NextPBM: a platform to study cell-specific transcription factor binding and cooperativity

Abstract

High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM ( n uclear ext ract p rotein- b inding m icroarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.

Customizable high-throughput platform for profiling cofactor recruitment to DNA to characterize cis-regulatory elements and screen non-coding single-nucleotide polymorphisms

“…High-throughput methods for studying TF-DNA binding (e.g., MITOMI, SMiLE-seq, CSI, PBM, SELEX-seq, ATI, nextPBM, etc.) [15][16][17][41][42][43][44][45][46][47] have had a tremendous impact on our understanding of TF function and genome-scale analysis of gene regulation, leading to large databases of widely-used TF binding models [48][49][50][51] . However, these methods have not been applied to the study of TF-COF complexes.…”

“…However, these methods have not been applied to the study of TF-COF complexes. Approaches such as ATI 47 and nextPBMs 17 have been used to study TF-DNA binding in a more native cellular context by using TFs directly from cell nuclear extracts instead of using purified TF samples. However, COF recruitment has not been examined using these approaches.…”

“…The nuclear extract protocols are as previously described 17 Microarray DNA double stranding and PBM protocols are as previously described [15][16][17] .…”

“…Each REF/SNP pair was screened for differential recruitment of p300, SMARCA4, TBL1XR1, RBBP5, and GCN5 as well as differential binding of representative ETS factor PU.1 nextPBM experimental results were preprocessed as above. Z-scores were obtained for each probe as previously described 57 Motif modeling using single variant (SV) probes was performed as previously described 17,57,61 for the SNP-QTL sites profiled in detail using CASCADE. For the multi-tile design used to model extended loci such as the LPS-responsive CXCL10 promoter segment, a weighted mean approach was applied as follows to overlapping positions in order to integrate 21 results across sequential tiles: all variant probes corresponding to a given nucleotide at a given position within the promoter segment were averaged using each probe's corresponding seed (reference genomic) z-score as a weight.…”

“…PBMs are double-stranded DNA microarrays that allow protein-DNA binding to be assayed to thousands of DNA sequences 15,16 . Recently, we developed the nuclear extract protein-binding microarray (nextPBM) approach 17 to study DNA binding of TFs present in nuclear extracts. However, TFs function by recruiting COFs, which subsequently alter gene expression through diverse mechanisms such as histone modification or chromatin remodeling (Figure 1a) 18 .…”