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

Description

Despite the unique ability of pioneer transcription factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called ChIP-ISO to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that 1) FOXA1 binding is strongly affected by co-binding TFs AP-1 and CEBPB, 2) FOXA1 and AP-1 show binding cooperativity in vitro, 3) FOXA1’s binding is determined more by local sequences than chromatin context, including eu-/heterochromatin, and 4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.

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Immunofluorescence experiments were performed according to a protocol from Yoney et al. 202279. The following primary antibodies and dilutions were used: FLAG (mouse monoclonal, Millipore-Sigma, F1804, 1:1000), FOXA1 (rabbit polyclonal, GeneTex, GTX100308, 1:500), and FOSL1 (mouse monoclonal, Santa Cruz Biotechnology, sc-28310, 1:50). The following secondary antibodies and dilutions were used: goat anti-mouse IgG(H+L) (Alexa Fluor 594, ThermoFisher, A-11005, 1:1000), and goat anti-rabbit IgG(H+L) (Alexa Fluor 488, ThermoFisher, A-11008, 1:500). Immunostained A549 ePB tet-on A-FOS cells were imaged using Leica DMI6000 with Hamamatsu ORCA-R2 C10600 camera and SOLA SE light source. Images were acquired in phase contrast, GFP, and Texas Red channels, and with 40x/1.30 objective. Immunostained WT A549, MCF-7 and HepG2 cells were imaged using Zeiss Axio Observer 7 with camera Axiocam 705 mono. Images were acquired in DIC, AF594, AF488 and DAPI channels, and with 20x/0.8 objective. The average fluorescence intensity within the nuclei of each cell in the field was calculated using the Zeiss Bio Apps Gene Expression tool. The measurement area was limited to the cell nucleus, which was detected from the signal in the DAPI-stained channel. Average fluorescence intensity in the green/red was then measured for each cell in the field and normalized to the DAPI intensity in the same cell to correct for differences in cell permeability across cell types.

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