Goutam RS et al. (2024), Cdx1 and Gsc distinctly regulate the transcript...

XB-ART-60641

Mol Cells 2024 Mar 23;474:100058. doi: 10.1016/j.mocell.2024.100058.

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Cdx1 and Gsc distinctly regulate the transcription of BMP4 target gene ventx3.2 by directly binding to the proximal promoter region in Xenopus gastrulae.

Goutam RS , Kumar V , Lee U , Kim J .

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A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide ChIP-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral (VMZ) and dorsal (DMZ) marginal zones, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the VMZ and DMZ by directly binding to the promoter region during Xenopus gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 cdx1 gsc myc smad1 tbxt ventx1 ventx1.2 ventx2 ventx2.2 ventx3 ventx3.2
GO keywords: gastrulation [+]
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Phenotypes: Xbo Wt + gsc MO + cdx1 MO (VMZ) (Supp. Fig 4 E) [+]

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	Graphical Abstract
	Fig. 1. Zygotic transcription and protein identity comparison of ventx family members.(A) Temporal expression patterns of ventx genes in whole embryos, as determined using RT-qPCR. (B) Protein sequences downloaded from “Xenbase”, compared for identity using Clustal omega, and schematically drawn. (C) Amino acid sequences of conserved homeodomains (HD) selected and compared for identity using the “Clustal omega Pairwise alignment tool” and schematically drawn. Solid color lines depict identity within the same class, whereas dotted lines indicate identity within different classes of the ventx family.
	Fig. 2. Expression of Cdx1 and Gsc ectopically modulates ventx3.2 transcription differentially. (A) A schematic of the methodology for the ventral gene expression assay. (B) RT-qPCR results for ventral-specific ventx1.1, ventx2.1, and ventx3.2 in samples injected with Cdx1. (C) RT-qPCR results for the above-mentioned genes in samples injected with Gsc. Fold enrichment was utilized for RT-qPCR analysis, and experiments were performed thrice. p 0.001 and p 0.0001 are the significant values assigned in (B and C). Abbreviations: Gsc, goosecoid; Cdx1, caudal-type homeobox 1.
	Fig. 3. Chromatin immunoprecipitation-sequencing and reporter gene assay mapping of the response elements of Gsc and Cdx1 within the ventx3.2 promoter region. (A) A plot of 3Flag-Gsc coverage within the ventx3.2 promoter region. (B) A map of serially deleted ventx3.2 promoter constructs. (C–D) Relative promoter activities of serially deleted constructs of ventx3.2 promoters injected with or without Gsc (C) and Cdx1 (D) mRNA at the one-cell stage. (C, D) ⁎⁎⁎⁎p ≤ 0.00001 indicates statistical significance; ns indicates non-significant values. Abbreviations: Gsc, goosecoid; Cdx1, caudal-type homeobox 1; RLU, relative luciferase unit.
	Fig. 4. Site-specific mutations of Gsc response element (GRE) and Cdx1 response element (CRE) within the ventx3.2 promoter eliminates Gsc and Cdx1 activities. Systemic representation of mutated GREs (A) and CREs (C) within the ventx3.2 promoter (targeted nucleotides are shown in italics and underlined). (B) Relative luciferase activity at embryonic Stage 11 for ventx3.2(-642), ventx3.2(-642)mGRE2, ventx3.2(-197), and ventx3.2(-197)mGRE1 (40 pg/embryo) without or with Gsc mRNA (500 pg/embryo) injected at the one-cell stage. (D) Relative luciferase activity for ventx3.2(-197)mCRE and ventx3.2(-197)mCRE injected at 40 pg/embryo with or without Cdx1 mRNA (1 ng/embryo) and harvested at Stage 11. (B,D) ⁎⁎⁎⁎p ≤ 0.00001 indicates statistical significance; ns indicates non-significant values. Abbreviations: Gsc, goosecoid; Cdx1, caudal-type homeobox 1; RLU, relative luciferase unit.
	ig. 5. Gsc and Cdx1 bind to the ventx3.2 proximal promoter region. (A) Schematic representation of common ChIP- PCR primers F (forward) and R (reverse) as well as the location of GRE and CRE in the ventx3.2 promoter. (B, C) ChIP-PCR results showing the interaction between Flag-Gsc and GRE and between Myc-Cdx1 and CRE. Common ChIP primers (containing both GRE and CRE) were used for amplification, while ventx3.2 CDS (exon 3) primers served as the negative control for both. (D) ChIP-qPCR results showing the occupancy of Myc-Cdx1 on CRE and Flag-Gsc on GRE. Fold enrichment was utilized to normalize ChIP-qPCR readings. ⁎⁎⁎⁎ p ≤ 0.0001. Abbreviations: Gsc, goosecoid; Cdx1, caudal-type homeobox 1; CRE, Cdx1 response element; GRE, Gsc response element; ChIP-PCR, chromatin immunoprecipitation-polymerase chain reaction.
	Fig. 6. Gsc knockdown induces ventx3.2 transcription, whereas Cdx1 knockdown reduces it in the DMZ and VMZ, respectively. (A) Endogenous ventx3.2 expression in the VMZ in Xenopus embryos, with xBra as the pan-mesodermal marker. (B) Schematic representation of the experimental design for VMZ and DMZ analyses. (C) Gsc knockdown at dorsal blastomeres at the four-cell stage induces ventx3.2 transcription in the DMZ during gastrulation. (D) Cdx1 knockdown in ventral blastomeres at the four-cell stage reduces ventx3.2 transcription in the VMZ. p 0.1, p 0.01, p 0.001, and p 0.0001 are considered statistically significant. Gsc, goosecoid; VMZ, ventral marginal zone; DMZ, dorsal marginal zone; Cdx1, caudal-type homeobox 1; Xbra, Xenopus brachyury.
	Fig. 7. Model proposed for the regulation of ventx3.2 transcription by Gsc and Cdx1. Schematic representation of the suggested molecular mechanism underlying ventx3.2 transcriptional regulation in Xenopus gastrulae. Abbreviations: Gsc, goosecoid; Cdx1, caudal-type homeobox 1.
	Supplementary Fig. 1. Stagewise expression patterns of Cdx1 and Gsc in Xenopus laevis. Whole embryos at different stages were harvested for expressional analysis. RT-qPCR was performed to analyze the stage-dependent expression of (A) Cdx1 and (B) Gsc.
	Supplementary Fig. 2. Ectopic expression of Cdx1 and Gsc in cap explants. (A) RT-PCR showings ventral and dorsal gene expression in the Cdx1-injected animal cap explants. (B) RT-PCR showings ventral and dorsal gene expression in Gsc -injected animal cap explants. Ornithrine de-carboxylase (Odc) was used as house-keeping gene.
	Supplementary Fig. 3. Morpholino information and phenotypic effects on Xenopus. (A) MO sequences and their target sites in the Gsc and Cdx1 Coding sequence regions.. (B) Gsc knockdown phenotype. Gsc MO (70 ng) was injected into each dorsal- blastomere at the four-cell stage, and its phenotypic effect was observed at NF stage 30. (C) Cdx1 knockdown phenotype. Cdx1 MO (38 ng) was injected into each ventral- blastomere at the 4-cell stage and the phenotype was observed at NF stage 37.
	Supplementary Fig. 4. Double knockdown of Cdx1 and Gsc in Xenopus laevis. (A) Embryos were injected dorsally (D) with 70ng Gsc MO and 20ng Cdx1 MO and ventrally (V) with 38ng Cdx1 MO and 40ng Gsc at 4-cell stage. DMZ and VMZ explants were dissected at stage 10 and were culture till stage 11-11.5. (B) qRT-PCR analysis of Cdx1/Gsc depleted DMZ. blastomere at the four-cell (C) qRT-PCR analysis of Cdx1/Gsc depleted VMZ (D) Phenotype of Cdx1/Gsc-MO dorsally injected. (E) Phenotype of Cdx1/Gsc-MO ventrally injected.