ZSWIM4 regulates early embryonic patterning and BMP signaling pathway by promoting nuclear Smad1 degradation

doi:10.21203/rs.3.rs-1817709/v1

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ZSWIM4 regulates early embryonic patterning and BMP signaling pathway by promoting nuclear Smad1 degradation

https://doi.org/10.21203/rs.3.rs-1817709/v1

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The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. We found that Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 caused ventralization. Overexpression of zswim4 decreased, whereas knockdown of zswim4 increased, the expression levels of ventrolateral mesoderm marker genes. Mechanistically Zswim4 attenuates the BMP signal by decreasing the protein stability of Smad1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of Zswim4. Indeed, Zswim4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of Smad1 in the nucleus. Our study identified a novel mechanism that restricts BMP signaling in the nucleus.

Zswim4

BMP

Smad1

Spemann-Mangold organizer

Xenopus

Cullin RING ubiquitin ligase

Embryonic patterning is a stepwise process requiring precise temporal and spatial regulation of multiple signaling pathways, such as Wnt, Nodal, and BMP signaling ^{1, 2, 3, 4}. The coordination of these pathways leads to the activation of cohorts of genes at the appropriate time and position in embryos and governs cell fate specification. The formation and function of the Spemann-Mangold organizer (hereafter referred to as the organizer) during gastrulation and subsequent neural induction are two distinct but closely related landmarks during embryonic development. These processes received particular attention because of their unique roles in the embryonic induction ^{5, 6}.

The BMP signaling pathway plays an essential role in dorsoventral patterning during gastrulation ⁷. The activation of BMP signaling is triggered by the binding of BMP ligands to its receptors, which induces the phosphorylation of receptor-regulated Smads (R-Smad 1/5/8); the phosphorylated R-Smads interact with Smad4 to form a complex, which then enters the nucleus and initiate transcription of the target genes. A dorsoventral gradient of BMP signaling is established by the cooperation of BMP ligands produced on the ventral side and BMP antagonists, such as Chordin and Noggin, secreted from the organizer ^{8, 9, 10}. A high level of BMP signal on the ventral side induces the epidermis and ventral mesoderm formation via activating xhox3, vent1, while a low level of BMP signal on the dorsal side favors the formation of neuroectoderm and dorsal mesoderm ^{8, 11, 12}. In addition to the level of available BMP ligands, BMP signaling is also regulated by controlling the stability of signaling components. Smurf1 and Smurf2, two HECT type E3 ubiquitin ligases, were reported to induce the ubiquitination and degradation of R-Smads and BMP type I receptors during Xenopus embryonic development ^{13, 14, 15, 16, 17}. ZC4H2 was reported to stabilize Smad1 by antagonizing its ubiquitination by Smurf ¹⁸.

Kctd15 plays important roles in Xenopus and zebrafish embryonic development ^{19, 20}. We identified Zinc finger SWIM-type containing 4 (zswim4) as the target gene of Kctd15 ²¹. The cellular function of Zswim4 and its roles during embryonic development are largely unknown. We found that Zswim4 is a maternal factor, and then specifically expressed in the organizer at the onset of gastrulation and during the formation of the neural plate. Gain-of-function and loss-of-function assays consistently suggest that Zswim4 restricts the ventrolateral mesoderm formation and maintains the neural tissue. Mechanically, Zswim4 physically interacts with Smad1 and the Cul2-RING ubiquitin ligase complex to induce the ubiquitination and degradation of Smad1, leading to the attenuation of BMP signaling outputs. Our study reveals a new regulatory mechanism of BMP signaling by Zswim4.

Zswim4 is expressed in the organizer with nuclear localization.

We previously identified zswim4 as a Kctd15 downstream target gene ²¹. The Zswim4 harbors a nuclear localization signal at the N terminus and a SWIM domain named after SWI2/SNF and MuDR transposases. The SWIM domain is followed by a cut8/STS1 domain which is related to the proteasomes in the nucleus (Fig. 1a) ^{22, 23, 24}. The zinc finger SWIM domain-containing protein family is a novel protein family. Although Zswim8 has been reported to regulate the degradation of targeted microRNA, and functions as a ubiquitin ligase ^{25, 26}, the functions of this protein family remain largely unknown. Protein alignment indicates that Zswim4 is highly conservative among different species (Supplementary Fig. 1A). The X. laevis Zswim4 shares 69.9% and 74.3% identities at the protein level with the human and mouse homologs, respectively (Supplementary Fig. 1b).

We first examined the temporal and spatial expression patterns of zswim4. Zswim4 is a maternal factor with high expression levels at the cleavage stage (Fig. 1b). During blastula stages, zswim4 expression is enriched in the animal hemisphere (Fig. 1c). At the onset of gastrulation, intensive zswim4 signals appeared in the dorsal blastopore lip, the organizer, and were then enriched at the dorsal ectoderm at mid-gastrula stages (Fig. 1d-f). Its expression was detected in the anterior edge of the neural plate at neural stages, suggesting a possible role in neural differentiation and brain formation (Fig. 1g). Zswim4 signals were detected in the lens, brain, and some cranial nerves in the tailbud stage (Fig. 1h, i). Sections of the stained embryos also revealed zswim4 signals in the foregut, neural tube, and the region surrounding the notochord (Fig. 1j, k). The temporal expression pattern revealed by quantitative RT-PCR showed that zswim4 is highly expressed and maintained at a constant level before gastrulation (Fig. 1l). However, the expression of zswim4 is sharply decreased after gastrulation (Fig. 1l). We also examined the expression pattern of Zswim4 in mouse embryos. Mouse Zswim4 is expressed in the brain area, including the telencephalon, midbrain and hindbrain, and spinal cord (Fig. 1m-r). Distinct expression of Zswim4 can also be detected in the eyes of the E11.5 embryos (Fig. 1o). In addition to the central nervous system, Zswim4 is also expressed in the limb bud (Fig. 1o). An analysis of tissue sections revealed more details of Zswim4 expression in mouse internal organs, including convoluted gut, umbilical blood vessels, and livers (Fig. 1r). Cellular localization of Zswim4 was also examined in Hela cells transfected with ZSWIM4-FLAG or ZSWIM4-GFP. Immunostaining showed that ZSWIM4 is predominantly localized in the nucleus (Fig. 1s; Supplementary Fig. 2).

We next performed animal cap assays to study the regulation of zswim4 transcription. We injected either bmp4, fgf8, or wnt3a mRNA into both blastomeres of embryos at the two-cell stage. The animal caps were dissected at stage 9 and cultured for two hours. The expression of zswim4 was examined by RT-PCR. Indeed, the overexpression of either bmp4, fgf8, or wnt3a suppressed the expression of zswim4 (Fig. 1t). The injections were effective, as the expression of epidermal keratin (epiker) and xbra for bmp4, the expression of xbra for fgf8, and nr3 for wnt3a were successfully induced (Fig. 1t). We also injected chordin mRNA into embryos at the two-cell stage. Similar to sox2, which is a pan neural marker gene ²⁷, the zswim4 expression is increased upon overexpression of chordin in animal cap assay (Fig. 1u). Xbra expression was not detected, suggesting no mesodermal tissue contamination in the dissected animal caps (Fig. 1u). The response of zswim4 transcription is consistent with its expression in the dorsal blastopore lip and neural plate during embryonic development.

Dysregulation of zswim4 disturbs anterioposterior axis formation.

To investigate Zswim4 function during early embryonic development, zswim4 mRNA was injected into X. laevis embryos at the two-cell stage with three increasing doses. Zswim4 mRNA (20 pg/embryo) can cause an apparent disturbance of body axis formation in embryos when examined at the tailbud stages. These embryos showed a phenotype characterized by a shortened, and bent body axis with tails curved to the dorsal side. Severely affected embryos showed strong inhibition of head formation (Fig. 2a). As the injection dose increased, the relative number of embryos with moderate and severe malformations increased (Fig. 2b).

For the loss-of-function assay, we designed two antisense morpholino (MO) oligonucleotides (zswim4MO1 and zswim4MO2) to knock down zswim4 in X. tropicalis embryos. Zswim4MO1 is a translation blocking MO targeting the 5’ untranslated region of zswim4. To test the blocking efficiency of zswim4MO1, 100 pg zswim4-FLAG mRNA with or without 10 ng zswim4MO1 was injected respectively into X. tropicalis embryos at the one-cell stage. Western blot showed that zswim4MO1 effectively blocked the translation of zswim4-FLAG harboring the zswim4MO1 binding sequence (Fig. 2c) in X. tropicalis. The zswim4MO2 is an mRNA splicing MO designed to target the junction region of zswim4 intron 1 and exon 2 (Fig. 2d). The mis-splicing of zswim4 pre-mRNA caused by zswim4MO2 results in the skipping of exon 2, and the joining of exon 1 to exon 3 directly. A pair of primers bridging exon 1 and exon 3 was designed to test the efficiency of zswim4MO2 (Fig. 2d). After injection with zswim4MO2 (10 ng/embryo), the embryos were collected for RNA extraction. RT-PCR results showed that the mis-spliced band of zswim4 (200-bp band) appeared in the sample from the zswim4MO2 injected embryos, while the level of wild-type band of zswim4 (400-bp band) was evidently reduced compared with the uninjected embryo (Fig. 2e), suggesting the high efficiency of zswim4MO2. Both bands were confirmed by sequencing (Supplementary Fig. 3a, b). The injection of zswim4MO1 or zswim4MO2 caused a similar phenotype of the anterior axis inhibition and shortened body axis in embryos at stage 28 with varying severities (Fig. 2f). Embryos in the moderate group just showed only a shortened body axis, while those in severe group showed a ventralization phenotype with an extremely short body axis and the loss of head structures (Fig. 2f). Rescue experiments were performed using zswim4 mRNA without the 5’ untranslated region. When zswim4MO1 or zswim4MO2 was co-injected with the zswim4 mRNA, the relative number of embryos in the severe and moderate groups is decreased, while the relative number of embryos in the mild and normal groups increased (Fig. 2g, h), suggesting that the phenotype caused by zswim4MOs can be partially rescued and is specific.

CRISPR/Cas9 mediated genomic editing was used to introduce mutations to the Xenopus zswim4 gene. We first designed four sgRNAs targeting zswim4 (sgRNA1-4 in Supplementary Fig. 4) and co-injected them with Cas9 protein into X. tropicalis embryos ²⁸. T7E1 assay was used to assess the gene disruption efficiency (Fig. 2i). In more than half of the embryos of the tail-bud stage, CRISPR/Cas9 disruption of zswim4 caused short tail and trunk, as well as loss of head structures, including eyes and brain. This phenotype was highly similar to the phenotype induced by the MOs (Fig. 2i). Taken together, these results indicate the critical roles of Zswim4 in body axis formation and head development.

We further chose two sgRNAs targeting the first exon of X. tropicalis zswim4 (sgRNA4 and 5 in Supplementary Fig. 4) to generate a zswim4 mutant line. The two sgRNAs were injected separately with Cas9 protein into the Xenopus embryos at the one-cell stage. After confirming the gene disruption by T7E1 assay and Sanger sequencing, the injected embryos and their wild-type siblings were raised into adults (Supplementary Fig. 5a-c). Female F0 frogs (zswim4F0) were then crossed with wild-type male frogs (Fig. 2j). In two independent crosses, approximately 26% of the offspring (33 out of 126) showed a ventralization phenotype, i.e., a shortened body axis, an enlarged ventral side, and suppression of the head. This phenotype was also observed in zswim4MO- or CRISPR/Cas9 injected embryos (Fig. 2k). Most of the embryos showing the phenotype died at the later stages, and the larvae that survived have relatively normal body axis, but with cyclopia, or various degrees of eye field split failure (Fig. 2l). Three representative embryos at stage 25 and five larvae at stage 42 were collected for the T7E1 assays. All of these embryos harbor mutated alleles and should be heterogeneous (Fig. 2k, l).

After raising the mutant embryos to adults, we obtained the F1 generation of zswim4 mutant X. tropicalis frogs containing a 10-bp deletion, zswim4^+/− (Supplementary Fig. 5d). The mutant allele of zswim4^+/− contains a premature stop codon and encodes a truncated form of Zswim4 without most of its protein domains, including the cut8 and zswim domains (Supplementary Fig. 5d). We further crossed two zswim4^+/− mutants, and the offspring was named zswim4F2, including wild-type, zswim4^+/− and zswim4^−/− embryos. Maternal zygotic zswim4^−/− mutant embryos (MZzswim4^−/−) were also obtained by crossing two zswim4^−/− frogs. However, we observe no obvious morphological defects in the zswim4F2 embryos or MZzswim4^−/− embryos until the tailbud stages. We reason that other members of the Zswim protein family compensate for the loss of the Zswim4, as CRISPR/Cas9 may induce the genetic compensation during the process of the establishment of zebrafish mutant line ²⁹. To confirm this, we examined the expression of homologous genes in the Zswim family with quantitative RT-PCR. Indeed, the expression of zswim5, zswim6, and zswim7 was increased approximately two-fold in MZzswim4^−/− embryos compared with wild-type embryos (Supplementary Fig. 5e). In zswim4F2 embryos, all of the zswim genes except zswim9 exhibited increased expression levels compared with their levels in wild-type embryos, with zswim7 showing an eight-fold increase (Supplementary Fig. 5e). These data suggest that the genetic compensation accounted for the phenotype discrepancy between F0 embryos and F2 embryos of the zswim4-knockout line. Such genetic compensation was not observed in F0 embryos simultaneously injected with Cas9 protein and four sgRNAs targeting zswim4 (Fig. 2i; Supplementary Fig. 5e).

Zswim4 restricts the ventrolateral mesoderm and promotes neural formation by inhibiting BMP signaling.

As Zswim4 affects body axis formation and is expressed in the organizer and the forming neural plate, we sought to determine whether the marker genes for the ventral mesoderm and neural ectoderm were affected by the dysregualtion of Zswim4. Whole-mount in situ hybridization results showed that overexpression of zswim4 severely inhibited the expression of sizzled and vent1 in X. laevis embryos injected with zswim4 mRNA into two ventral blastomeres at the four-cell stage (Fig. 3a-d). Consistent with this observation, quantitative RT-PCR also showed a reduction in the levels of the two ventral mesoderm markers in embryos after the injection of zswim4 mRNA (Fig. 3e). The expression of neural ectoderm marker genes, otx2 and sox2, was both decreased after the knockdown of zswim4 with zswim4MO1 (Fig. 3f-i). At the neurula stages, the signal of the pan-neural marker, sox2, was reduced in half of the embryos injected with zswim4MO1 (Fig. 3j, k). Quantitative RT-PCR results showed that the expression of sox2 was reduced after the injection of Xenopus embryos with different doses of zswim4MO1. Meanwhile, the ventral mesoderm marker genes, sizzled and vent1, were increased in a dose-dependent manner (Fig. 3l). Collectively, these results indicate that Zswim4 is required to restrict the ventral mesoderm formation and promote neural fate in the early gastrula.

As the BMP gradient along the dorsoventral axis plays an important role in Xenopus embryonic patterning, we next examined whether Zswim4 regulates embryonic development by antagonizing BMP signaling. To test whether Zswim4 inhibits BMP signaling transduction, we injected bmp4 mRNA (300 pg/embryo) into both blastomeres of embryos at the two-cell stage and then performed animal cap assays. The activation of BMP signaling was confirmed by the up-regulation of xhox3, xbra, and wnt8, three BMP responsive genes ³⁰. The expression levels of these genes were attenuated by the co-injection of a high dose of zswim4 mRNA (200 pg/embryos) (Fig. 3m), suggesting the suppression of BMP signaling by zswim4. Consistent with this observation, the overexpression of zswim4 alone in the animal cap induced the expression of neural markers, sox2, sox3, and otx2, suggesting that the endogenous BMP signal was also attenuated by zswim4 (Fig. 3n). To examine whether endogenous Zswim4 also plays a role in antagonizing BMP signal, we overexpressed bmp4 in embryos, resulting in 37% of the injected embryos with strong expression of sizzled. After co-injection of zswim4MO1 with bmp4 mRNA, 69% of the injected embryos showed strong sizzled expression (Fig. 3o, p). These results suggested that Zswim4 restricts ventral mesoderm formation by antagonizing BMP signaling. Chordin antagonizes the BMP signaling pathway, and injection of chordin induces a secondary axis when ventral blastomeres are targeted ^{9, 31}. Twenty percent of the embryos injected with chordin mRNA (3 of 15) had a secondary axis, while after co-injection of zswim4 mRNA (200 pg/embryo) and chordin mRNA, 52% (11 of 21) of the embryos had a secondary axis (Fig. 3q, r). Phosphorylated Smad1 (p-Smad1) is an essential mediator of BMP signal activation. Overexpression of zswim4 in the whole embryo (200 pg/embryos) apparently reduced the level of p-Smad1 and total Smad1 (Fig. 3s). Conversely, the knockdown of zswim4 caused a moderate increase in total Smad1 levels and an apparent elevation of p-Smad1 levels (Fig. 3t). These results indicated that Zswim4 inhibits BMP signaling activity in both whole embryos and animal caps.

ZSWIM4 attenuates BMP signaling in the HEK293T cell line.

To examine whether the inhibitory role of Zswim4 on BMP signaling is conserved in mammalian cells, we performed luciferase assays in HEK293T cells transfected with a BMP responsive element (BRE)-luciferase reporter. BMP4-induced luciferase activity was inhibited by transfected ZSWIM4 in a dose-dependent manner (Fig. 4a). Similarly, the expression of ID1 and ID2 induced by BMP treatment was also downregulated in ZSWIM4 transfected cells (Fig. 4b, c). Thus, overexpression of ZSWIM4 inhibits BMP signaling in HEK293T cells. We next knocked down endogenous ZSWIM4 through transfection of siRNAs targeting human SWIM4. All three siRNAs can effectively attenuate ZSWIM4 expression, resulting in approximately a 60% reduction in ZSWIM4 mRNA levels compared with the levels in control cells (Supplementary Fig. 6). Knockdown of ZSWIM4 promoted BMP4-induced luciferase activity, and the expression of ID1 and ID2 (Fig. 4d-f). Moreover, we established ZSWIM4 mutant HEK293T cell lines using CRISPR/Cas9. Two ZSWIM4 mutant clones, Z7 (Δ25/Δ16) and Z9 (Δ2/+375), exhibited increased ID1 expression with or without BMP treatment compared with wild-type cells (Fig. 4g-j). Consistent with this observation, the levels of p-Smad1 and total Smad1 were upregulated in the two mutant cell lines (Fig. 4k, l). Collectively, these results indicate that the endogenous ZSWIM4 functions to attenuate the BMP signaling in HEK293T cells.

Given that Zswim4 is localized in the nucleus, we performed cell fractionation assays to determine whether only the nuclear Smad1 phosphorylation was affected by Zswim4. After transfection of ZSWIM4-Myc and treatment with BMP4, we performed cell fractionation to separate cytosol and nuclear proteins. A decrease in the total SMAD1 level was observed in the nuclear fraction after overexpression of ZSWIM4, compared with the levels in the nuclear fraction of control cells, while in the cytosol, the SMAD1 level remained at a similar level after overexpression of ZSWIM4 (Supplementary Fig. 7). The phosphorylation of SMAD1 in the nuclear portion was reduced to a much greater extent than in the cytosolic fraction upon ZSWIM4 overexpression (Supplementary Fig. 7). These results suggest that ZSWIM4 mainly reduced the total level of Smad1 and the level of phosphorylated SMAD1 in the nuclear fraction, which is consistent with its nuclear localization.

ZSWIM4 promotes the ubiquitination and degradation of SMAD1.

To examine the regulatory level of ZSWIM4 in the BMP signaling hierarchy, we transfected HEK293T cells with either constitutively active BMP receptor, BMPR1A_CA ³², or constitutively phosphorylated SMAD1, SMAD1-DVD ³³. Both of the components increased the BRE-luciferase activity, and this effect was blocked by overexpression of ZSWIM4 (Fig. 5a). This result suggests that ZSWIM4 acts downstream of SMAD1 phosphorylation, and its function is independent of de-phosphorylation process of Smad1, or at least, does not entirely rely on SMAD1 de-phosphorylation. Considering that the total Smad1 protein levels were increased in the ZSWIM4 mutant cell lines, Z7 and Z9, it is possible that ZSWIM4 attenuates BMP signaling by reducing SMAD1 protein stability. Indeed, co-transfection of Myc-tagged SMAD1 with increasing doses of zswim4 caused a dose-dependent reduction of SMAD1-Myc protein (Fig. 5b). In Xenopus zswim4 morphants, the endogenous Smad1 protein was also upregulated compared with that in control embryos (Fig. 5c). Thus, Zswim4 decreases Smad1 protein levels in both Xenopus embryos and mammalian cells.

The mRNA levels of smad1 were not affected by zswim4 overexpression in either Xenopus embryos or HEK293T cells (Supplementary Fig. 8a, b). We then examined whether ZSWIM4 is involved in regulating SMAD1 protein stability. SMAD1 protein stability was measured following the treatment of HEK293T cells with cycloheximide (CHX) to inhibit protein synthesis. Indeed, the protein stability of SMAD1-FLAG was markedly reduced in the presence of ZSWIM4 compared with its stability in control cells (Fig. 5d, e). We conclude that ZSWIM4 regulates the protein stability of SMAD1.

The ubiquitination and proteasome-mediated degradation of SMAD1 represents an important regulatory mechanism in BMP signal. To examine the ubiquitination of Smad1, we co-transfected HA-tagged SMAD1 and Myc-tagged Ubiquitin into HEK293T, and immunoprecipitation was performed. Western blots showed that the overexpression of ZSWIM4 significantly increased the ubiquitination of SMAD1 (Fig. 5f), while SMAD1 ubiquitination was reduced in the ZSWIM4 mutant cell lines, Z7 or Z9 (Fig. 5g, h). Taken together, these results indicate that Zswim4 attenuates BMP signaling by promoting the ubiquitination and degradation of SMAD1.

The ubiquitin ligases SMURF1 and SMURF2 have been reported to promote the ubiquitination and degradation of SMAD1 ^{14, 15}. To examine whether ZSWIM4 is required for SMURF1 or SMRUF2 to regulate SMAD1 stability, we co-transfected SMAD1-FLAG and SMURF1 or SMURF2 into either the ZSWIM4 mutant cell line, Z7, or wild-type HEK293T cells. The levels of SMAD1-FLAG were reduced by SMURF1 or SMURF2 overexpression in both cell lines, suggesting that ZSWIM4 is not involved in the degradation of SMAD1 mediated by SMURF1 or SMURF2 (Supplementary Fig. 8c).

ZSWIM4 forms a complex with SMAD1 and Elongin B/C-containing ubiquitin ligase.

As ZSWIM4 regulates the ubiquitination of SMAD1, we next investigated whether it can physically interact with SMAD1. The FLAG-tagged SMAD1 and Myc-tagged ZSWIM4 were transfected into HEK293T cells, and co-immunoprecipitation (Co-IP) was performed with either anti-Myc or anti-FLAG antibodies. Strong interactions between SMAD1 and ZSWIM4 were observed in co-transfected groups (Fig. 5i). Co-IP was also performed using Xenopus embryos injected with zswim4-FLAG mRNA with anti-FLAG or anti-Smad1 antibodies. An interaction between Zswim4-FLAG and endogenous Smad1 was also detected in Xenopus embryos (Fig. 5j). The Smad1 protein comprises three domains, i.e., the MH1and MH2 domains, and a linker region. To identify the domain responsible for the interaction with ZSWIM4, we co-transfected HEK293T cells with ZSWIM4-Myc and different deletion mutants of SMAD1 with a FLAG tag, respectively (Fig. 5k). After Co-IP, ZSWIM4-Myc was only co-precipitated with the SMAD1 deletion mutants containing an intact MH1 domain, indicating that the MH1 domain in SMAD1 is essential for its interaction with ZSWIM4 (Fig. 5l). Accordingly, immunofluorescence coupled with confocal microscopy revealed the co-localization of ZSWIM4 and SMAD1 in the nuclei of the HeLa cells (Fig. 5m). Thus, Zswim4 specifically interacts with Smad1 in both Xenopus embryos and mammalian cell lines. In contrast, no interaction was detected between ZSWIM4 and SMAD2, the transducer protein of the Nodal signaling pathway (Supplementary Fig. 9).

To identify the possible interaction partners of Zswim4 in regulating SMAD1 stability, stable isotope labeling by amino acid in cell culture (SILAC)-IP was performed (Fig. 6a)^{34, 35}. After LC-MS/MS analysis of the immunoprecipitates, ELONGIN B (ELOB) and ELONGIN C (ELOC) were identified among the 10 most abundant proteins in forward and reverse SILAC (Fig. 6b). The interaction between Zswim4 and ELOB or ELOC was then confirmed by Co-IP in HEK293T cells transfected with FLAG-tagged zswim4 and Myc-tagged ELOB or ELOC (Fig. 6c, d). The ELOB and ELOC have been identified as positive regulators of RNA polymerase II ^{36, 37}. They also interact with Cullin and the RING domain protein Rbx to form Cullin-RING ligase (CRL) complex, which promotes the ubiquitination of its targets by binding to different substrate-recognition components ³⁸. ZSWIM4 was predicted to be a member of the Cul2-RING ubiquitin ligase complex using the online tool, STING. Previous studies showed that ZSWIM8 functions as a substrate-recognition subunit of the CRL complex ^{25, 26}. We then performed an alignment between Zswim4 proteins from different species and human ZSWIM8, and a conserved BC-box and Cul2-box were identified in the N-terminus (Fig. 6e) ³⁹. Indeed, we found that Zswim4 has a physical interaction with CUL2, the scaffold protein in the CRL complex (Fig. 6f). To examine whether ZSWIM4 interacts with ELOB or ELOC through the BC-box, we generated a ZSWIM4 deletion mutation, ΔN, by removing the N-terminal amino acids 1–99 containing the BC-box (Fig. 6g). Indeed, ΔN exhibited a much weaker binding ability to ELOB and ELOC than did wild-type ZSWIM4 (Fig. 6h, i). Thus, ZSWIM4 interacts with the components of the CRL complex, and the amino acids 1–99 are essential for binding to ELOB and ELOC. Taken together, these results indicate that ZSWIM4 forms a complex with CRL and SMAD1.

ZSWIM4 functions in the CRL complex to regulate SMAD1 stability.

As ZSWIM4 interacts with SMAD1 and CRL, it may function as a substrate recognition unit for the CRL complex. To examine whether ZSWIM4 regulates SMAD1 protein degradation by cooperating with CRL, we co-expressed Zswim4 and the components of the CRL complex. Compared with the single transfection of ZSWIM4-Myc or cul2, co-transfection of both showed a stronger effect at reducing the stability of Myc-tagged SMAD1 (Fig. 7a). Further co-transfection of ELOB and ELOC caused the highest degree of degradation of SMAD1-Myc (Fig. 7b). Consistent with these results, the ubiquitination assay of SMAD1-HA showed the strongest signals in the cells co-overexpressing Zswim4-FLAG, Cul2, ELOB, and ELOC (Fig. 7c). Furthermore, luciferase assays were performed to examine the effects of these factors on BMP signaling activity. Transfection of a low dose of zswim4 or cul2 had little effects on the luciferase activity much, while their co-transfection apparently reduced the luciferase activity. Luciferase activity was further reduced when ELOB and ELOC were also transfected, either in the presence or absence of BMP2 (Fig. 7d). Next, an animal cap assay was performed to study whether the CRL complex can attenuate the BMP signal in Xenopus embryos. Considering the high expression level of zswim4 in animal pole (Fig. 1c), bmp4 mRNA was co-injected with cul2 or human ELOB and ELOC mRNA. Overexpression of cul2 or ELOB and ELOC efficiently attenuated the bmp4-induced expression of epiker and msx1 (Fig. 7e), suggesting a conserved role of CRL in attenuating BMP signaling. Taken all together, these results indicate that ZSWIM4 and the CRL complex work synergistically to attenuate SMAD1 protein stability and BMP signaling.

Zswim4 is a conserved protein, but its functions are largely unknown. In this study, we investigated the function of Zswim4 during early embryonic development and elucidated its function in mediating BMP signaling transduction. Our results indicate that it attenuates BMP signaling and is essential for axis formation during gastrulation. Indeed, Zswim4 is a novel BMP inhibitor that is involved in the Smad1 degradation in the nucleus.

Zswim4 is expressed in the organizer and then enriched in the dorsal ectoderm at the mid-gastrula stages. The organizer plays an essential role during the neural induction, the development of the anterioposterior ⁴⁰ and dorsoventral pattern ⁴¹. Our observations on Zswim4 are consistent with these functions. The gain-of-function of zswim4 led to a shortened anteroposterior axis and the repression of the head formation. Knockdown of zswim4 in X. tropicalis embryos resulted in ventralization, which was also observed in zswim4 mutant F1 generation embryos (Fig. 2k). The organizer attenuates the Wnt, BMP, and Nodal signaling during gastrulation by secreting their antagonists. Chordin and Noggin are the two secreted BMP antagonists that help establish a BMP gradient along the dorsoventral axis by blocking the binding of BMP ligands to their receptors ⁴². Meanwhile, Zswim4 localized in the nucleus attenuates BMP signaling by inducing the degradation of nuclear Smad1. The phosphorylation of Smad1, as an indicator of BMP signaling activation, was also downregulated by Zswim4, which may be mainly due to the degradation of the nuclear Smad1. The BRE luciferase activity induced by a constitutively active BMP receptor or by constitutively phosphorylated Smad1-DVD was attenuated by ZSWIM4 overexpression to nearly the same extent (Fig. 5a), suggesting that direct de-phosphorylation of Smad1 by Zswim4 plays a minor role, if any, in this process. Thus, in addition to the secreting antagonists and inhibitory SMADs, we identified a mechanism by which ZSWIM4 regulates BMP signaling.

Ubiquitin-mediated proteasomal degradation of SMAD1 represents an important mechanism to regulate BMP signaling. Smurf1 and Smurf2 were reported to induce the ubiquitination and degradation of R-Smads in BMP signaling pathway ^{14, 15}. The SMURF1/2 mediated SMAD1 ubiquitination was maintained at a similar level in ZSWIM4 mutant cells to that in WT HEK293T cells, suggesting ZSWIM4 regulates SMAD1 stability by another mechanism. Indeed, ELOB and ELOC, the two components of the CRL complex, were identified as ZSWIM4 interaction partners by SILAC assay. We also confirm the physical interaction between the ZSWIM4 and CUL2. CRL complexes utilize the CUL2 as the scaffold and recruit diverse substrate-receptors to target different proteins ³⁸. ZSWIM4 interacts with SMAD1 and the CRL components, CUL2, ELOB and ELOC, and co-expression of them synergistically promotes SMAD1 ubiquitination and degradation. These observations support the model that ZSWIM4 works as a substrate-recognition subunit of CRL to regulate SMAD1 stability (Fig. 8). Confocal imaging showed that ZSWIM4 proteins were localized in the nucleus, and the endogenous SMAD1 signals were enriched in those regions (Fig. 4m), suggesting that ZSWIM4 functions in the nucleus to induce SMAD1 degradation. In line with the model, a decrease in the total Smad1 level was only observed in the nuclear portion after overexpression of ZSWIM4, but not in the cytosol (Supplementary Fig. 7).

The Zswim family consists of at least nine members. To date, only a few studies have reported the functions of the family members. ZSWIM1 is a novel biomarker in T helper cell differentiation ⁴³. A mutation in ZSWIM6 causes acromelic frontonasal dysostosis (AFND) which is characterized by craniofacial, brain, and limb malformations, perhaps by inhibiting Hedgehog signaling ⁴⁴. Zswim6 also contains a Cul2-box ⁴⁵, and may function as an E3 ubiquitin ligase. In C. elegans, the Zswim8 homolog, EBAX-1 regulates axon guidance through functioning as a substrate-recognition subunit in the CRL complex ⁴⁶. Two recent studies also showed that ZSWIM8 is involved in target-directed miRNA degradation in cells of different species by functioning in the CRL complex to degrade Argonaute proteins, with Zswim8 acting as a substrate receptor ^{25, 26}. In our study, ZSWIM4 was found to cooperate with the CRL complex to regulate the ubiquitination and degradation of SMAD1 protein. Moreover, ZSWIM4 was found to regulate BMP signaling and early embryonic development in Xenopus embryos. Although Zswim family members are involved in various biological processes, studies by us and others suggest that they function, at least in part, as substrate adaptors for the CRL complex using similar molecular mechanisms. This may explain the strong genetic compensation effect observed in our X. tropicalis zswim4F2 and MZzswim4^−/− embryos (Supplementary Fig. 5e), in which other Zswim family members were upregulated, and may have compensated for Zswim4 function in the zswim4 mutant embryos. Taken together, we identified ZSWIM4 as a novel BMP inhibitor that promotes SMAD1 ubiquitination in the nucleus, and plays an essential role during embryonic body axis formation.

Plasmid constructs

The open reading frames of human and Xenopus zswim4, cul2, and human ELOB, ELOC, SMAD1 were amplified by PCR and subcloned into pCS2⁺ or pCS2⁺-MT, pCS2⁺-FLAG, pCS2⁺-HA vectors. Truncated mutants of human ZSWIM4 were generated by PCR and subcloned into pCS2⁺-MT. All the constructed plasmids were confirmed by sequencing. pSpCas9(BB)-2A-GFP (PX458) was purchased from Addgene (Addgene plasmid # 48138), and was used to generate ZSWIM4 mutant cell line ⁴⁷. pCMV5-hBMPR-1A CA (# 49527) and DR274 (# 42250) were also obtained from Addgene.

Embryo manipulations and injection

X. laevis embryos were obtained by in vitro fertilization ⁴⁸, and X. tropicalis embryos were obtained by natural mating. To prime the frogs, the male and female X. tropicalis were injected with 20 units of hCG into the dorsal lymph sac. On the next day, 200 units of hCG were injected for induction of full ovulation. Embryos were staged according to Nieuwkoop and Faber ⁴⁹. Capped mRNAs were synthesized in vitro using the mMessage mMachine kit (Thermo Fisher Scientific). Zswim4MOs were purchased from Gene Tools, Inc. (zswim4MO1: 5'-GGGACGGATATAGCAGCGAACCC-3'; zswim4MO2: 5'-CTCCTCGACCTGAAACAGAGGGAAC-3'). mRNAs or MOs were injected at the two-cell or four-cell stage. For CRISPR-mediated gene disruption, the sgRNAs were in vitro transcribed using the MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) with DR274 as the template, and then co-injected with CAS9 protein (PNA Bio) into the one-cell stage X. tropicalis embryos.

Whole-mount in situ hybridization and sectioning

Xenopus embryos were collected at the indicated stage, fixed in HEMFA ⁵⁰, and whole-mount in situ hybridizations were performed as previously described ^{51, 52}. After in situ hybridization, the embryos were embedded and sectioned according to the protocol used in our previous study ⁵⁰. Mouse in situ hybridization was performed using a previously published protocol ⁵³.

Animal cap assay, RT-PCR, and real-time PCR

For animal cap assay, indicated mRNAs or MOs were injected into the animal pole of X. laevis embryos at the two-cell stage. When the embryos developed to stage 9, the animal caps were dissected, and incubated in the L-15 medium for two hours ⁵⁴. RNA was then extracted using TRIzol reagent, and reverse transcriptions were performed using Superscript III (Thermo Fisher Scientific). Real-time PCR was performed using a ViiA 7 Real-Time PCR system (Applied Biosystems) using TB Green PCR premix (Takara). The Primers used for semi-quantitative RT-PCR and quantitative RT-PCR are listed in Supplementary Table 1.

Luciferase assay

HEK293T cells were seeded into a 24-well plate and transfected with a BRE reporter plasmid, Renilla luciferase pRL, and the indicated plasmids. One day after transfection, the cells were serum-starved for two hours, and then BMP2 or BMP4 was added. Luciferase activity was measured 18 hours later using Dual-Luciferase Assay system (Promega).

Co-immunoprecipitation (co-IP)

Co-IP was performed as previously described ⁵⁵. Briefly, the lysates of Xenopus embryos or HEK293T cells were incubated with the indicated antibody at 4°C overnight, followed by incubation with protein G-Dynabeads (Thermo Fisher Scientific) for one hour and then washing in lysis buffer five times. The precipitants and whole cell lysates were separated by SDS-PAGE and transferred to the nitrocellulose membrane for blotting.

Immunofluorescence

Cells were plated on poly-lysine coated glass slides. Twenty-four hours after transfection, the glass slides were washed twice with cold 1× PBS. The cells adhered on the glass slides were fixed with 2% PFA in 1× PBS and then incubated with the desired antibodies for 1 hour. Alexa Fluor 488- or 568-conjugated secondary antibodies were used to stain the first antibody. Fluorescence signals were captured by using an Olympus FV3000 confocal microscope.

Stable Isotope Labeling by Amino acid in Cell culture (SILAC)-IP for ZSWIM4 interactomics

Samples were prepared for SILAC using the SILAC Protein Quantitation Kit (Thermo Fisher Scientific #A33972) according to the manufacturer’s protocol. Briefly, HEK293T cells were cultured in DMEM, and 10% dialyzed fetal bovine serum, with L-Arginine monohydrochloride and L-Lysine monohydrate (Light amino acids) or ¹⁵N₂¹³C₆-lysine and ¹⁵N₄¹³C₆-arginine (Heavy amino acids). After eight passages, cells were collected from the heavy samples to verify the efficiency of isotope incorporation by mass spectrometry. For forward SILAC assays, the Zswim4-FLAG expression construct was transfected into the heavy sample, and the construct with a FLAG tag only was transfected into the light sample. For reverse SILAC assays, the Zswim4-FLAG construct was transfected into the light sample. IP was then performed with anti-FLAG antibodies as in the Co-IP protocol described above. The eluted precipitants were quantified and digested with trypsin, and then desalted and dried in a vacuum evaporator. The precipitants from light and heavy samples were combined in a 1:1 ratio for LC-MS/MS analysis.

Proteins, antibodies, and siRNAs

Recombinant BMP2 or BMP4 proteins were purchased from Cell Signaling Technology (CST). The antibodies used in this study are listed in Supplementary Table 2. Small interfering RNAs (siRNA) targeting ZSWIM4 were purchased from Genepharma (Shanghai, China). The sequences of the synthetic siRNAs are siRNA1 5’GCGUGUUGCAAGUGGGAUUTT3’, siRNA2 5’GGAGGAGACACUUACCCUUTT3’, and siRNA3 5’GCCCGCAAGCCCUGAUGAATT3’.

Statistical analysis

Statistical analysis was performed using either an unpaired two-tailed t-test or an unpaired two-tailed Chi-squared test with a P value less than 0.05 considered statistically significant (*P < 0.05, **P < 0.01; ns, no significant difference). Data represent mean ± SD.

Acknowledgment

This work was supported by the National Key R&D Program of China, Grant/Award Number: 2016YFE0204700, the Research Grants Council of Hong Kong (14112618 and 14119120) to HZ, and CRF equipment grant C5033-19E. Additional support was provided by the Hong Kong Branch of CAS Center for Excellence in Animal Evolution and Genetics to HZ. We thank our laboratory colleagues for their helpful discussion in this project.

Contributions

C.W., Z.L., H.I.U. performed the Xenopus experiments and analyzed the data. C.W., Y.Z. performed biochemical experiments. L.Z., Y.Z. carried out SILAC-IP and LC-MS/MS analysis. Q.L. performed Immunofluorescence. H.Z. conceived the project. C.W., H.Z. wrote the manuscript. X.Q., D.C., B.M., G.L., J.S., Y.Y., Y.D., Q.ZHAO, B.F., Q.ZHOU, W.Y.C. edited the manuscript and contributed to discussions.

Competing interests

The authors declare no competing interests.

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ZSWIM4 regulates early embryonic patterning and BMP signaling pathway by promoting nuclear Smad1 degradation

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