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Abstract
Early embryonic cell fates are specified through coordinated integration of transcription factor activities and epigenetic states of the genome. Foxh1 is a key maternal transcription factor controlling the mesendodermal gene regulatory program. Proteomic interactome analyses using FOXH1 as a bait in mouse embryonic stem cells revealed that FOXH1 interacts with PRC2 subunits and HDAC1. Foxh1 physically interacts with Hdac1, and confers transcriptional repression of mesendodermal genes in Xenopus ectoderm. Our findings reveal a central role of Foxh1 in coordinating the chromatin states of the Xenopus embryonic genome.
Figure 1. E14 mESCs stably expressing recombinant mFOXH1-SFB. (a) A diagram of the recombinant protein with SFB triple tags, S-protein, FLAG, and streptavidin-binding peptide (SBP) at C-terminus. (b) E14 cells transduced by lentivirus after puromycin selection. Black scale bar, 10 μm. (c) Western blot of E14 cells expressing mFOXH1–SFB recombinant proteins using anti-FLAG antibody. β-Tubulin: loading control. (d) Immunofluorescence images of mFOXH1-SFB localized to the nucleus of E14 cells. The cytosolic signals of FLAG represent background. White scale bar, 10 μm. (e) Western blot showing the protein levels of mFOXH1–SFB in control E14 (UT), heterogeneous cell pool (HP), and monoclonal lines MC1, MC2, MC3, and MC4. mFOXH1: anti-FLAG antibody; β-Tubulin: loading control. (f) RT-qPCR analyses of pluripotent and early germ layer markers in E14 mFOXH1–SFB cells. These gene expression levels were normalized to gapdh. Error bars: standard deviation values between two technical replicates
Figure 2.Foxh1 interacts with PRC2 subunits and Hdac1. (a) Gene Ontology enrichment analysis of 342 potential putative Foxh1 interacting proteins. (b) Interactome diagram illustrating Foxh1 interacting proteins in chromatin regulation in Cytoscape (Shannon et al., 2003). The sizes of circles reflect p-values. (c) The list of FOXH1 interacting proteins belonging to chromatin organization (GO:0006325), SMAD2, and SMAD3. The numbers indicate the mean of total peptides detected among three biological replicates. (d) Western blot showing the interaction between Foxh1 and PRC2 core subunits Ezh2, Eed, and Suz12 in HEK293T cells. EtBr, ethidium bromide. (e) Interaction between Foxh1 and Hdac1 in HEK293T cells
Figure 3. Mesendodermal gene suppression in the ectoderm by Foxh1–Hdac1. (a) Dissection of animal cap for RNA-seq. (b) Foxh1 morphants and rescued embryos. Numbers of embryos with the representing phenotype are listed. (c) The heatmap representing differentially expressed genes in Foxh1 morphant versus control ectodermal tissues. (d) Violin plot showing the expression levels of Foxh1 regulated genes in different germ layers. AC, animal cap; MZ, marginal zone; VG, vegetal mass. (e) Clustered heatmaps depicting the genomic binding signals of Foxh1 in st8 (mid blastula), st9 (late blastula), and st10.5 (early gastrula) embryos with a 5 kb window. Signals are centered on Foxh1 peak summits. (f) Venn diagram showing genes co-repressed by Foxh1 and Hdac1 in the ectoderm. The expression levels of 12 repressed genes in each germ layers are represented in the heatmap. (g) Model of Foxh1 and other unidentified TFs recruiting Hdac1 to suppress the mesendodermal gene regulatory program in the ectoderm
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