Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming.
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Genomic data have been deposited in the Gene Expression Omnibus database under accession number GSE134465.
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We thank R. Dunbrack (Fox Chase Cancer Center) for the secondary structure analysis, C. Ducker for the crosslinking studies, S. Hipkens for the mouse embryonic stem cell culture and gene targeting in the Transgenic Mouse/Embryonic Stem Cell Shared Resource, R. McCarthy for data review and the University of Pennsylvania Flow Cytometry and Cell Sorting Facility. M.I. was supported by postdoctoral fellowships from Japan Society for the Promotion of Science Foundation (H26-683), Naito Foundation (RYU10000032), Astellas Foundation for Research on Metabolic Disorders (K0076) and Uehara Memorial Foundation (H24-20124). P.S. was supported by grant nos. SAF2016-75531-R (MICINN/FEDER, UE) and B2017/BMD-3724 (Comunidad de Madrid). The research was supported by NIH grant no. GM36477 to K.S.Z.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Schematic of crosslinking of histone octamers used as input and FOXA1. SDS-PAGE analysis of FOXA1 or FOXA1 crosslinked to core histones, stained with Coomassie blue. Crosslinked products of a mobility expected for FOXA1 and core histone together are noted as band A and band B. The full blot gel is presented in the Source Data files. b, Underlined sequences are identified by peptide mass matching while the subset highlighted by green are used for relative peptide quantification in Extended Data Fig. 2. c, Strategy to map candidate interaction sites, explaining how crosslinked peptides gain a much greater mass and become depleted from the m/z spectrum.
Extended Data Fig. 2 Mass spectrometry identification of FOXA1 peptides depleted by crosslinking to core histones.
Relative quantification of FOXA1 peptides in crosslinked bands A and B shown in Extended Data Fig. 1a. The integrated intensities of ions corresponding to peptides YPHAKPPYSYISLITMAIQQAPSK and ASQLEGAPAPGPAASPQTLDHSGATATGGASELK were used for normalizing tabulated intensities of other FOXA1 peptide ions. Those peptide intensities found unaltered in bands A and B (Extended Data Fig. 1a) are shown in the blue panel while those whose intensities changed in the FOXA1:Histone x-linked bands are shown in the red panel, and noted by red arrows, as discussed in the main text. Lower right, quantitation of peptide signals of aa415–443 over control peptide signals of aa313–246 within the same respective spectrum, demonstrating a diminution of aa415–442 in band A due to blockage at K414.
a, A putative α-helical region is conserved in FOXA1 and FOXA2 homologs. Conserved regions II and III are highlighted in teal and the α-helical region around K414 (orange) is highlighted in green. b, FOXA2 wild-type (WT) protein and delta-helix (ΔHx) mutant bound to Sepharose beads in a pulldown assay to assess binding to histone octamers. The bar graph represents mean ± s.e.m. of four replicates. P values are from a one-sided paired t-test, comparing the ratio of the core histones H3/H2A/H2B as a group and H4 to the recovered amount of full length FOXA2 in the same lane (n = 4 experiments; *, P < 0.05 different from WT). Dashed lines indicate partial FOXA2 degradation products from the C-terminus of the 6×-his tagged FOXA2 proteins, which were tagged on the N-terminus. The full gel is presented in the Source Data files.
Extended Data Fig. 4 Deficiency in activation of endogenous FOXA1 liver target genes by FOXA- ΔHx and FOXA1-PP mutant proteins.
a, Schematic of functional assay for FOXA1 wild-type and mutants in H2.35 liver cells. b, Apoa1 and Ttr1 expression analysis by RT-qPCR relative to expression levels in the control. Results shown as mean ± s.e.m. of four biological replicates. P values are from two-sided Student’s t-test. c, Western blot analysis of two biological replicates for endogenous and exogenous FOXA proteins, demonstrating similar amounts of FOXA-ΔHx and FOXA1-PP mutant proteins as FOXA1-WT, and thus indicating an intrinsic deficiency in the mutants’ abilities to restore expression of endogenous liver genes in a Foxa1 knock-down background. Two experiments were repeated independently with similar results. Full blots are presented in the Source Data files.
a, Generation of Foxa2WT-tRFP and Foxa2∆Hx-tRFP knock-in alleles with targeting and exchange cassettes. b, Frequency of genotypes resulting from heterozygous intercrosses of Foxa2WT-tRFP/WT at embryonic stages. c, Box and whisker plots show stage distribution at wild-type, heterozygous, and homozygous of Foxa2WT-tRFP and Foxa2∆Hx-tRFP embryos at E8.5. The bottom and top of the boxes correspond to the 25th and 75th percentiles, and the internal band is the 50th percentile (median). The ends of the whiskers represent 1.5 times the IQR. The points represent outliers. The indicated P-values are obtained by one-sided Wilcoxon rank sum test.
a, Bright field images of Foxa2∆Hx-tRFP/WT and Foxa2∆Hx-tRFP/∆Hx-tRFP (with a gross phenotype) littermate at E12.5 from heterozygous intercrosses. Images are representative of the numbers of embryos indicated in Fig. 3a. b, Representative FACS pattern of FOXA-tRFP-high (P5 gate), -middle (P4 gate), and -negative (P3 gate) cells from Foxa2tRFP/tRFP E7.5 embryos. Foxa2WT/WT and Foxa2tRFP/WT samples were loaded only for setting the gates, but not for the sorting. The FOXA2-tRFP-middle (P4 gate) was set by avoiding autofluorescence and including up to the maximum tRFP intensity of heterozygous (Foxa2tRFP/WT) cells expressed. The FOXA2-tagRFP-high (P5 gate) exhibited a higher tagRFP signal than heterozygous cells. The FACS experiments were repeated more than 40 times independently with similar results.
a, Heatmaps show DESeq adjusted RNA-seq counts for all differentially expressed genes with adjusted p-value < 0.1 (by one-sided Wald test with FDR correction at 10%). The individual replicates of wild-type (Foxa2WT-tRFP /WT-tRFP) and ∆Hx (Foxa2∆Hx-tRFP/∆Hx-tRFP) in FOXA2-tRFP-high and -middle cells were presented. n = 3 biologically independent RNA-seq datasets per group. b, RNA-seq tracks of each biological replicate of FOXA2-tRFP-mid cells from E7.5 Foxa2WT/WT (green) and Foxa2∆Hx-tRFP/∆Hx-tRFP (gray) embryos at down-regulated gene loci in Foxa2∆Hx-tRFP/∆Hx-tRFP.
Extended Data Fig. 8 Deletion of α-helical region of FOXA2 alters the accessible chromatin sites in E7.5 embryos.
a, de novo motif enrichment analysis at differential open chromatin sites (ATAC-seq peaks) between FOXA2[WT]-tRFP-high cells and FOXA2[WT]-tRFP-middle cells. P-value (by one-sided Monte Carlo simulation with FDR controlled at 5%) and % targets are indicated in parentheses. n = 2 biologically independent ATAC-seq datasets per group. b, de novo motif enrichment analysis at differential open chromatin sites (ATAC-seq peaks) of “wild-type (Foxa2WT-tRFP /WT-tRFP)-specific”, “∆Hx (Foxa2∆Hx-tRFP/∆Hx-tRFP)-specific”, and “wild-type and ∆Hx common” open chromatin sites in E7.5 FOXA2-tRFP-high cells and FOXA2-tRFP-middle cells. P-value (by one-sided Monte Carlo simulation with FDR controlled at 5%) and % targets are indicated in parentheses. n = 2 biologically independent ATAC-seq datasets per group.
Extended Data Fig. 9 Deletion of α-helical region of FOXA2 alters gene expression and accessible chromatin landscapes in E7.5 embryos.
a, GO term enrichment analysis of downregulated and upregulated genes in FOXA2-tRFP-high and FOXA2-tRFP-middle cells. P-value (by one-sided EASE/Fisher’s exact test) and gene count are indicated in parentheses. n = 3 biologically independent RNA-seq datasets per group. b, The distribution of WT-specific, ∆Hx-specific, and WT-∆Hx common open chromatin sites at non-overlapped genomic features.
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Iwafuchi, M., Cuesta, I., Donahue, G. et al. Gene network transitions in embryos depend upon interactions between a pioneer transcription factor and core histones. Nat Genet 52, 418–427 (2020). https://doi.org/10.1038/s41588-020-0591-8
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