GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Series GSE247431

Query DataSets for GSE247431

Status

Public on Nov 09, 2023

Title

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

Organism

synthetic construct

Experiment type

Other

Summary

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.

Overall design

Electrophoretic mobility shift assay with high-throughput sequencing (EMSA-seq) on a synthetic oligonucleotide library incubated with 1-60 nM purified FOXA1 and separated on a native polyacrylamide gel to separate FOXA1 bound and unbound oligonucleotides for purification followed by high-throughput sequencing

Contributor(s)

Xu C, Kleinschmidt H, Yang J, Leith E, Johnson J, Tan S, Mahony S, Bai L

Citation missing

Has this study been published? Please login to update or notify GEO.

Submission date

Nov 09, 2023

Last update date

Nov 10, 2023

Contact name

Lu Bai

E-mail(s)

lub15@psu.edu

Organization name

Penn State University

Street address

406 South Frear Building

City

University Park

State/province

ZIP/Postal code

16802

Country

USA

Platforms (1)

GPL32628

NextSeq 2000 (synthetic construct)

Samples (24)

More...

GSM7889858	1nM FOXA1, Free Band, Rep 1
GSM7889859	1nM FOXA1, Shifted Band, Rep 1
GSM7889860	1nM FOXA1, Free Band, Rep 2

This SubSeries is part of SuperSeries:

GSE247432

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

Relations

BioProject

PRJNA1037550

Download family	Format
SOFT formatted family file(s)	SOFT
MINiML formatted family file(s)	MINiML
Series Matrix File(s)	TXT

Supplementary file	Size	Download	File type/resource
GSE247431_FOXA1_EMSA-seq_AllProcessedData.xlsx	7.2 Mb	(ftp)(http)	XLSX
GSE247431_RAW.tar	9.4 Mb	(http)(custom)	TAR (of TXT)
SRA Run Selector
Raw data are available in SRA