Systematic dissection of sequence features affecting the binding spcificity of a pioneer factor

Open Access
- Author:
- Xu, Cheng
- Graduate Program:
- Molecular, Cellular, and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 15, 2024
- Committee Members:
- Robert Paulson, Outside Unit & Field Member
Xiaojun Lian, Major Field Member
Shaun Mahony, Major Field Member
Lu Bai, Chair & Dissertation Advisor
Melissa Rolls, Program Head/Chair - Keywords:
- Gene regulation
Transcription factor
Pioneer factor
Binding specificity
synthetic oligonucleotide library
FOXA1
AP-1
TF cooperativity
ChIP-ISO - Abstract:
- Sequence-specific transcription factors (TFs), which recognize their cognate DNA motifs, are central players in regulation of gene expression. In higher eukaryotes, TFs only bind to a small proportion of their motifs across the genome, partially because of the inhibition of nucleosomes. Strong binding selectivity is also observed for pioneer factors (PFs) despite their ability to bind to nucleosomal DNA, and the underlying mechanism is not well understood. Here, we designed a high-throughput assay named Chromatin Immunoprecipitation with Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect local 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 next-generation amplicon sequencing. We found that within the same sequence background, FOXA1 binding is strongly affected by its motif strength, clustering of motifs, and co-binding TFs including AP-1 and CEBPB. AP-1 is particularly important for enhancing FOXA1 binding, which is further illustrated by inhibition of AP-1 binding and genome-wide studies. Comparison among different cell lines and RNA-seq analysis reveal that AP-1 contributes to the cell-type-specific binding and functions of FOXA1. In vivo and In vitro studies further confirmed the interdependency and cooperativity between FOXA1 and AP-1 binding, although FOXA1 binding to naked DNA depends more on its core motifs. Finally, by moving sequences originated from different genomic loci to the same chromatin background and measuring FOXA1 binding, we showed that FOXA1’s binding specificity is more determined by the local sequence than chromatin background, including H3K9me3 or H3K27me3-marked heterochromatin. Our conclusions are consistent with a convolutional neural network (CNN) analysis of FOXA1 ChIP-seq data. In summary, our study provides insights of the genetic rules underlying PF binding specificity and reveals a potential mechanism for regulating its binding events during cell differentiation. Our study also establishes an experimental framework for understanding TF binding specificity and cis-regulatory logic.