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Ventx1.1 as a Direct Repressor of Early Neural Gene zic3 in Xenopus laevis

  • Umair, Zobia ;
  • Kumar, Shiv ;
  • Kim, Daniel H. ;
  • Rafiq, Khezina ;
  • Kumar, Vijay ;
  • Kim, SungChan ;
  • Park, Jae-Bong ;
  • Lee, Jae-Yong ;
  • Lee, Unjoo ;
  • Kim, Jaebong
  • Received : 2018.08.13
  • Accepted : 2018.09.14
  • Published : 2018.12.31

Abstract

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

Keywords

neurogenesis;transcriptional regulation;Ventx1.1;Xenopus;zic3

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Fig. 1. Ventx1.1 represses early and late neural genes in Xenopus whole embryo and animal cap explants.

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Fig. 2. Genome-wide Xenopus ChIP-Seq of Ventx1.1 identifies two Ventx1.1 response cisacting elements (VRE1 and VRE2) on the zic3 promoter.

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Fig. 3. Site-directed mutagenesis of VRE1 and VRE2 within the zic3 promoter-reporter construct abolishes the repressional activity of Ventx1.1. on the reporter.

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Fig. 4. VRE1 mutation partially abolishes negative and positive effects of Xcad2 and FoxD5b-En, respectively.

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Fig. 5. Schematic diagram depicting Ventx1.1 as a key protein in the neural inhibitory circuit of BMP4/Smad1/Xcad2/Ventx1.1 and the reciprocal inhibitory circuit of FoxD5b/Ventx1.1.

Table 1. Primers used for serially-deleted reporter gene constructs

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Table 2. Primers used for RT-PCR amplification

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Table 3. Primers used for site-directed mutagenesis gene constructs

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Table 4. Primers used for ChIP-PCR assay

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Acknowledgement

Supported by : National Research Foundation of Korea (NRF), Hallym University