Dataset Information

Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 [RNA-seq]

ABSTRACT: Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247414 | GEO | 2023/11/09

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

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.

Dataset Information

Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 [RNA-seq]

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure