Xbra and Smad-1 response elements cooperate in PV.1 promoter to inhibit the early neurogenesis in Xenopus embryos

Crosstalk of signaling pathways plays crucial roles in cell fate determination, cell differentiation and proliferation. Both BMP-4/Smad-1 and FGF/Xbra signaling induce the expression of PV.1, leading to neural inhibition. However, BMP-4/Smad-1 and FGF/Xbra signaling crosstalk in the regulation of PV.1 transcription is still largely unknown. In this study, Smad-1 and Xbra physically interacted and regulated the PV.1 transcriptional activation in a synergistic manner. Xbra and Smad-1 directly bound within the proximal region of the PV.1 promoter and cooperatively enhanced the binding of an interacting partner within the promoter. Maximum cooperation was achieved in the presence of intact DNA binding sites of both Smad-1 and Xbra. Collectively, BMP-4/Smad-1 and FGF/Xbra signal crosstalk was required to activate the PV.1 transcription, synergistically. Suggesting that crosstalk of BMP-4 and FGF signaling facilitates the fine-tuning regulation of PV.1 transcription to inhibit neurogenesis during embryonic development of Xenopus. Summary statement FGF/Xbra positively regulates the PV.1 expression in the Xenopus via an unknown mechanism. Our study shows that both BMP-4/Smad-1 and FGF/Xbra exhibits a signaling crosstalk to regulate PV.1 transcription activation, promoting to ectoderm and mesoderm formation and inhibiting the early neurogenesis in Xenopus.

To examine the role of both cis-acting elements (BRE and XbRE) in PV.1 (-180) promoter construct, we mutated both cis-acting consensus XbRE and BRE within PV.1 (-180) promoter construct as shown in Figure 3F. We co-injected doubly mutated PV.1  promoter construct with Smad-1, and Xbra, in combination or separately. The results showed that Xbra and Smad-1-mediated stimulation in relative promoter activity of PV.1 (-180) promoter construct was completely abolished in PV.1 (-180)m(BRE+XbRE) ( Figure 4F) while concomitant overexpression of Smad-1 and Xbra increased up to 16-fold, 8.5-fold and 2.5-fold relative promoter activity with wild-type PV.1 (-180), PV.1 (-180)mXbRE and PV.1 (-180)mBRE, respectively ( Figure 4B, 4D and 4E, bar 4). The results indicated that both consensus cis-acting BRE and XbRE were required for maximum activation of PV.1 transcription in synergistic manner. Moreover, BMP-4/Smad-1 and FGF/Xbra signaling crosstalk was played a critical role in a PV.1-dependent neural inhibition during early Xenopus development. We thus postulated whether Xbra and Smad-1 physically interact to regulate the synergistic activation of PV.1. We co-injected Xbra with the different construct of Smad-1; Smad-1 (wild-type), C-terminal phospho-mimic Smad-1 (3SD), and C-terminal phospho-dead mutant Smad-1 (3SA) and harvested injected embryos till stage 11 to perform immunoprecipitation assay ( Figure 4G). The results showed that Xbra physically interacted with Smad-1 (wild-type) and phospho-mimic Smad-1 (3SD) while the interaction of Xbra with phospho-dead mutant Smad-1 (3SA) was not detected ( Figure 4H). This result suggested that Cterminal phosphorylation of Smad-1 played a significant role in Xbra-Smad-1 interaction and notably required for synergistic regulation of PV.1 transcription activation. Moreover, the results showed that Smad-1-Xbra interaction was critically required for establishment of BMP-4/Smad-1 and FGF/Xbra-mediated signaling crosstalk which regulated the transcriptional activation of PV.1. This result supported to the previous study that Smad-1 C-terminal phosphorylation is remarkably required for Smad-1-Xbra interaction for stimulating the expression of the ventral genes (Messenger et al., 2005). For further confirmation of Smad-1 and Xbra-mediated synergistic regulation of PV.1 transcription, we assumed whether both Smad-1 and Xbra stimulates DNA binding of their interacting partner within the endogenous PV.1 promoter region. We co-injected Myc-Xbra mRNA with and without Smad-1 to perform the ChIP-PCR assay with anti-Myc antibody (Blythe et al., 2009). The result showed that ectopic expression of Smad-1 stimulated Xbra binding within the proximal region of the endogenous PV.1 promoter ( Figure 5A and B). Thus, we further asked whether Xbra stimulates Smad-1 binding within endogenous PV.1 promoter region and restores the inhibition of Smad-1, mediated by FGF/MAPK signaling (Schier, 2001;Pera et al., 2003). We co-injected Flag-Smad-1 mRNA with and without Xbra and performed ChIP-PCR assay with anti-Flag antibody. Surprisingly, this result showed that Xbra robustly enhanced the Smad-1 binding within the proximal region of endogenous PV.1 promoter (-180bp to -162bp) upstream of putative transcription initiation site ( Figure 5C and D). Further, we postulated whether the physical interaction of Smad-1 and Xbra regulates expression of other gene of the Vent family, we tested the Xvent promoter region with Smad-1 and Xbra both in separately, and in combination. Results exhibited that Smad-1 and Xbra increased relative promoter activity of Xvent2 (-1031) promoter construct and induced the expression of Xvent2, separately, but the concomitant injection of Smad-1 and Xbra was not able to regulate the expression of Xvent2 in a synergistic manner (Supplementary Figure 1). These results collectively indicated that both Xbra and Smad-1 directly bound within the proximal promoter region of PV.1. However, the direct binding of Xbra and Smad-1 within PV.1 promoter region was not critically required for synergistic regulation of PV.1 transcription. These results strongly suggested that Smad-1 played a significant role in a synergistic regulation of PV.1 transcription and also in BMP-4/Smad-1 and FGF/Xbra-mediated signaling crosstalk. Moreover, Xbra not only positively regulated the PV.1 transcription activation but also strongly stimulated the BMP-4/Smad-1 signaling. Also, the result suggests that synergistic effect of Smad-1 and Xbra may restore the activity of FGF/MAPK-mediated inhibition of BMP-4/Smad-1 signaling.
Furthermore, suggested that PV.1 may be a novel target of Smad-1 and Xbra interaction in a Vent family.

Discussion
Studies demonstrated that both BMP-4/Smad-1 and FGF/Xbra signaling positively regulates the transcription of the BMP-4 downstream target gene, PV.1 to promote embryos ventralization and ventral mesodermal formation (Lee et al., 2011;Yoon et al., 2014b). These studies established a signaling crosstalk between BMP-4/Smad-1 and FGF/Xbra signaling. However, the significance and mechanism of this signaling crosstalk are still mostly awaited to delineate. BMP-4/Smad-1 facilitates the ectoderm formation and inhibits the neural induction during early Xenopus development. Inhibition of BMP signaling is required for inducing the early neurogenesis in Xenopus development (Hawley et al., 1995;Wilson and Hemmati-Brivanlou, 1995;Xu et al., 1995;Hemmati-Brivanlou and Melton, 1997). Furthermore, FGF/Xbra signaling actively participates in the lateral mesodermal formation and A-P patterning of neural tissue (Kim et al., 1998;Gamse and Sive, 2000;Weisinger et al., 2008). Previous studies demonstrated that FGF/Xbra signaling catalyzes the inhibitory phosphorylation of Smad-1 at linker region and inhibits the BMP-4/Smad-1 signaling (Schier, 2001;Pera et al., 2003). Surprisingly, FGF/Xbramediated inhibition of BMP-4 signaling alone is not sufficient to trigger the neurogenesis in ectodermal explant (Wilson et al., 2001;Delaune et al., 2005). A recent study documented that   ,Ngnr,and (Figure 1A and 1B). This finding supports our previous study that Xbra induces the PV.1 expression and inhibits the early neurogenesis (Yoon et al., 2014b). However, this is still questionable that whether Xbra-mediated neural inhibition is facilitated in a PV.1-dependent manner. We demonstrated that PV.1 MOs-mediated knockdown of PV.1 restores the Xbra-mediated inhibition of early neurogenesis in ectodermal animal cap explants. Moreover, the ectopic expression of PV.1 MOs increases the expression of early and late neural marker genes, including FoxD5a, Ngnr, N-CAM, and Otx2, which decreased by Xbra, and PV.1 MOs also decreases the Xbra-mediated induction of PV.1 (Figure 2A and 2B). In addition, co-injection of PV.1 MOs with Xbra also increases the expression of early and late neural marker genes, including FoxD5a, Ngnr, N-CAM and Otx2 and also decreases the PV.1 expression (data not shown). These results significantly evidenced that Xbra catalyzes the inhibition of early neurogenesis in a PV.1-dependent manner. Moreover, these results suggested that PV.1 may be a direct target of Xbra and PV.1 promoter region may contain positive Xbra response elements (XbRE) to facilitate the PV.1-dependent neurogenesis inhibition.

Identification of Xbra response element (XbRE) within the PV.1 promoter.
To identify whether PV.1 promoter region contains cis-acting XbRE elements within 5'-flanking region of PV.1 and positively regulates PV.1 transcription activation, we found that Xbra induces the PV.1 transcription while DN-Xbra reduces the PV.1 transcription ( Figure 3A and 3B). These results provide evidence that PV.1 promoter region contains putative cis-acting XbRE and positively regulates PV.1 transcription activation to inhibit the early neurogenesis. In reporter assay of different serially deleted promoter construct found that Xbra increases the relative promoter activity of different serially deleted promoter constructs of PV.1 up to 1.5-4-fold ( Figure 3C-3E). These results strongly evidence that PV.1 (-103) promoter construct contains cis-acting XbRE to trigger the PV.1 transcriptional activation and inhibits the early neurogenesis.
A study documented the conserved cis-acting binding response elements (TCACACCT) for the T-box domain containing transcription factor in Drosophila (Conlon et al., 2001). In addition, recent study observed that Xbra modulates the expression of its target genes in Drosophila embryonic cells in a dose-dependent manner through binding within a consensus sequence Kusch et al., 2002). We further mapped out the cisacting XbRE within PV.1 (-103) promoter constructs and observed that PV.1 (-103) promoter construct contains a putative cis-acting XbRE (ATCACACTT, within -70bp~-62bp) upstream of putative transcription initiation site ( Figure 3F). To test whether putative XbRE positively regulates the PV.1 transcription activation, the loss-of-function study resulted that putative XbRE positively regulates the PV.1 transcription activation ( Figure 3G and 3F). These results collectively provide evidence that PV.1 (-103) promoter construct contains cis-acting XbRE which actively participates in transcriptional activation of PV.1 and inhibits the early neurogenesis. Thus, we assumed that whether Xbra directly binds within PV.1 promoter region to activate PV.1 transcription. The ChIP assay analysis confirmed that Xbra directly binds within the proximal region of the endogenous PV.1 promoter ( Figure 3H and I). These results collectively evidenced that PV.1 promoter comprises cis-acting XbRE which positively regulates the PV.1 transcription and Xbra directly binds within the proximal region of the PV.1 promoter to inhibit the early neurogenesis.

Xbra and smad-1 synergistically regulate the PV.1 transcription activation.
Our previous study documented that PV.1 promoter region contains the cis-acting BRE promoter. Using this hypothesis, we assumed that Xbra and Smad-1 mediates a signaling crosstalk between BMP-4/Smad-1 and FGF/Xbra signaling in context to PV.1 transcription activation. Our study provides evidence that Xbra and Smad-1 strongly increase the promoter activity of PV.1 (-180) promoter construct, which contains both BRE and XbRE, and cooperates synergistically to activate the PV.1 transcription for inhibiting early neurogenesis ( Figure 4A and 4B). We further hypothesized that whether XbRE plays a role in the synergistic regulation of PV.1 transcription. The loss-of-function study of XbRE resulted that Xbra and Smad-1-mediated synergistic activation of PV.1 (-103) promoter construct is completely abolished with PV.1 (-103)mXbRE ( Figure 4C). This finding suggested that XbRE plays a significant role in a synergistic activation of PV.1 transcription.
We further raised questions that whether XbRE or BRE, which one plays a more crucially role in synergistic activation of PV.1 transcription to inhibit the neurogenesis. Thus, our study evidenced that relative promoter activity of BRE mutated PV.1 (-180)mBRE (up to 2.5-fold) declines more remarkably in Xbra-Smad-1-injected embryos as compared to PV.1(-180)mXbRE (up to 8.5-fold) ( Figure 4D and 4E). These results concluded that BRE plays a more significant role in a synergistic regulation of PV.1 transcription activation as compared to XbRE. Moreover, these finding collectively suggested that BRE-mediated transcriptional activation of PV.1 inhibits early neurogenesis more efficiently and plays a crucial role in signaling crosstalk of BMP-4/Smad-1 and FGF/Xbra during Xenopus embryos. Moreover, the doubly loss-of-function study in PV.1 (-180) promoter construct showed that Smad-1 and Xbra-mediated synergistic activation of PV.1 transcription are completely abolished with PV.1 (-180)m(BRE+XbRE) promoter construct ( Figure 4F). These results concluded that both XbRE and BRE cis-acting response elements cooperate synergistically to regulate the PV.1 transcription activation and inhibit the early neurogenesis in the ectoderm. In addition, BRE plays a more profound role in signaling crosstalk, initiated by BMP-4/Smad-1 and FGF/Xbra in context to transcriptional activation of PV.1. Additionally, PV.1 may be a novel target of Smad-1 and Xbra interaction in a Vent family because the physical interaction of Smad-1 and Xbra does not regulate the Xvent2 expression in a synergistic manner (S1).
A study documented that C-terminal phosphorylation of Smad-1 plays a significant role in the physical interaction of Smad-1 and Xbra. Smad-1 physically interacts with an N-terminal domain of Xbra while C-terminal phosphorylated mutant Smad-1 ( S378N, Y336D, and Y343D) does not interact with Xbra (Messenger et al., 2005). Thus, we assumed that whether Xbra and Smad-1 interact with each other to establish the signaling crosstalk for synergistic regulation of PV.1 transcription activation. The immunoprecipitation assay showed that Xbra physically interacts with C-terminal phosphorylated Smad-1 and phospho-mimic 3SD (S462D, S463D, and S465D) Smad-1 while Xbra interaction with phospho-dead 3SA (S462A, S463A, and S465A) Smad-1 is not reported (Figure 4G and 4H). This study concludes that Smad-1 C-terminal phosphorylation plays a significant role in Xbra and Smad-1 interaction and establishes a signaling crosstalk between BMP-4/Smad-1 and FGF/Xbra to activate the PV.1 transcription in a synergistic manner for inhibiting the early neurogenesis in the ectoderm.
Physical binding of Smad-1 and Xbra stimulates promoter activity of PV.1 and is dependent on DNA binding, but not absolutely require DNA binding for cooperative stimulation.
Above collective findings showed the evidence that Xbra and Smad-1 cooperate synergistically to regulate the transcriptional activation of PV.1 and establishes a signaling crosstalk between BMP-4/Smad-1 and FGF/Xbra in context to activation of PV.1 transcription. Thus, we hypothesized that whether Smad-1 and Xbra increase the DNA binding of their respective interacting partners with the endogenous PV.1 promoter region for synergistic regulation of PV.1 transcription. Moreover, whether DNA binding of Xbra and Smad-1 is absolutely required for synergistic activation of PV.1 transcription. In ChIP assay found that Smad-1 increases Xbra binding with the endogenous promoter region of PV.1 ( Figure 5A and B). Moreover, we observed that Xbra could also increase Smad-1 binding with the endogenous promoter region of PV.1 ( Figure 5C and D). These results suggested that Smad-1 and Xbra not only triggers the promoter activity of PV.1 transcription in a synergistic manner but also promotes the DNA binding of their interacting partner and facilitates a signaling crosstalk in context to PV.1 transcriptional activation. Surprisingly, we observed that Smad-1 and Xbra binding to the endogenous PV.1 promoter are only required for transcriptional activation of PV.1, but this binding is not absolutely required for cooperative stimulation of PV.1 transcriptional activation.
Taken together, all collective findings suggested that the 5'-flanking region of putative transcription start site of PV.1 contains cis-acting XbRE within -70bp~-62bp upstream of putative transcription initiation site of PV.1. Furthermore, we found that Xbra is a positive regulator of PV.1 transcription and inhibits the early neurogenesis in a PV.1-dependent manner.
Moreover, Xbra and Smad-1 cooperates synergistically to regulate the PV.1 transcription activation positively and inhibits the early neurogenesis in ectoderm explant. In addition, BRE plays a more significant role in synergistic regulation the PV.1 transcription rather than XbRE and inhibits the early neurogenesis more efficiently. We also observed that FGF/Xbra and BMP-4/Smad-1 establishes a signaling crosstalk to inhibit the early neurogenesis. Additionally, Xbra and Smad-1 directly binds within PV.1 promoter region and enhances the binding of their respective interacting partner with the endogenous PV.1 promoter, but this promoter DNA binding is not absolutely required for synergistic regulation of PV.1 transcription activation during Xenopus development.

Ethics Statement
Institutional Animal Care and Use Committee (IACUC) approval is not required for the experimental use of amphibians or reptiles in Korea. All members of our research group are

PV.1 promoter constructs
The 2.5 kb of 5'-flanking region of positive clone was subcloned into the pGL-2 basic plasmid (Promega, Madison, WI) and was designated the -2525bp construct. Serially deleted PV.1 promoter mutants and triple-repeat BMP-4-response element (BRE) were generated from -2525bp construct and subcloned into a pGL-2 basic plasmid by PCR amplification (Table 1) according to Lee et al. 2011(Lee et al., 2011.

RNA isolation and RT-PCR
Xbra mRNA (1 ng) was injected into the animal pole at 1-cell stage of Xenopus embryos and harvested into 30% MMR solution with respect to control non-injected embryos till stage 8.
Animal caps were then dissected from the injected and non-injected embryos and incubated until stage 11 or 24 into 1X L-15 growth medium. Total RNA was isolated from whole embryos or animal caps using RNA-bee reagent following the manufacturer's instructions (TEL-TEST, Friendwood, Texas) and treated with DNase I remove gDNA contamination. RT-PCR was performed with Superscript II (Invitrogen, Carlsbad, CA), as described by the manufacturer, with 2 mg total RNA per reaction. PCR was performed according to the following conditions: 30 seconds at 94 0 C, 30 seconds at each annealing temperature, 30 seconds at 72 0 C; 20-28 cycles of amplification (Table 2).

Chromatin immune-precipitation (ChIP)
Chromatin immunoprecipitation assay was performed as described ( Primers were shown in Table 1 and 2.

Morpholino Oligos (MOs)
PV.1 morpholino oligos (MOs, Genetools, LLC) is an anti-sense oligodeoyynucletides were used for loss-of-function study. This morpholino was designed against 5' UTR and/or the start site of transcription initiation. The sequence of PV.1 MOs was as follows: PV.1 MOs: 5'-AATCTTTGTTTGAACCATGCTGAAGG -3' MOs were warmed at 55 0 C for 5 min and keep at 37 0 C until injection the embryos to avoid the clogging of microinjection needles. MOs were injected with 10ng per embryos.

Nucleotide sequence accession number
The PV.1 (accession number; AF133122) cDNA sequence has been submitted to GenBank.

Whole mount in-situ hybridization
Embryos were injected with mRNAs at one-cell stage and performed whole-mount in situ hybridization at stage 11 using standard methods with anti-sense probes for Xvent2 (Yoon et al., 2014a).

Conflicts of authors:
There is no author conflict to disclose.

Funding: This research was supported by National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2016R1D1A1B02008770)
and (NRF-2016M3A9B8914057). ( 2  0  0  2  )  '  A  n  t  i  m  o  r  p  h  i  c  P  V  .  1  c  a  u  s  e  s  s  e  c  o  n  d  a  r  y  a  x  i  s  b  y  i  n  d  u  c  i  n  g  e  c  t  o  p  i  c  o  r  g  a  n  i  z  e  r  '  ,   B  i  o  c  h  e  m  B  i  o  p  h  y  s  R  e  s  C  o  m  m  u  n   2  9  2  (  4  ) :     completely diminish with doubly mutated PV.1 (-180)m(BRE+XbRE) as compared to Figure   4B, 4D, and 4E. (G and H) 1ng of Smad-1-Flag, phospho-mimic Smad-1 3SD-Flag, and phosphorylation dead-mutant Smad-1 3SA-Flag were co-injected with Xbra-Myc (1ng) at 1-cell stage and collected the total protein at stage 11 to perform the immuno-precipitation with anti-Flag antibody. All relative promoter activity experiments performed in triplicate. All relative promoter activity data are shown as mean ± SE.