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Nucleation of DNA repair factors by FOXA1 links DNA demethylation to transcriptional pioneering

Nature Genetics volume 48, pages 1003–1013 (2016)Cite this article

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Abstract

FOXA1 functions in epigenetic reprogramming and is described as a 'pioneer factor'. However, exactly how FOXA1 achieves these remarkable biological functions is not fully understood. Here we report that FOXA1 associates with DNA repair complexes and is required for genomic targeting of DNA polymerase β (POLB) in human cells. Genome-wide DNA methylomes demonstrate that the FOXA1 DNA repair complex is functionally linked to DNA demethylation in a lineage-specific fashion. Depletion of FOXA1 results in localized reestablishment of methylation in a large portion of FOXA1-bound regions, and the regions with the most consistent hypermethylation exhibit the greatest loss of POLB and are represented by active promoters and enhancers. Consistently, overexpression of FOXA1 commits its binding sites to active DNA demethylation in a POLB-dependent manner. Finally, FOXA1-associated DNA demethylation is tightly coupled with estrogen receptor genomic targeting and estrogen responsiveness. Together, these results link FOXA1-associated DNA demethylation to transcriptional pioneering by FOXA1.

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Figure 1: Identification of FOXA1-associated proteins.
Figure 2: Characterization of endogenous FOXA1 DNA repair complex.
Figure 3: Hierarchical architecture of the FOXA1 DNA repair complex.
Figure 4: Genomic landscape of the FOXA1 DNA repair complex.
Figure 5: DNA methylation dynamics after FOXA1 depletion.
Figure 6: FOXA1 overexpression evokes POLB-dependent demethylation.
Figure 7: The FOXA1 DNA repair complex in estrogen response.

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Acknowledgements

This work was supported by grants (91219201, 81530073, and 81130048 to Y.S. and 81300254 to Y.Z.) from the National Natural Science Foundation of China and a grant (973 Program: 2014CB542004 to J.L.) from the Ministry of Science and Technology of China.

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  1. Yu Zhang and Di Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Yu Zhang, Di Zhang, Qian Li, Jing Liang, Luyang Sun, Xia Yi, Zhe Chen, Ruorong Yan, Guojia Xie, Wanjin Li, Shumeng Liu, Bosen Xu, Lei Li, Jianguo Yang, Lin He & Yongfeng Shang

  2. Department of Biochemistry and Molecular Biology, Key Laboratory of Hormones and Development (Ministry of Health), Tianjin Key Laboratory of Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

    Yongfeng Shang

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Contributions

Y.S. and Y.Z. conceived the project. Y.S. and Y.Z. conceived and designed the experiments. Y.Z. and D.Z. performed the experiments. Y.Z. performed computational analysis. Y.S. and Y.Z. wrote the manuscript. Y.S., Y.Z., D.Z., J.L., and Q.L. analyzed the data. Y.Z., D.Z., J.L., Q.L., L.S., X.Y., Z.C., R.Y., G.X., S.L., B.X., W.L., L.L., J.Y., and L.H. performed material preparation.

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Integrated supplementary information

Supplementary Figure 1 Antibodies used in detection of the components of the FOXA1 DNA repair complex.

Note the lower band in the DNA-PKcs blot possibly resulted from degradation during sample preparation owing to the large size of DNA-PKcs (~460 kDa). Specificity of the major components of the FOXA1 DNA repair complex, including FOXA1, POLB, LIG3, Ku80, and PARP1, was further supported by their specific depletion in the targeting RNAi– transfected cells.

Supplementary Figure 2 Composition of the FOXA1 DNA repair complex.

(a) Left, schematic of FOXA1 mutants. Middle and right the indicated proteins purified from E. coli were analyzed by SDS–PAGE followed by Coomassie blue staining. (b) MCF-7 cells were transfected with siNS or siRNAs targeting FOXA1 DNA repair factors (siFOXA1, siPOLB, siPARP1, siKu80, or siLIG3). The effect of RNAi on the mRNA levels of ERα, FOXA1, and PARP1 was examined by real-time RT–PCR. Data are displayed as means ± s.d. for triplicates (*P < 0.01, one-way ANOVA). (c) FOXA1 has no discernible effects on PARP1 binding. qChIP was performed to examine the binding pattern of FOXA1 and PARP1 at the indicated sites in MCF-7 cells transfected with siNS or siFOXA1. Data are displayed as means ± s.d. for triplicates (*P < 0.01, t test for the qChIP data in siFOXA1-transfected cells versus siNS-transfected cells). enh, enhancer. (d) Whole-cell lysates from MCF-7 cells in the presence or absence of DNase I were subjected to immunoprecipitation with FOXA1 antibodies followed by immunoblotting using antibodies against POLB.

Supplementary Figure 3 The efficiency of RNAi in decreasing the protein expression of the subunits of the FOXA1 DNA repair complex.

MCF-7 cells were transfected with siNS or siRNAs targeting FOXA1 DNA repair factors (siFOXA1, siPOLB, siPARP1, siKu80, or siLIG3). The RNAi effect was examined by immunoblotting.

Supplementary Figure 4 Enrichment of the FOXA1 DNA repair complex at distinct epigenetic states.

Enrichment of the FOXA1 complex at distinct epigenetic states. (a) Chromatin state annotation was produced from ChromHMM using ChIP-seq data on H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K36me3, and H3K27ac in MCF-7 cells. This model distinguished seven broad classes of chromatin state, referred to as follows: state E1, Polycomb-repressed state enriched for characteristic H3K27me3; state E2, heterochromatin state without enrichment for any of these marks; state E3, repressive state enriched for H3K9me3; state E4, transcribed state enriched with H3K36me3 at gene bodies; state E5, enhancer state strongly enriched for the enhancer markers H3K27ac and H3K4me1; state E6, enhancer state weakly enriched for H3K27ac; state E7, promoter state strongly enriched for H3K4me3 and moderately enriched for H3K27ac. Emission and transition parameters for the multivariate hidden Markov model are displayed as a heat map. (b) The heat map indicates the relative percentage of the genome represented by each chromatin state and the relative fold enrichment of the full set of FOXA1 complex binding sites for the states determined in a. (c) The entire set of FOXA1 complex binding sites were grouped on the basis of their intersection with the epigenomic states determined in a. Average profiles of FOXA1 and POLB in these seven subgroups were plotted.

Supplementary Figure 5 Average ChIP-seq read density profile (log2 transformed) of the indicated proteins at FOXA1-binding sites or p300-associated enhancers without FOXA1 binding under the indicated conditions.

BS, binding sites.

Supplementary Figure 6 Sequential ChIP for the co-binding of the FOXA1 DNA repair complex subunits at control regions.

Data are displayed as means ± s.d. for triplicates.

Supplementary Figure 7 DNA methylation profiles of one locus on chromosome 2 and one locus on chromosome 10.

Tracks of two biological replicates of bisulfate sequencing and ChIP-seq density of FOXA1 are shown.

Supplementary Figure 8 DNA methylation patterns at cell-lineage-specific FOXA1-binding sites.

(a) Cell-lineage-specific FOXA1-binding sites in MCF-7 cells or HepG2 cells or sites shared by the two cell lines are shown. (b) The biolin plot shows the density of average CpG levels at tissue-specific FOXA1-binding sites and control regions. (c) Average profiles of FOXA1 at tissue-specific FOXA1-binding sites and control regions.

Supplementary Figure 9 DNA methylation changes at three clusters.

(a–c) The scatterplots of the indicated clusters show mean 5mC abundance in the indicated windows (350 bp) in siNS-transfected MCF-7 cells (x axis) against its counterpart in siFOXA1-transfected cells (y axis). Loci with significant methylation changes in the scatterplot are identified as off-diagonal points. (d) 5mC abundance differences in MCF-7 cells transfected with siFOXA1 versus siNS are measured from each library and plotted as a box plot.

Supplementary Figure 10 DNA methylation changes at the central groups.

(a) The sites from cluster I with a centralized demethylation pattern were further stratified into five subgroups by CpG counts within their LCHs. (b) Average number of CpG dinucleotides in each subgroup of central cluster I. (c) The scatterplots of the indicated subgroups show mean 5mC abundance within the 200-bp window centered on LCH midpoints in siNS-transfected MCF-7 cells (x axis) plotted against its counterpart in siFOXA1-transfected cells (y axis). Loci with significant methylation changes in the scatterplot are identified as off-diagonal points.

Supplementary Figure 11 Multiple-track plots showing profiles of DNA methylation, histone modifications, and ChIP-seq read density of FOXA1 and ERα in the indicated conditions at one locus on chromosome 12 and another locus on chromosome 19.

Methylated CpG differences in MCF-7 cells transfected with siFOXA1 versus siNS were calculated with combined replicated libraries. The densities of H3K4me1, H3K4me3, and H3K27ac signals are shown in a heat map with yellow indicative of strong signals and blue indicative of weak signals.

Supplementary Figure 12 Average profile of the indicated histone modifications at each group.

Groups were determined in Figure 5.

Supplementary Figure 13 Sole knockdown of POLB has no discernible impact on DNA methylation at FOXA1-binding sites in MDA-MB-231 cells.

DNA methylation was examined by bisulfite sequencing of cloned PCR products. The BSP results in MDA-MB-231 cells transfected with siNS or siPOLB together with empty vector are shown as open or closed circles representing unmethylated and methylated CpGs, respectively. The CpG sites within the red boxes highlight the closest CpG dinucleotides surrounding FOXA1 binding motifs as in Figure 6b.

Supplementary Figure 14 DNA methylation and FOXA1 complex binding were examined in FOXA1- or POLB-depleted MCF-7 cells.

(a) DNA methylation levels at the indicated regions (Fig. 7a) were measured by MEDIP-coupled quantitative real-time PCR. Data are displayed as means ± s.d. for triplicates (*P < 0.05, one-way ANOVA). (b) qChIP was performed in MCF-7 cells transfected with siNS, siFOXA1, or siPOLB with antibodies against FOXA1 (top) and POLB (bottom). Data are displayed as means ± s.d. for triplicates (*P < 0.05, one-way ANOVA).

Supplementary Figure 15 Dependency of induced estrogen responsiveness on the FOXA1 DNA repair complex.

MDA-MB-231 cells overexpressing the indicated genes were further challenged with siNS or siPOLB. (a) Expression of these proteins was measured by immunoblotting. (b–d) In these cells, we further measured the mRNA levels of pS2 in the presence or absence of estrogen using real-time RT–PCR (b), the time course of ERα and FOXA1 binding at the pS2 promoter using qChIP (c), and the DNA methylation status of a CCGG site in the pS2 promoter using the methods described in Figure 7d (d). Data are displayed as means ± s.d. for triplicates (*P < 0.01, one-way ANOVA).

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Zhang, Y., Zhang, D., Li, Q. et al. Nucleation of DNA repair factors by FOXA1 links DNA demethylation to transcriptional pioneering. Nat Genet 48, 1003–1013 (2016). https://doi.org/10.1038/ng.3635

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