Kumar S et al. (2019), Ventx1.1 competes with a transcriptional activa...

XB-ART-56631

BMB Rep 2019 Jun 01;526:403-408.

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Ventx1.1 competes with a transcriptional activator Xcad2 to regulate negatively its own expression.

Umair Z , Kumar V , Lee U , Choi SC , Kim J .

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Dorsoventral patterning of body axis in vertebrate embryo is tightly controlled by a complex regulatory network of transcription factors. Ventx1.1 is known as a transcriptional repressor to inhibit dorsal mesoderm formation and neural differentiation in Xenopus. In an attempt to identify, using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we observed that Ventx1.1 associates with its own 5'-flanking sequence. In this study, we present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevented Xcad2 from binding to the Ventx1.1 promoter, and vice versa. Taken together, these results suggest that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo. [BMB Reports 2019; 52(6): 403-408].

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Species referenced: Xenopus laevis
Genes referenced: cdx2 myc ventx1 ventx1.2 ventx2.2

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	Fig. 1. Ventx1.1 represses expression of ventral-specific genes. (A) One-cell stage embryos were injected in the animal pole region with Flag-Ventx1.1 mRNA (500 pg), and animal caps were dissected from injected or uninjected embryos at stage 8 and cultured to stage 11.5 for RT-PCR analysis. EF1Î± serves as a loading control. WE, stage 11.5 whole embryo. AC, animal cap. âˆ’RT, control in the absence of reverse transcriptase. (B) Quantification of expression levels of ventral genes (normalized to EF1Î±) from three independent experiments for (A). Error bars indicates standard error (SE).
	Fig. 2. Identification of a Ventx1.1-responsive element (VRE) in its own promoter region. (A) Diagram of Ventx1.1 promoter deletion constructs. The length that each promoter fragment extends upstream of the major Ventx1.1 transcription initiation site is indicated at left. Each promoter fragment was fused to luciferase (LUC) reporter gene. (Bâ€“E) Embryos were injected at one-cell stage with wild-type or putative VRE-mutated Ventx1.1 promoter deletion constructs alone or with Flag-Ventx1.1 mRNA (500 pg) as indicated, cultured to stage 10.5 (B) or 11.5 (Bâ€“E), and harvested for luciferase reporter assays. All relative promoter activity data are shown as mean Â± standard error (SE). (F) ChIP-PCR analysis showing the occupancy by Ventx1.1 of its own promoter region. Ventx1.2, a negative control for PCR, was amplified using its coding region-specific primers. IgG, a negative control IgG.
	Fig. 3. Xcad2 up-regulates Ventx1.1 transcription by occupying VRE1. (A) Animal caps from embryos injected or not with Flag-Ventx1.1 (500 pg) and/or Myc-Xcad2 (1 ng) mRNA as indicated were subjected to RT-PCR analysis at stage 11.5. (Bâ€“E) One-cell stage embryos were injected with indicated combinations of âˆ’951-bp reporter, âˆ’951-bp-mVRE1 reporter, Flag-Ventx1.1 (500 pg), and Myc-Xcad2 (1 ng) and cultured to stage 11 for ChIP-PCR (C, D) or stage 11.5 for luciferase reporter assay (B, E). (D) Quantification of relative intensities of bands in lanes 2, 5, and 6 shown in (C).
	Fig. 4. Ventx1.1 and Xcad2 co-occupy Ventx1.1 promoter region in competition with each other. (Aâ€“D) Embryos were injected with a combination of Flag-Ventx1.1 (0.5 ng) and increasing doses of Myc-Xcad2 (0.5, 1 ng) or Myc-Xcad2 (1 ng) and increasing doses of Flag-Ventx1.1 (0.25, 0.5 ng) as indicated and cultured to stage 11.5 for ChIP-PCR analysis. (B, D) represent quantification of relative intensities of bands in lanes 1, 2, and 3 in (A, C), respectively. (âˆ’), no injection of Myc-Xcad2 or Flag-Ventx1.1. Ventx2.1, a negative PCR control, was amplified with its coding region-specific primers. (E) A proposed model for Ventx1.1 or Xcad2-mediated control of Ventx1.1 transcription.
	Fig. 1. Ventx1.1 represses expression of ventral-specific genes. (A) One-cell stage embryos were injected in the animal pole region with Flag-Ventx1.1 mRNA (500 pg), and animal caps were dissected from injected or uninjected embryos at stage 8 and cultured to stage 11.5 for RT-PCR analysis. EF1α serves as a loading control. WE, stage 11.5 whole embryo. AC, animal cap. −RT, control in the absence of reverse transcriptase. (B) Quantification of expression levels of ventral genes (normalized to EF1α) from three independent experiments for (A). Error bars indicates standard error (SE).
	Fig. 2. Identification of a Ventx1.1-responsive element (VRE) in its own promoter region. (A) Diagram of Ventx1.1 promoter deletion constructs. The length that each promoter fragment extends upstream of the major Ventx1.1 transcription initiation site is indicated at left. Each promoter fragment was fused to luciferase (LUC) reporter gene. (B–E) Embryos were injected at one-cell stage with wild-type or putative VRE-mutated Ventx1.1 promoter deletion constructs alone or with Flag-Ventx1.1 mRNA (500 pg) as indicated, cultured to stage 10.5 (B) or 11.5 (B–E), and harvested for luciferase reporter assays. All relative promoter activity data are shown as mean ± standard error (SE). (F) ChIP-PCR analysis showing the occupancy by Ventx1.1 of its own promoter region. Ventx1.2, a negative control for PCR, was amplified using its coding region-specific primers. IgG, a negative control IgG.
	Fig. 3. Xcad2 up-regulates Ventx1.1 transcription by occupying VRE1. (A) Animal caps from embryos injected or not with Flag-Ventx1.1 (500 pg) and/or Myc-Xcad2 (1 ng) mRNA as indicated were subjected to RT-PCR analysis at stage 11.5. (B–E) One-cell stage embryos were injected with indicated combinations of −951-bp reporter, −951-bp-mVRE1 reporter, Flag-Ventx1.1 (500 pg), and Myc-Xcad2 (1 ng) and cultured to stage 11 for ChIP-PCR (C, D) or stage 11.5 for luciferase reporter assay (B, E). (D) Quantification of relative intensities of bands in lanes 2, 5, and 6 shown in (C).
	Fig. 4. Ventx1.1 and Xcad2 co-occupy Ventx1.1 promoter region in competition with each other. (A–D) Embryos were injected with a combination of Flag-Ventx1.1 (0.5 ng) and increasing doses of Myc-Xcad2 (0.5, 1 ng) or Myc-Xcad2 (1 ng) and increasing doses of Flag-Ventx1.1 (0.25, 0.5 ng) as indicated and cultured to stage 11.5 for ChIP-PCR analysis. (B, D) represent quantification of relative intensities of bands in lanes 1, 2, and 3 in (A, C), respectively. (−), no injection of Myc-Xcad2 or Flag-Ventx1.1. Ventx2.1, a negative PCR control, was amplified with its coding region-specific primers. (E) A proposed model for Ventx1.1 or Xcad2-mediated control of Ventx1.1 transcription.

References [+] :

Ault, The homeobox gene PV.1 mediates specification of the prospective neural ectoderm in Xenopus embryos. 1997, Pubmed, Xenbase