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FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

ABSTRACT: FOXA1 is a FKHD family protein that translates epigenetic signatures at target enhancers to lineage-specific transcription and differentiation. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of this epigenetic signature, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding at its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. FOXA1 binding events were markedly reduced following TET1 depletion. Together, our results support that FOXA1 is not only a reader, but also a writer, of the epigenetic signatures at lineage-specific enhancers and that TET1 and FOXA1 form a feed-forward regulatory loop for enhancer activation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE73363 | GEO | 2016/07/22

SECONDARY ACCESSION(S): PRJNA296797

REPOSITORIES: GEO

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Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. To determine the genome-wide distribution of Tet1 and 5hmC in mouse ES cells, as well as identify the gene transcription changes after Tet1 depletion. GSM706669-GSM706671: We used GST pull-down followed by deep sequencing to map the DNA bound by the Tet1 CXXC domain in vitro. We made two mutants that have a single point mutation (Cys574 to Ala or Cys586 to Ala) in the core CXXC domain to ascertain the essential role of the CXXC domain in DNA binding by comparing the sequencing profile of DNA bound by wild type CXXC with the profiles of the CXXC mutants. GSM706672-GSM706673: Tet1 ChIP-seq was performed to identify the genome-wide distribution of Tet1 in mouse ES cells. GSM706674-GSM706679: We performed hydroxymethylated DNA immunoprecipitation (hMeDIP)-seq combined with a shRNA-mediated gene depletion strategy. To identify the loci specific 5hmC regulation by Tet1, we compared the 5hmC genome-wide distributions in control (Luc shRNA) and Tet1-depleted (Tet1 shRNA2863) mouse ES cells. GSM706680-GSM706682: To identify the gene regulation network by Tet1, we compared the gene expression profiles of control (scramble shRNA) and Tet1-depleted (Tet1 shRNA 2863 and Tet1 shRNA 3387) mouse ES cells determined by mRNA-seq.

Project description:FOXA1 is a pioneer factor that is important in hormone dependent cancer cells to stabilise nuclear receptors, such as estrogen receptor (ER) to chromatin. FOXA1 binds to enhancers regions that are enriched in H3K4mono- and dimethylation (H3K4me1, H3K4me2) histone marks and evidence suggests that these marks are requisite events for FOXA1 to associate with enhancers to initate subsequent gene expression events. However, exogenous expression of FOXA1 has been shown to induce H3K4me1 and H3K4me2 signal at enhancer elements and the order of events and the functional importance of these events is not clear. We performed a FOXA1 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells in order to identify FOXA1 interacting partners and we found histone-lysine N-methyltransferase (MLL3) as the top FOXA1 interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition, in line with our observation that MLL3 associates with an enhancer specific protein. We performed MLL3 ChIP-seq in breast cancer cells and unexpectedly found that MLL3 binds mostly at non-promoter regions enhancers, in contrast to the prevailing hypothesis. MLL3 was shown to occupy regions marked by FOXA1 occupancy and as expected, H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. Motif analysis and subsequent genomic mapping revealed a role for Grainy head like protein-2 (GRHL2) which was shown to co-occupy regions of the chromatin with MLL3. Regions occupied by all three factors, namely FOXA1, MLL3 and GRHL2, were most enriched in H3K4me1. MLL3 silencing decreased H3K4me1 at enhancer elements, but had no appreciable impact on H3K4me3 at enhancer elements. We identify a complex relationship between FOXA1, MLL3 and H3K4me1 at enhancers in breast cancer and propose a mechanism whereby the pioneer factor FOXA1 can interact with a chromatin modifier MLL3, recruiting it to chromatin to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.

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FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

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