31). Zebrafish kctd15a and -b are ex- 1To whom correspondence should be addressed. E-mail: [email protected]. pressed at the neural plate border immediately lateral to the forming This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NC domain. We proposed that the inhibitory function of Kctd15 on 1073/pnas.1300203110/-/DCSupplemental.

2870–2875 | PNAS | February 19, 2013 | vol. 110 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1300203110 Downloaded by guest on September 27, 2021 AP-2α occurs in at least three isoforms varying at the N ter- minus; our standard construct is based on isoform 1b (35). To test whether Kctd15 inhibition is isoform specific, we tested zebrafish isoform 1a in the reporter assay; isoform 1a was active and inhibited by Kctd15 to a similar extent as isoform 1b (Fig. S2D). There are five AP-2 family members encoded by separate genes in vertebrate organisms (22). In Xenopus NC formation AP-2α is the dominant factor, whereas in zebrafish both AP-2α and AP-2γ are involved (20, 23, 28–30). We found that reporter activation by AP-2α,-2β,and-2γ is inhibited effectively by Kctd15 (Fig. S3A). We further used animal cap explants to test Kctd15 function in an additional context. AP-2α can cooperate with other factors to induce NC markers in this system (20, 29), and Kctd15 was able to inhibit this induction (Fig. S3 B and C).

AP-2 Binding to DNA Is Not Inhibited by KCTD15. Multiple steps in AP-2 expression and function might be inhibited by KCTD15. Using HEK293T cells, we asked ﬁrst whether KCTD15 reduces expression of AP-2α, the rationale being that some KCTD proteins are cofactors of ubiquitin ligases, leading to degradation of their substrates (36, 37). Eliminating this possibility we found that the level of AP-2α protein in the cell was not affected by KCTD15 (Fig. S4A). Regulation of intracellular localization controls the activity of many transcription factors, but we found that AP-2α remains predominantly nuclear in the presence of KCTD15 (Fig. S4B). AP-2α functions as an obligatory dimer in DNA binding and transcriptional activation. Using differentially BIOLOGY DEVELOPMENTAL

Fig. 1. Kctd15 and tfap2 expression overlaps in zebraﬁsh embryos, and the protein products interact physically. (A)Expressiondomainsoftfap2a and kctd15 (both blue) are compared with foxD3 (red). Anterior to the left, midline to bottom. (B) KCTD15 and AP-2α interact in mammalian cells. KCTD15 tagged with FOS and TFAP2a were cotransfected into HEK293T cells. Immunoblotting (IB) was carried out with Flag, AP-2α, and tubulin antibodies. (C) Co-IP of endogenous AP-2α and Kctd15 in one-somite-stage embryo extracts. Lanes are as follows: 1: AP-2α marker from co-IP of zAP-2α and Kctd15 expressed in HEK293T cells; 2: beads plus embryo extract, used as control; 3: beads plus embryo extract and anti-Kctd15 antibody; 4: beads plus buffer; 5: empty; 6: input, embryo lysate. Lanes 2 and 3 contain extract from 100 embryos, whereas lane 6 could be loaded with 3 embryos only because of high protein levels; therefore no band is seen. Background bands indicated by asterisks.

reporter in HEK293T cells, whereas cotransfection with zKctd15 dramatically reduced activity (Fig. 2B). Strong inhibition was also seen after coinjection into zebrafish embryos (Fig. 2C). Titration of Kctd15 levels highlights the fact that its inhibition of AP-2α activity is highly efficient (Fig. 2B), and human KCTD15 is even more efficient (Fig. S2 A and B). To compare protein expression levels we used one antibody to detect both proteins by their epitope tags. Kctd15 was highly expressed, with the zebrafish version approximately eightfold and the human version approximately 50-fold higher than AP-2α (Fig. 2D and Fig. S2C). Fig. Fig. 2. KCTD15 represses AP-2 function. (A) AP2-Luc reporter, containing S2D shows that zKctd15-FOS is as effective as the untagged three AP-2 consensus sites driving expression of luciferase (Luc) (24, 34). (B) B The reporter was strongly stimulated by zebrafish AP-2α (200 ng) and dra- version (Fig. 2 ) in inhibiting reporter activity, validating the use matically inhibited by zKctd15 (levels in ng indicated). RLU, relative light of tagged proteins to judge expression levels. On this basis we units. (C) Reporter activity in zebrafish embryos was inhibited by Kctd15. (D) estimate that at equimolar levels of Kctd15 and AP-2α protein Cells were transfected with FOS-tagged zebrafish AP-2α and Kctd15, and the reporter is inhibited by approximately 50%. lysates were blotted. Kctd15 is expressed more efficiently than AP-2α.

Zarelli and Dawid PNAS | February 19, 2013 | vol. 110 | no. 8 | 2871 Downloaded by guest on September 27, 2021 tagged constructs we showed that KCTD15 does not prevent The AP-2α PRD contains a PPxY sequence (here PPPY), known AP-2α dimer formation (Fig. S4C). as a protein motif interacting with the WW domain (39), preceded To test the possible influence of KCTD15 on binding of AP-2 by a similar four-residue motif (PPYF) (Fig. 4A and Fig. S5A). to its cognate sites in the DNA, we explored NC genes that are Because this motif is highly conserved among vertebrate AP-2 also expressed in HEK293T cells, such as MSX1, PAX3, FOXD3, proteins except AP-2δ (22), we systematically mutated proline and and SNAI2, components of the NC gene regulatory network (4). tyrosine residues in the PRD-Gal4 fusion construct and tested We checked the sequence of 1 kb upstream of the transcription Kctd15 interaction and transcriptional activity (Fig. 4 A–C and start site and chose one region for each gene that contains a ca- Fig. S6A). The activation function of the AP-2α PRD was not nonical AP-2 binding site. To carry out ChIP experiments we abolished by mutating individual or all proline residues within the transfected expression constructs for AP-2α alone or together eight-residue sequence (Fig. 4C and Fig. S6A). Rather, the activity with KCTD15 into the cells, because the endogenous content of was increased several fold above WT. In contrast, Kctd15 binding these two proteins is low. The ChIP results show that the chro- and inhibition were affected differentially. The P59A mutant in matin regions containing cognate sites did bind AP-2α, both in which the first proline of the PPxY motif was mutated could not the absence and presence of KCTD15 (Fig. 3A). In some cases bind Kctd15 (Fig. 4B), and reporter activation by this mutant was the presence of KCTD15 increased AP-2α binding, but such an largely unaffected by Kctd15; although up to a twofold inhibition effect was not seen in all regions tested. An additional ChIP was seen, we consider it insignificant compared with the 80- to experiment was carried out on cells transfected with AP2-Luc 200-fold inhibition of other constructs (Fig. 4C). The other single- reporter (Fig. 2A) to provide abundant optimized binding sites. proline mutants tested were bound and inhibited by Kctd15 as well The reporter DNA showed binding to AP-2α that was unaffected as the WT (Fig. 4 B and C and Fig. S6A). Likewise, mutation of the by the presence of KCTD15 (Fig. 3B). Thus, it seems that conserved tyrosine in the PPxY motif (Y62A) retained activity and KCTD15 does not prevent AP-2α binding to chromatin. Kctd15 sensitivity. Further, the 2A mutant in which both prolines Specificity of AP-2/DNA interactions was further tested in an of the degenerate PPYF motif were mutated (P55/56A) main- oligonucleotide pull down assay. Biotinylated double-stranded tained activity and Kctd15 sensitivity. In contrast, the 4A and 6A oligonucleotides containing an AP-2 binding site were incubated mutants, which involved multiple residues including P59, lost their with lysates of AP-2α–expressing cells, the complexes isolated, and affinity for and sensitivity to Kctd15 (Fig. 4 B and C and Fig. S6A). the eluted proteins visualized by gel electrophoresis and immu- To test whether these results depend on the artificial Gal4 fusion noblotting. Using two different oligonucleotides we find that AP- and use of UAS reporter we introduced the P59A mutation, alone 2α is bound and is effectively competed by WT but not by binding- or as part of the 4A mutant, into full-length AP-2α and tested site mutant nonbiotinylated oligonucleotides (Fig. 3C). Again, these mutant proteins against the AP2-Luc reporter. As shown in binding of AP-2α was unaffected by the presence of KCTD15 in Fig. S6B, the experiment confirmed the results based on the PRD- one case, whereas being slightly strengthened in the second case. Gal4 fusion. Although WT AP-2α was Kctd15 sensitive, both AP- 2α (P59A) and (4A) were active but Kctd15 insensitive. Fig. S6 C KCTD15 Inhibition Requires Proline 59-Dependent Binding to the and D shows that the various mutant constructs were expressed at AP-2α Activation Domain. AP-2α contains an activation domain closely similar levels. in its N-terminal region and a DNA binding and dimerization These results lead to the following conclusions. In agreement domain in its C-terminal region (22, 24, 38) (Fig. S5 A and B). with and extension of the data of Wankhade et al. (38), who Using constructs that express the two regions of zebrafish AP-2α mutated two of the residues we tested, we conclude that the we found that the N-terminal domain binds KCTD15 about as prolines and the tyrosine in the PPxY motif in AP-2α are not well as the full-length molecule, whereas no binding could be required for its activation function. However, P59 alone among detected with the C-terminal domain (Fig. S5B). Previous studies the residues tested is critical for binding of the activation domain have shown that the activation function of AP-2α is concentrated to Kctd15 and its sensitivity to inhibition. The tight correlation of in an ∼60–70 amino acid region that is proline-rich, although it binding with functional impairment implies that Kctd15 inhibits does not correspond to a typical proline-rich activation sequence AP-2α by direct interaction with a region in the activation do- (24, 38) (Fig. S5A). A fusion of the proline-rich domain (PRD) to main. Because P59 is not required for activity, we suggest that the GAL4 DBD interacts with Kctd15 and further strongly acti- Kctd15 binding occludes a functionally critical region or leads to vates an upstream activation sequence (UAS)-Luc reporter, as conformational changes that preclude transcriptional activation. shown previously (24, 38) (Fig. 4 A–C). Most importantly in the present context, the reporter activity elicited by the PRD-Gal4 Discussion fusion is inhibited by Kctd15 as effectively as AP-2α–dependent Kctd15 Inhibits AP-2 Activity. We originally focused our attention activity (Fig. 4C; compare with Fig. 2 B and C). Thus, Kctd15 on Kctd15 because of its highly effective inhibition of NC for- affects the activation function of AP-2α. mation in zebrafish and Xenopus embryos (31). The observed

Fig. 3. KCTD15 does not affect speciﬁc binding of AP-2 to DNA. (A) ChIP assay showing binding of AP- 2α to promoter regions of PAX3, MSX1, SNAI2, and FOXD3 in HEK293T cells. A region of approximately 200 bp containing an AP-2 binding site was ampli- ﬁed. Cells were transfected with AP-2α alone or together with KCTD15, as shown. PAX3 exon 5 was used as negative control. (B) ChIP assay of AP2-Luc. The AP2-Luc reporter construct was overexpressed in HEK293T cells alone (L), together with AP-2α (L-A), KCTD15 (L-K), or all (L-A-K). KCTD15 did not inhibit AP-2α binding to its cognate sites. (C) Oligonucleo- tide binding assay. Two oligos, each containing an AP-2 binding site, were used. Extracts of cells overexpressing AP-2α (A), KCTD15 (K), or both (A-K) were incubated with WT 3′-biotinylated (WT*) double-stranded oligos. The oligo–protein complexes were isolated, proteins eluted, separated by gel electrophoresis, and blotted with anti-AP-2α. Competition was carried out with WT or mutant (mut) unlabeled oligos (absence of asterisk indicates no biotinylation).

2872 | www.pnas.org/cgi/doi/10.1073/pnas.1300203110 Zarelli and Dawid Downloaded by guest on September 27, 2021 Fig. 4. Critical role of P59 for Kctd15 interaction and inhibition of the PRD. (A) Mutants in the conserved PPxY motif within the AP-2α PRD, and two additional mutants in the adjoining region are shown. PRDs were fused to the Gal4-DBD and the FOS tag. (B) Kctd15 interaction depends on P59. WT and mutant PRD fusion constructs were expressed in HEK293T cells alone or with Kctd15, and complexes were pulled down using Strep-Tactin matrix. PRDs could bind Kctd15 except in the constructs containing the P59A mutation (P59A, 4A, and 6A). (C) PPxY-mutants activate the UAS-Luc reporter two- to ﬁvefold higher than WT PRD-Gal4. PRD-Gal4 WT and P60A, Y62A, and 2A were dramatically inhibited by zebraﬁsh Kctd15, whereas P59A and mutants containing this change were inhibited twofold or BIOLOGY

less. The constructs were expressed at similar levels DEVELOPMENTAL (B and Fig. S6 C and D).

interference with Wnt activity provides a partial explanation of possibility that the interaction is mediated by other proteins in the effect, which is complemented by blockage of AP-2 function the extract, binding in extracts of mammalian cells and zebrafish in the embryo, as described in the present report. Canonical Wnt embryos is consistent with direct binding. Physical interaction in activity is not only essential for NC induction, it also acts very cell extracts between AP-2 and the related protein KCTD1 has high within the signaling hierarchy that initiates this process, and been reported previously, as was the inhibition of reporter acti- expression of tfap2a itself depends on a Wnt signal (29). Because vation (32), but no biological role is known for KCTD1. The Kctd15 inhibits tfap2a expression in the NC domain one might extent of inhibition seen with KCTD15 was substantially higher, argue that Kctd15 affects an upstream event, except for the fact but it is not clear whether this reflects inherent differences be- that the NC regulatory network is not a linear pathway due to tween the proteins or experimental conditions. The inhibitory multiple feedback loops. Thus, disruption of any one component effect was similar with all three AP-2 family members tested and can have both direct and indirect effects. This is particularly true was seen both in cultured human cells and in zebrafish embryos. of AP-2, which acts at different levels of the regulatory hierarchy Kctd15 inhibition of AP-2 activity is not based on a reduction in (20). Thus we suggest that Kctd15 affects NC formation through nuclear abundance of the transcription factor or its ability to bind both the Wnt and AP-2 modules. to cognate sites in the DNA. Instead, the effect depends on a Although our interest in Kctd15 and AP-2 derives from their role specific binding of Kctd15 to the proline-rich activation domain in NC formation, AP-2 has many biological roles, including the (PRD) within the N-terminal half of AP-2α. This binding involves activation of genes such as metallothionein (24), p21WAF1/CIP1 a specific proline residue that is part of a PPxY motif. Mutations in (40), several epidermal genes in Xenopus (18), and others (22, 27). any or all prolines in this region resulted in active AP-2α mole- A large number of target genes have been identified by genome- cules, as indicated previously (32, 38), but just a single proline wide approaches, including genes activated and suppressed by mutant abolished the sensitivity to Kctd15 (Fig. 4 and Fig. S6). AP-2 (26, 41, 42). It remains to be studied whether Kctd15 This striking effect emphasizes the specific structural requirements inhibits gene regulation by AP-2 in general or only in a certain for Kctd15 binding and inhibition of AP-2. It is possible that context. Although the expression patterns of AP-2 and Kctd15 Kctd15 binding affects the interaction of AP-2 with coactivators or overlap in some regions, they show considerable differences (23, corepressors. Although such factors have been described (45–47), 29, 31, 43, 44), and thus not all AP-2 functions are likely to be it is likely that additional cofactors exist that interact with AP-2 affected by Kctd15. and have a role in Kctd15 regulation. The PPxY motif is not known to bind to the BTB domain, the Physical and Functional Interactions Between Kctd15 and AP-2. We characteristic feature of the Kctd family. Rather, PPxY is a widely find that AP-2α and Kctd15 can bind to each other when coex- distributed motif that binds to the WW domain and in this man- pressed in cultured cells. Although we have not excluded the ner mediates multiple biologically important interactions (39).

Zarelli and Dawid PNAS | February 19, 2013 | vol. 110 | no. 8 | 2873 Downloaded by guest on September 27, 2021 Because there is no WW domain in Kctd15 and no similarity be- (American Type Culture Collection) were transiently transfected by using tween the BTB and WW domain structures (39, 48), the structural Lipofectamine 2000 (Invitrogen). HEK293T cells were transfected using basis for the AP-2/Kctd15 interaction remains to be elucidated. Xtreme-GeneHP(Roche).

Multiple Functions in the Kctd Protein Family. The Kctd family, with Pull Down, Immunoprecipitation, and Immunoblotting. Cells or embryos were more than 20 members, is characterized by a BTB domain with lysed in cold lysis buffer [0.5% Triton X-100, 50 mM Tris·HCl (pH 7.5), 150 mM similarity to potassium channels (36, 48), but Kctd proteins do NaCl, 5 mM EDTA] supplemented with complete Mini protease inhibitor not function as channels. BTB domain-containing proteins were mixture (Roche). General procedures were followed as previously described fi (57). NuPAGE Bis-Tris Gel system 4–12% (Invitrogen) was used for pro- classi ed into four subgroups with variable properties, yet the tein separation. SuperSignal West Pico or Dura Chemiluminescent Sub- basic folding structure of BTB domains remains similar (48). The strates from Thermo Scientific were used for detection of HRP-conjugated Kctd factors belong to the T1 subfamily of BTB domain-con- antibodies. taining proteins that are believed to assemble as tetramers (48), For immunoprecipitation of endogenous complex, one-somite embryos and a tetrameric form has been suggested for KCTD11 (37). were dechoryonated and lysed in RIPA buffer containing protease inhibitors. However, the only structure solved for a family member, KCTD5, Anti-Kctd15 antibody (2.5 μg; LifeSpan Biosciences) was bound to the beads shows a pentameric assembly, albeit with maintenance of the (Protein G Sepharose 4 Fast Flow; GE Healthcare). Precleared lysates corre- common BTB fold (49). Because AP-2 is known to dimerize, it is sponding to 100 embryos were used in each sample and incubated overnight likely that the complex between AP-2 and Kctd15 represents a at 4 °C. General procedures were followed as indicated above. The complex higher-order structure. was eluted using 2× sample buffer with 50 mM DTT for 5 min at 95 °C. A biological role has been determined for only a few of Kctd family members. Kctd5 and Kctd11 are adaptors for Cullin E3 Subcellular Fractionation. This was performed using NE-PER Nuclear and Cy- ubiquitin ligases, and Kctd11 affects the hedgehog pathway (36, 50). toplasmic Extraction Reagents (Thermo Scientific) according to the manu- Copy number variation of a chromosomal region that includes facturer’s instructions. KCTD13 is associated with neurological defects, and overexpression of KCTD13 in zebrafish induced microcephaly resembling the hu- mRNA Microinjection. mRNAs were synthesized from linearized constructs man condition, suggesting an involvement of KCTD13 in brain using the mMESSAGE mMACHINE kit (Ambion) according to the manu- ’ development (51). The related factors Kctd12.1 (also named Left- facturer s instruction. RNA quality was checked on formaldehyde gels. RNA over), Kctd12.2 (Right on), and Kctd8 (Dexter) show asymmetric or plasmids were injected into the yolk or the cell of one-cell-stage embryos. fi Embryos were collected at bud or one- to two-somite stage for in situ hy- expression in the left and right habenula in zebra sh and have been bridization (ISH) or luciferase assay. valuable markers for studying brain asymmetry (52). As mentioned above, Kctd1 interacts with AP-2 (32, 33). Kctd15, which we have Whole-Mount ISH. Zebrafish tfap2a ISH probe was prepared by subcloning shown to inhibit NC development and affect Wnt signaling and AP- ′ + Xenopus the 3 UTR into pCS2 after BamHI/EcoRI digestion. The foxD3 probe has been 2 function, is regulated by FGF in , and an enhancer trap previously described (31). Antisense riboprobes were generated according to line that mimics kctd15 expression in zebrafish has been described the manufacturers’ instructions (Roche). Whole-mount ISH was performed as (43,44).Further,theKCTD15 gene is associated with obesity in previously described (59). whole-genome association studies (53, 54), but no molecular basis for such a link has been reported. Factors important in adipogenesis ChIP Assay. Human genome browsers and PromoSer predictor (http://biowulf. are regulated by AP-2 (55-56), suggesting a possible molecular basis bu.edu/zlab/PromoSer) were used to identify putative AP-2-binding sites in for the observed linkage of KCTD15 to obesity. PAX3, MSX1, FOXD3,andSNAI2 genes within 1 kb upstream of the transcription start site. Primer sets were designed to amplify a 180- to 200-bp fragment Materials and Methods containing the consensus sites (Table S1). HEK293T cells were transfected with cDNAs and Plasmids. ORFs from ESTs of human TFAP2a isoform 1b (National TFAP2a alone or together with KCTD15. ChIP was performed as previously fi Center for Biotechnology Information reference sequence: NM_001032280) described (60) with small modi cations (61). Cross-linked chromatin was ∼ was obtained from Open Biosystems; zebrafish Tfap2a isoform 1b (Mamma- sheered to 300 bp using the Bioruptor Next Gen (Diagenode). Monoclonal α lian Gene Collection: 76915; IMAGE: 6524319) and Tfap2a isoform 1a anti-AP-2 3B5 (DSHB) antibody and control IgG were used for immunopre- (NM_176859.2, AF457191) amplified from cDNA of 24-h embryos were subcl- cipitation. PCR was performed using AccuPower PCR PreMix (Bioneer Inc.). oned into pCS2+ vector. Human Kctd15 ORF (1–283 aa) was obtained from HEK293T cells by RT-PCR. Zebrafish Kctd15a has been previously described Oligonucleotide-Binding Assay. Two 40mer oligonucleotides containing AP-2 (31). Human KCTD15,zebrafish Kctd15,andTfap2a (isoforms a and b and consensus sites (Tables S1) were 3′-biotynilated (WT*). WT and mutant un- domains) were subcloned into pcDNA3.1/myc-His containing the C-terminal labeled forms were used in competition experiments. Hybridized oligos FOS tag, generously provided by J. Magadan [Eunice Kennedy Shriver National (10 nmol) were mixed with precleared whole-cell extracts overexpressing AP- Institute of Child Health and Human Development (NICHD), National Institutes 2α, KCTD15, or both. Binding buffer containing 10 mM Tris·HCl (pH 7.5), 50 + of Health (NIH), Bethesda, MD] (57). zKctd15-FOS was transferred into pCS2 mM NaCl, 1 mM DTT, 0.3 mg/mL BSA, 10% (vol/vol) glycerol, and 10 μg vector for producing RNA for embryo injection. Xenopus Tfap2a and -b were polydI/dC per sample (Sigma) was used. WT oligos at 10×,50×, and 200×, generously provided by T. Sargent (NICHD, NIH, Bethesda, MD) and AP2-Luc and mutant oligos at 50× and 200× were used for competition. NeutrAvidin reporter by H. Hurst (Queen Mary University, London, UK). GFP-TFAP2a was resin (Sigma) was used to pull down the complexes. General procedures generated in the pEGFP-C1 vector and TFAP2a-Flag in the pFLAG-CMV-6c were as previously described (62). vector (Sigma-Aldrich). Wild-type and mutant zebrafish AP-2α PRD fused to the Gal4-DBD, and full-length Tfap2 mutants were subcloned into pcDNA3.1/ Luciferase Assay. pGL3-AP2-Luc and pGL2-Gal4-UAS-Luc reporters were used myc-His. 6×-Gal4-UAS-Luc reporter (58) was from Addgene (plasmid 33020). to study AP-2 or PRD and mutant activity. HEK293T cells were transfected with 250 ng of AP2-Luc or 6×-UAS-Luc reporter, 25 ng of pRL-CMV (renilla), Antibodies. Anti-AP-2α 3B5 and anti AP-2α 5E4 were purchased from DSHB, 200 ng of Tfap2a (full length or PRD and mutants), and 100 ng of Kctd15 and anti-zebrafish AP-2α antibody from LifeSpan Biosciences (LS-C87212/ unless specified. AB*/TL embryos were injected with 50 pg AP2-Luc reporter 25863). Anti-FLAG clone M2 antibody was from Sigma, and anti-GFP-HRP and 5 pg pRL-CMV DNAs, 50 pg Tfap2a mRNA alone or together with 80 pg from Miltenyi Biotec. Human KCTD15 was detected with anti-KCTD15 Kctd15 mRNA. Injected embryos (one-somite stage) or HEK293T cells (24 h MaxPab mouse polyclonal antibody (H00079047-B01P; Abnova) or rabbit after transfection) were lysed in 1× Passive Buffer (Promega). Luciferase anti-human KCTD15 polyclonal antibody (LS C110024/25441; LifeSpan Bio- assay was performed using the Dual Luciferase Reporter Assay System from sciences). The latter was also used to detect zebrafish Kctd15. Anti-α-tubulin Promega. Each luciferase activity was measured at least three times. antibody (Calbiochem) was used as control. HRP-conjugated secondary antibodies were from Jackson Laboratories. Animal Cap Assay. These were carried out as previously described (31, 63).

Cell Culture and Transfection. HEK293T and HeLa cells were grown in Dul- ACKNOWLEDGMENTS. We thank Martha Rebbert for assistance with Xen- becco’smodiﬁed Eagle’s medium supplemented 10% FBS. HeLa cells opus experiments, and Sunit Dutta, Ramanujan Hegde, Helen Hurst, Javier

2874 | www.pnas.org/cgi/doi/10.1073/pnas.1300203110 Zarelli and Dawid Downloaded by guest on September 27, 2021 Magadan, Hyunju Ro, Tom Sargent, Trevor Williams, and Minho Won for Program of the Eunice Kennedy Shriver National Institute of Child Health advice and reagents. This work was supported by the Intramural Research and Human Development.

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