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. Author manuscript; available in PMC: 2023 Aug 4.
Published in final edited form as: Dev Growth Differ. 2022 Aug 4;64(6):297–305. doi: 10.1111/dgd.12799

Foxh1 engages in chromatin regulation revealed by protein interactome analyses

Jeff Jiajing Zhou 1, Paula Duyen Pham 1, Han Han 1, Wenqi Wang 1, Ken WY Cho 1,2,3,*
PMCID: PMC9474667  NIHMSID: NIHMS1824494  PMID: 35848281

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.

Keywords: Xenopus, pioneer transcription factors, HDAC, PRC2, epigenetics, proteomics

Introduction

Early embryos undergo drastic genetic reprogramming accompanied by epigenetic remodeling to robustly establish various embryonic cell fates. Extensive studies have identified many essential genes that confer either positive or negative regulation on developmental programs (ENCODE Project Consortium 2012& 2020). In Xenopus, Foxh1, a maternal forkhead-box transcription factor (TF), is a critical cofactor in forming a nodal-induced Smad-Foxh1 complex that directs mesodermal and endodermal cell lineages (Zhou et al., 1998; Hill, 2001; Whitman, 2001; Howell et al., 2002; Kofron et al., 2004; Chiu et al., 2014). Foxh1 binds to embryonic genome as early as the 32~64 cell stage, which is significantly earlier than the first major wave of zygotic genome activation (ZGA) (Charney et al., 2017). Time-course ChIP-seq analyses revealed that Foxh1 persistently binds to many putative mesendodermal cis-regulatory modules, which subsequently gain well-characterized active histone modifications such as H3K4me1 and H3K27ac during ZGA and onward. These observations raise the question of whether Foxh1 directly recruits epigenetic modifiers like histone-modifying enzymes during ZGA.

In this study, we characterize FOXH1-associated protein complexes in mouse embryonic stem cells (mESCs) through a proteomic approach. Our data suggest that Foxh1-interacting proteins can regulate gene expression by altering chromatin architectures. Among them, the protein-protein interactions between Foxh1-PRC2 core subunits and Foxh1-Hdac1 appear to be notable. We highlight that Foxh1 and Hdac1 co-repress a small subset of mesendodermal genes in the ectoderm during Xenopus germ layer specification, suggesting the role of Foxh1 in safeguarding misactivation of mesendodermal genes in the ectoderm.

Materials and Methods

DNA plasmid constructs

Mouse FOXH1 cDNA (F: ATGGCCTCGGGCTGGGACCT, R: TTACATGCTGTACCAGGAAAGGAGCCAGCCT) was cloned into the destination vector containing C-terminal triple (S tag-Flag tag-SBP tag, or SFB) tags (Wang et al., 2014). The resulting mFOXH1-SFB is cloned into pLV-EF1a-IRES-Puro (Addgene, 85132) for lentivirus production. Ezh2 (F: ATGGGCCAGACGGGCAAGAA, R: TCAGGGGATTTCCATTTCTCTCTCAATACC), Eed (F: ATGTCCGAAGCTTCCGGTC, R: TCACCGCAGTCTGTCCCAG), Suz12 (F: ATGGCCCCTCAGAAGCACG, R: TCAGGGCTTCTGCTTTTTGCTGT), Hdac1 (F: ATGGCGCTGAGTCAAGGA, R: TCAGGCAGATTTGGTCTCT) cDNAs were cloned into pCS2+ (modified from Addgene 102860) to generate HA tagged fusion proteins. Foxh1 cDNA (F: ATGAGAGACCCCTCCAGTCTG, R: CTACATTAGACCTTGCCTGCTTGG) was cloned in pCS2+ to generate 3X FLAG tagged fusion protein.

Cell culture and Lentiviral transduction

E14 mESC cells were cultivated in KnockOut D-MEM with FBS, GlutaMAX (Thermo Fisher), MEM non-essential amino acids, penicillin-streptomycin, b-mercaptoethanol, and murine LIF (Millipore Sigma). Lentivirus containing pLV-EF1a-mFOXH1-SFB-IRES-Puro was packaged by psPAX2 (Addgene) and pMD2.G (Addgene), and transduced on mESCs. Puromycin (5ug/ml) was applied consecutively to select target clones. After 14 days, single clones were expanded. HEK293T cells were cultured in DMEM with FBS and penicillin-streptomycin. HEK293T cells were transfected with polyethylenimine (Longo et al., 2013).

Animal Model and Morpholinos

Xenopus tropicalis embryos were fertilized and cultured in 1/9X Marc’s modified Ringers. Embryos were dissected at the late blastula stage (6 hpf) and cultured to the early gastrula (7 hpf) for harvest. Foxh1 morpholino injections were performed at the 1-cell stage. 22.5 ng of foxh1 (foxh1 MO: 5′-TCATCCTGAGGCTCCGCCCTCTCTA-3′; Chiu et al., 2014) or standard control (Genetools) was used. For a rescue experiment, foxh1 MO and in vitro transcribed foxh1 mRNA (30 pg) from pCS2-3X FLAG-foxh1 were co-injected into an embryo.

Immunoprecipitation and co-Immunoprecipitation

For mFOXH1 mass spectrometry, 3 × 107 mFOXH1 mESCs were harvested and followed as described (Wang et al., 2014). The purification eluate was analyzed by Harvard Taplin MS facility for LC-MS. For co-immunoprecipitation, transfected cells were lysed in cell lysis buffer and subjected to IP using anti-FLAG (Millipore-Sigma) or anti-HA (Millipore-Sigma) with Pierce protein A/G magnetic beads (Thermo Scientific). Beads were washed with lysis buffer, followed by the elution using 2X SDS loading buffer. The protein eluate was analyzed using western blotting. For benzonase treatment, 100 units of benzonase (Sigma-Aldrich) were added to each IP sample. The final concentrations of ethidium bromide used are 50, 100 and 400ng/ul for each IP sample.

Western Blotting

Cells were directly harvested in 1X SDS loading buffer, boiled at 95°C, and subjected to western blotting using anti-FLAG, anti-Tubulin (Sigma, T5168), and anti-HA.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with 0.5% Triton X-100 in PBS for 5 min, and then blocked with 0.1% Tween-20 (in TBST) and 2% Goat Serum (Vector Laboratories, NC9270494) for 1h at RT. A dilution of 1:200 anti-FLAG (Millipore-Sigma) antibody was incubated with cells for 1h at RT, and washed three times with TBST. Cells were incubated with AlexaFluor488 (Thermo Fisher) for 1h, washed three times in TBST, and stained with Hoechst (Sigma). Confocal images were acquired using the Nikon A1R point scanning confocal microscope.

Gene Expression Analysis and RNA-seq

For RT-qPCR, reverse-transcription assays were performed using Maxima reverse transcriptase (Thermo Fisher). RT-qPCR was performed using LightCycler 480 SYBR Green I master mix (Roche). RT-qPCR primer sequences are as follow. Eomes F: ACCGGCACCAAACTGAGA, R: AAGCTCAAGAAAGGAAACATGC; Foxa2 F: CCATCAGCCCCACAAAATG, R: CCAAGCTGCCTGGCATG; Foxh1 F: ATTATCCGTCAGGTCCAGGC, R: TAGAGGAAAGGTTGTGGCGG; Nanog F: CCAACCCAACTTGGAACAAC, R: TATGGAGCGGAGCAGCAT; Oct4 F: TTGCAGCTCAGCCTTAAGAAC, R: TCATTGTTGTCGGCTTCCCT; Pax6 F: CACCAGACTCACCTGACACC, R: ACCGCCCTTGGTTAAAGTC; Tbxt F: CAGCCCACCTACTGGCTCTA, R: GAGCCTGGGGTGATGGTA. For quantification of gene expression, the 2^ −ΔΔCt method was used. gapdh was used as a control gene for normalization. For RNA-seq, total RNA from dissected animal cap explants was extracted using Trizol (Amin et al., 2014). mRNA was isolated using NEBNext PolyA mRNA Magnetic Isolation Module. Sequencing libraries were prepared using NEBNext Ultra II RNA library prep kit and sequenced by the Illumina NovaSeq 6000. All experiments were done in 2 biological replicates. Sequencing samples were aligned using STAR v2.7.3a (Dobin et al., 2013) to Xenopus tropicalis genome v10.0 (http://www.xenbase.org/, RRID:SCR_003280). Differentially expressed genes were identified using edgeR v3.36.0 (Robinson et al., 2010) with parameters fold change >2 and false discovery rate (FDR) < 0.05 in R v4.1.2 (R Core Team, 2022). Gene ontology analysis was performed using Metascape (Zhou et al., 2019) with default parameters (min overlap= 3, p-value cutoff= 0.01, and min enrichment= 1.5) and visualized by Cytoscape (Shannon et al., 2003).

Additional Analysis

Mass spectrometry: List of proteins detected in three independent spectrometry experiments (using heterogeneous pooled, monoclonal 1, and monoclonal 2 cells) were compared, and overlapping proteins were identified after removing identified proteins from the negative control (wildtype E14 mESCs). Spatial expression analysis: Sequencing data from dissected Xenopus gastrula embryonic tissues (Blitz et al., 2017) were aligned using STAR v2.7.3a (Dobin et al., 2013) and quantified by RSEM v1.3.3 (Li and Dewey, 2011) to obtain TPM values. TPMs for marginal zones were calculated as the mean TPMs of dorsal, lateral and ventral marginal zones. ChIP-seq: Sequencing data were aligned to Xenopus tropicalis v10.0 genome (http://www.xenbase.org/, RRID:SCR_003280) using Bowtie2 v2.4.4 (Langmead and Salzberg, 2012). PCR duplicates were removed using Samtools v1.10 (Li et al., 2009). Irreproducibility discovery rate (IDR) analysis (Li et al., 2011) was used to identify high-confidence peaks called by Macs2 v2.7.1 (Zhang et al., 2008) between two biological replicates following ENCODE3 ChIP-seq pipelines (IDR threshold of 0.05). Clusters are made by Bedtools v2.29.2 (Quinlan and Hall, 2010). Clustered heatmaps were generated using DeepTools v3.5.0 (Ramírez et al., 2014). Genes suppressed by Hdac1: The gene list is obtained by overlapping an up-regulated gene list (855 genes) after HDAC inhibition in the ectoderm and a gene list (10,683 genes) bound by Hdac1 at early gastrula stage (Zhou et al., BioRxiv 2022).

Data Accessibility

RNA-seq datasets generated from this study are available at NCBI Gene Expression Omnibus using GSE204990. Publicly available datasets used are available at NCBI GEO: GSE53654 (Foxh1 ChIP-seq), GSE85273 (Foxh1 ChIP-seq), GSE81458 (RNA-seq of regionally isolated gastrula tissues), GSE198378 (Hdac1 ChIP-seq, HDAC inhibition RNA-seq).

Results and Discussion

Generation of E14 cells stably expressing protein mFOXH1-SFB

To minimize interference from large amounts of yolk granules present in Xenopus embryos, we generated E14 mESC lines stably expressing mFOXH1 with epitope tags encoding S-protein peptide, two FLAG peptides, and a streptavidin-binding peptide (SBP) (Wang et al., 2014) (Figure 1A). The S-protein peptide and SBP tags enable a sequential pulldown, while the FLAG tag allows for the detection of bait protein. A population of E14 cells transduced with lentivirus harboring mFOXH1-SFB were expanded, and shown to express mFOXH1-SFB (Figure 1B, 1C). This population of mFOXH1-SFB E14 mESCs consists of a heterogeneous pool of cells with different integration sites and copies of mFOXH1-SFB. Immunofluorescence staining of mFOXH1-SFB showed localized expression in the nucleus (Figure 1D). Clonally expanded mFOXH1-SFB E14 mESCs (monoclonal 1 and 2) exhibited more uniform expression of mFOXH1-SFB protein. Variable protein levels of mFOXH1-SFB were expressed in different clones of mFOXH1-SFB E14 mESCs (Figure 1E). Gene expression analyses showed high levels of mFoxh1 and pluripotency gene transcripts (e.g., Nanog), but not early germ layer marker gene transcripts (e.g., Tbxt, Eomes and Foxa2), suggesting that mFoxh1 overexpression does not affect the cellular pluripotency of mESCs (Figure 1F).

Figure 1: E14 mESCs stably expressing recombinant mFOXH1-SFB.

Figure 1:

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(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 microns. (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 microns. (E) Western blot showing the protein levels of mFOXH1-SFB in control E14 (UT), heterogeneous cell pool (HP), and monoclonal line 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.

FOXH1 participates in diverse chromatin regulation

Liquid chromatography-mass spectrometry (LC-MS) was performed on mFOXH1-SFB purification eluate. A total of 342 proteins consistently recovered from three independent samples (heterogeneous pool, monoclonal 1, and monoclonal 2 cells) are considered as putative mFOXH1 interactants (Table 1). Gene ontology enrichment analysis showed that mFOXH1 participates in diverse cellular processes (Figure 2A). We highlight that mFOXH1 interacts with proteins functioning in various aspects of chromatin regulation (Figure 2B), consistent with the notion that some critical TFs often recruit coregulators to influence the local chromatin states (Spitz and Furlong, 2012; Chen and Dent, 2013; Zaret, 2020). SMAD2 and SMAD3, known FOXH1 interactants through Activin/Nodal signaling (Massagué, 2012), are captured. Proteins positively or negatively regulating chromatin are identified as potential mFOXH1 interacting proteins (Figure 2C). For example, SMARCA5 and HELLS are known regulators related to SWI-SNF chromatin remodeling complexes (Oppikofer et al., 2017; Dennis et al., 2001). NSD1 is a histone methyltransferase depositing H3K36 methylation during transcription elongation (Lucio-Eterovic et al., 2010). TET1 and OGT are assembled into DNA methyltransferase complexes (Hrit et al., 2018). SUZ12, JARID2, and EZH2 are subunits of Polycomb repressive complex 2 (PRC2) that deposits H3K27me3 modification on inactive genes (Chammas et al., 2019). HDAC1 is a histone deacetylase removing active histone acetylation modifications (Seto and Yoshida, 2014). These results suggest that mFOXH1 participates in diverse epigenetic processes which can either positively or negatively regulate transcription.

Table 1:

342 high confidence interacting proteins identified from mass spectrometry

MYBBP1A MTDH RPS15A HNRNPA3 SSB SRSF6 GNAI2 HIST1H1E
TRIM28 ACTB KPNA2 EBNA1BP2 MCM5 RBM28 JARID2 ABCF2
NCL PABPC1 PNPLA6 RPS19BP1 ZSCAN4C DHX37 IMP3 DNAJC21
HNRNPU RPL8 ATP5C1 HNRNPC EXOSCIO IARS DNMT3L POLR2A
HSPA8 SYNCRIP EPRS RFC4 ATP1A1 TCP1 POLR2E RPF2
HSP90AB1 HNRNPH1 RPS24 ATP2A2 FRG1 RTCB SPTLC1 MRPS30
HNRNPM PTBP1 MDN1 NOL6 SKP1 URB2 DUS3L HDAC1
RPL7 TDH RPL21 DDX28 RBMXL2 ALYREF RPLP1 AP2A2
RPS4X CKAP4 GLYR1 SRSF3 C1QBP TUBB4B PISD LARP7
ATAD3 DDX1 ABCE1 MYEF2 DDX47 RPL34 PPAN AP2M1
DDX21 UTP20 RUVBL1 PRDX1 ESC02 NARS TMEM214 SPTLC2
RPL4 NAT 10 RPS7 TARDBP HSPD1 RPL35 RRP15 CDIPT
TRIM71 RCC2 CAD POLR1B FAM98B VCP DNAJB6 CCT3
PDCD11 RRP12 RPL26 TRIM25 SLC25A1 SSR1 HSP90B1 TRIP12
FOXH1 HSPA2 RPS26 PES1 RPS27 NSUN2 CANX FAU
RPS9 RPL23A RPL14 SGPL1 L1TD1 SMAD2 NOL9 DDX49
LBR RPL18 RPL18A VDAC2 NOP53 RPN2 YBX3 MRPL39
GNL3 NOP2 YBX1 RPL22 EXOSC9 NOP16 MRPL15 GTF3C1
RPS2 RPL30 SERBP1 POLR1E RBM39 TIMM23 CFL1 MRPS26
DDX5 RPL27 RPLP2 AP2B1 DDX10 HNRNPAB RSL24D1 IGF2BP3
RPL6 DRG1 ASPH DNAJA3 RPL36-PS3 PCBP3 DDX27 RBBP7
D1PAS1 DNAJA1 CSDE1 TECR RUVBL2 LYAR TUBA4A ERLIN2
NOP56 MCM3 HK2 ILF2 RRP1B BCAS2 CUL1 PRMT5
RPL7A RARS RPL19 GNL3L RBM19 ARMC10 PTCD3 STAU1
RPL3 RSL1D1 HELLS RPL17 NOP14 WDR46 RPS19 TEX10
RPS6 DDX17 DARS CAPRIN1 RFC2 NIFK FUS EIF2S3Y
RPL10A PRPF19 RACK1 RPL10L DDX56 SRPK1 SMAD3 CEBPZ
RPS11 DHX15 TSR1 PFKP MRPL21 NAP1L1 DDOST PCBP1
RPL13 EIF2S2 RPSA KPNB1 RFC5 AIMP1 GRSF1 NEMF
RPL13A TUFM ABCF1 NXF1 SLC25A3 NISCH G3BP1 POLRMT
IGF2BP1 DDX51 SUZ12 NOP58 RPL27A RFC1 GPX4 RARS2
RPL23 RPL24 POLR1C EXOSC8 RBMX EZH2 XRN2 MSH6
YME1L1 HDLBP FXR1 ANKRD17 WDR18 DDX52 DDX31 OGT
CDC5L POP1 RPS20 HNRNPR PHB HNRNPD RPS28 TRMT1L
NPM1 DHX30 SLC25A5 HNRNPAO GTPBP4 SRPRB SMARCA5 TET1
RPS18 FBL TIMM50 RPS4L CD3EAP RRBP1 RBM14 PDE12
HNRNPF RPL35A HNRNPA1 NVL AIFM1 CCT4 ZFR HIST1H1A
RPS16 RPL31 MTCH2 LAS1L SLC25A13 FKBP8 NSD1 MSH2
RPS13 RPS25 RPL15 RBFOX2 SF3B1 GTPBP1 KARS RIF1
HSD17B12 HSP90AA1 DHX9 MARS FTSJ3 RPL37A PRKCI LENG8
SPATA5 RPL10 RBM34 ILF3 AFG3L1 DNAJA2 SLC25A4 POLR1A
RPLPO RPS8 TUBB4A SF3B3 SENP3 EEF1G SRSF4
TMPO RPS23 RPN1 EIF4A2 CDKAL1 AIMP2 DNAJC9
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Figure 2: Foxh1 interacts with PRC2 subunits and Hdac1.

Figure 2:

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(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.

To confirm that similar interactions identified in mESCs can also occur among Xenopus counterparts, we performed co-immunoprecipitation (co-IP) experiments using respective Xenopus protein counterparts. To distinguish DNA/RNA-dependent and -independent association of these proteins, protein lysates were treated with either ethidium bromide to disrupt the structure of nucleic acids or Benzonase endonuclease to degrade nucleic acids. We showed that Foxh1 physically interacts with three core subunits (Ezh2, Eed, and Suz12) of PRC2 (Figure 2D). The interaction between Foxh1 and Hdac1 was also confirmed (Figure 2E). These validation studies suggest that Xenopus Foxh1 functions as a transcriptional repressor via the recruitment of PRC2 and Hdac1-containing complexes.

The role of Foxh1 in developing embryonic ectoderm

Nodal signaling is only active vegetally, whereas Foxh1 is expressed uniformly in early embryos. We speculate that Foxh1 bears a repressive function in the absence of Nodal signaling within the ectoderm. We tested the model whereby Foxh1 recruits a repressive epigenetic modifier Hdac1 to repress mesendodermal genes in the developing ectoderm.

First, we identified genes regulated by Foxh1 in the ectoderm. Xenopus embryos were injected with control or Foxh1 morpholino (MO) animally, and developed until the late blastula stage. Animal cap (prospective ectoderm) tissues were dissected and incubated to the early gastrula equivalent stage for RNA-seq (Figure 3A). Foxh1 morphants exhibited incomplete blastopore closure and impaired AP axis (80%, n=10 embryos). These abnormalities are partially rescued (70%, n=10) by ectopic expression of MO-resistant foxh1 mRNA in Foxh1 morphants (Figure 3B). Differential gene expression analysis of ectodermal tissues isolated from Foxh1 morphant and control embryos revealed 192 upregulated and 384 downregulated genes (Figure 3C). Spatial gene expression analyses (Blitz et al., 2017) comparing animal (ectoderm), marginal (mesoderm) and vegetal (endoderm) tissue fragments showed that differentially regulated genes by Foxh1 in ectoderm do not exhibit any germ layer enrichment (Figure 3D). Next, we examined the biological function of Foxh1-Hdac1 interaction. Foxh1and Hdac1 share similar binding profiles (Figure 3E), suggesting that Foxh1 and Hdac1 occupy similar genomic regions. A list of 513 Hdac1 suppressed genes were identified through combined analyses on genes induced after HDAC inhibitor (Trichostatin A, TSA) and Hdac1 bound genes (Zhou et al., BioRxiv 2022). Of 192 Foxh1 repressed genes, 12 genes were suppressed by Hdac1 (Figure 3F). Notably, 11 of the 12 Foxh1-Hdac1 co-repressed genes showed enriched expression in either mesoderm or endoderm, suggesting that Foxh1 and Hdac1 co-repress mesendodermal genes in the ectoderm. That a small percentage (6.3%) of genes are suppressed by Foxh1-Hdac1 indicates additional mechanisms (i.e., the involvement of other TFs for Hdac1 recruitment in the absence of Foxh1) playing a role in inhibiting the expression of other mesendodermal genes in the developing ectoderm (Figure 3G).

Figure 3: Mesendodermal gene suppression in the ectoderm by Foxh1-Hdac1.

Figure 3:

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(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, and 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.

In sum, our FOXH1 proteomic study identified epigenetic modifiers involved in different aspects of chromatin regulations (Figure 2B, 2C). The capture of SMARCA5 and HELLS, regulators related to SWI/SNF complex, provides a mechanism where Foxh1 may exploit the activity of SWI/SNF complex to alter nucleosome positions along the early embryonic genome. Foxh1 physically interacts with both PRC2 (Figure 2D) and Hdac1 (Figure 2E), presumably conferring repressive chromatin states through H3K27me3 deposition and histone hypoacetylation. We propose that the interaction between Foxh1 and Hdac1 occurs in the physiological context, causing transcriptional repression in the ectoderm of developing embryos (Figure 3G) based on the following evidence. Nodal signaling is absent in the ectoderm (Hill et al., 2001) while Foxh1 is ubiquitously expressed (Chiu et al., 2014; Charney et al., 2017) in early Xenopus embryos. Foxh1 physically interacts with PRC2 subunits and Hdac1 (Figure 2D, 2E). The genomic binding profile of Hdac1 highly overlaps with that of Foxh1 (Figure 3E). Importantly, 11 out of 12 Foxh1-Hdac1 co-repressed genes in the ectoderm (Figure 3F) are expressed in the mesoderm and endoderm. This supports the model that Foxh1 functions as a transcriptional repressor for mesendodermal genes in the ectoderm via the histone deacetylase activity of Hdac1. Since only a subset of Foxh1 target genes are affected by HDAC inhibition, it is tempting to speculate that other TFs function in a combinatory fashion (Ravasi et al., 2010). In light of the model, our previous studies in Xenopus showed that Foxh1 binds to genomic regions overlapped with multiple TFs such as Otx1, Vegt, Sox3, and Sox7 (Paraiso et al., 2019; Jansen et al., 2022; Zhou et al., BioRxiv 2022). Hence, a loss or reduction of Foxh1 alone may be insufficient to fully relieve this suppression. It will be useful to determine whether these maternal TFs function cooperatively with Foxh1 to suppress mesendodermal genes in the ectoderm.

Acknowledgments

We thank current Cho lab members for critical comments, the Genomic High Throughput Facility at University of California, Irvine for sequencing services, Xenbase (RRID:SCR_003280) for genomic and community resources, the National Xenopus Resource (RRID:SCR_013713) for Xenopus tropicalis, and Taplin Mass Spectrometry Facility at Harvard Medical School for LC-MS services. This work is supported by NIH R01GM126395, R35GM139617 and NSF 1755214 to K.W.Y.C., and NIH R01GM126048 and ACS RSG-18-009-01-CCG to W.W..

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

RNA-seq datasets generated from this study are available at NCBI Gene Expression Omnibus using GSE204990. Publicly available datasets used are available at NCBI GEO: GSE53654 (Foxh1 ChIP-seq), GSE85273 (Foxh1 ChIP-seq), GSE81458 (RNA-seq of regionally isolated gastrula tissues), GSE198378 (Hdac1 ChIP-seq, HDAC inhibition RNA-seq).

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