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Kumar V, Goutam RS, Umair Z, Park S, Lee U, Kim J.
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Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.
Figure 1. Ectopic expression of Foxd4l1.1reduced dorsal mesoderm (organizer) and ectodermal gene expression during gastrula. Xenopus embryos were injected with Foxd4l1.1 and Smad2 (1 ng/embryo) at the one-cell stage. RT expression was analyzed at stage 11 in whole embryos (WE): (A) RT expression of different germ-layer-specific markers was analyzed; (B and C) chrd(−2250)eGFP was injected alone or co-injected with Foxd4l1.1, Smad2, or both at the one-cell stage; eGFP fluorescent analysis was performed at stage 11: (B) quantification of chrd(−2250)eGFP fluorescent intensity (bar 1) co-injected with Foxd4l1.1 (bar 2), co-injected with Smad2 (bar 3), and Foxd4l1.1 plus Smad2 (bar 4); (C) chrd(−2250)eGFP fluorescence along with co-injection with Foxd4l1.1 or Smad2 or both, separately (at stage 10/10.5) as indicated. In the lower panel, inverted color images generate the pink color for eGFP fluorescent positive areas and reduce the background seen. -RT (No RT) served as a negative control, and uninjected whole embryo (WE) served as a positive control. *** p ≤ 0.001.
Figure 2. Ectopic presence of Foxd4l1.1 abolished chrd(−2250), ARE, and BRE mediated expression. Xenopus embryos were injected with chrd(−2250), ARE, and BRE (40 pg/embryo) with or without Foxd4l1.1, Smad2/3, or BMP4 mRNA (1 ng/embryo) at the one-cell stage and harvested at stage 11: (A) chrd(−2250) reporter activities with or without Foxd4l1.1 or Smad2/3; (B) ARE reporter activities with or without Foxd4l1.1 or Smad2/3; (C) BRE reporter activities with or without BMP4, Foxd4l1.1, or Smad2/3. The y-axis represents relative light units (RLUs). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001, n.s. denotes non-significant values.
Figure 3. Chrd promoter contains FRE1: (A) ChIP-sequencing coverage plot of Foxd4l1.1 within the chrd promoter region; (B) schematic representation of the serially deleted promoter constructs of chrd promoter. The numbering of promoter constructs (or cis-acting response elements) is given from the translational start site (TLS); (C) relative promoter activities of serially deleted promoter constructs of chrd(-2250) with or without Foxd4l1.1; (D) systemic representation of mutated FRE1, FRE2, and ARE2 (targeted sequences; underlined, italic, red color) within the region of −2250 bps chrd promoter construct; (E) relative luciferase activity for chrd(-2250), chrd(-2250)mFRE1, chrd(-2250)mFRE2, and chrd(-2250)mFRE3 with or without Foxd4l1.1. ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001, n.s. denotes non-significant values.
Figure 4. Foxd4l1.1 physically interacts with C-terminal domain of Smad2 and Smad3 proteins: (A,B) co-immunoprecipitation (Co-IP) assay was performed to examine the interaction of Flag-Foxd4l1.1 and HA-Smad2/3 recombinant proteins: (A) Co-IP was performed with an anti-Flag antibody, followed by Western blotting (WB) with anti-HA antibody; (B) Co-IP was performed with anti-HA antibody, followed by WB with anti-Flag antibody; (C) schematic representation of truncated Smad2 and Smad3 constructs; these include Smad2/3-ΔC, having the C-terminal domain deleted, and Smad2/3-C-ter, containing only the C-terminal domain of the original parental protein; (D) co-immunoprecipitation (Co-IP) assay was performed to examine the interaction between Flag-Foxd4l1.1 and HA-Smad2-ΔC, HA-Smad3-ΔC, HA-Smad2-C-ter, and HA-Smad3-C-ter. Co-IP was performed with anti-Flag antibody, followed by WB with anti-HA antibody.
Figure 5. Foxd4l1.1 is bound to FRE1 and blocks Smad2/3 binding to AREs (ARE1 and ARE2). For ChIP-PCR, Flag-Foxd4l1.1 with or without HA-Smad2 and HA-Smad3 mRNA were injected at the one-cell stage, then harvested at stage 11: (A) ChIP-PCR primer design and C 1, ARE1, ARE2, FRE1, and C 2 locations within the chrd promoter are shown with F as location of the forward primer and R as location of the reverse primer; (B) chromatin immunoprecipitation assay was performed to examine the interaction of Flag-Foxd4l1.1 with FRE1, the two internal negative controls (C 1 and C 2), and one external negative control (Ventx2.1); (C) chromatin immunoprecipitation assay was performed to examine the occupancy of either Smad2 or Smad3 with or without Foxd4l1.1. Ventx2.1 served as external negative control; (D), ChIP-qPCR was performed to examine the occupancy of HA-Smad2 and HA-Smad3 to AREs with or without Flag-Foxd4l1.1 (fold enrichment method used to normalize the ChIP-qPCR reads). **** p ≤ 0.0001.
Figure 6. Proposed model of Foxd4l1.1 mediated inhibition of chrd transcription. In dorsal mesoderm for the organizer, activin signaling activates chrd transcription mediated by Smad2 and Smad3. Each Smad interacts with ARE1 and ARE2 within the chrd promoter (−2130 to −2113 bps). The neuroectoderm-specific transcription factor Foxd4l1.1 is directly bound to the FRE1 region of the chrd promoter to inhibit chrd transcription (−2027 to −2021 bps). Foxd4l1.1 physically interacts with Smad2 and Smad3 and blocks binding to the AREs.
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