A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

Mutational processes in cancer preferentially affect binding of particular transcription factors

Protein binding microarrays provide comprehensive information about the DNA binding specificities of transcription factors (TFs), and can be used to quantitatively predict the effects of DNA sequence variation on TF binding. There has also been substantial progress in dissecting the patterns of mutations, i.e., the "mutational signatures", generated by different mutational processes. By combining these two layers of information we can investigate whether certain mutational processes tend to preferentially affect binding of particular classes of TFs. Such preferential alterations of binding might predispose to particular oncogenic pathways. We developed and implemented a method, termed "Signature-QBiC", that integrates protein binding microarray data with the signatures of mutational processes, with the aim of predicting which TFs’ binding profiles are preferentially perturbed by particular mutational processes. We used Signature-QBiC to predict the effects of 47 signatures of mutational processes on 582 human TFs. Pathway analysis showed that binding of TFs involved in NOTCH1 signaling is strongly affected by the signatures of several mutational processes, including exposure to ultraviolet radiation. Additionally, toll-like-receptor signaling pathways are also vulnerable to disruption by this exposure. This study provides a novel overview of the effects of mutational processes on TF binding and the potential of these processes to activate oncogenic pathways through mutating TF binding sites.

Zinc finger protein SALL4 functions through an AT-rich motif to regulate gene expression

SummaryThe zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, down-regulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive target for designing therapeutic strategies. However, whether SALL4 binds DNA directly to regulate gene expression is unclear and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) experiments, we identified and validated the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA-seq analyses after SALL4 knockdown, we discovered hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of Histone 3 Lysine 9-specific Demethylases (KDMs). Taken together, these results elucidated the mechanism of SALL4 DNA binding and revealed novel pathways and molecules to target in SALL4-dependent tumors.

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody availability and reliability are problematic. The Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies against human-presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field-tested. We therefore screened their ability in a variety of assays. 887 unique antibodies against 681 unique chromatin proteins, of which 605 are putative sequence-specific transcription factors (TFs), were assayed by ChIP-exo. Subsets were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. At least 6% of the tested antibodies were validated for use in ChIP-based assays by the most stringent of our criteria. An additional 34% produced data suggesting they warranted further testing for clearer validation. We demonstrate and discuss the metrics and limitations to antibody validation in chromatin-based assays.

A Rice Promoter Protein Binding Microarray for Cis-Acting Elements for Rice Transcription Factors

The authors have withdrawn this preprint due to author disagreement.

A Rice Promoter Protein Binding Microarray for Cis-Acting Elements for Rice Transcription Factors

BackgroundTranscription factors (TFs) regulate the expression of genes at the transcriptional level by binding a specific DNA sequence. Thus, predicting the DNA-binding motifs of TFs is one of the most important areas for the functional analysis of TFs in the postgenomic era. Although many methods have been developed for this challenge, there are still many TFs with unknown DNA-binding motifs.FindingsIn this paper, we designed an rice (Oryza sativa)-specific protein binding microarray (RPBM), and its probes are 40 bp long with 20 bp of overlap; there are 49 probes spanning the 1 kb promoter region before the translation start site of each gene. To confirm the efficiency of RPBM technology, we selected two TFs, OsWOX13 and OsSMF1. We identified the ATTGATTG DNA-binding sequence and 635 putative target genes of OsWOX13. OsSMF1 bound to GCTGACTCA and GGATGCC sequences and bound especially strongly to CCACGTCA. A total of 932 putative target genes were identified for OsSMF1.ConclusionsRPBM can be applicable in the analysis of DNA-binding motifs for TFs where binding is evaluated in extended natural promoter regions. The analysis can also be applicable to TFs that have single or multiple binding motifs. The technology might even be expanded for application to TFs that are heterodimers or form higher-order complexes.

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

Determining how DNA variants affect the binding of regulatory complexes to cis-regulatory elements (CREs) and non-coding single-nucleotide polymorphisms (ncSNPs) is a challenge in genomics. To address this challenge, we have developed CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements), which is a protein-binding microarray (PBM)-based approach that allows for the high-throughput profiling of cofactor (COF) recruitment to DNA sequence variants. The method also enables one to infer the identity of the transcription factor-cofactor (TF-COF) complexes involved in COF recruitment. We use CASCADE to characterize regulatory complexes binding to CREs and SNP quantitative trait loci (SNP-QTLs) in resting and stimulated human macrophages. By profiling the recruitment of the acetyltransferase p300 and MLL methyltransferase component RBBP5, we identify key regulators of the chemokine CXCL10, and by profiling a set of five functionally diverse COFs we identify a prevalence of ETS sites mediating COF recruitment at SNP-QTLs in macrophages. Our results demonstrate that CASCADE is a customizable, high-throughput platform to link DNA variants with the biophysical complexes that mediate functions such as chromatin modification or remodeling in a cell state-specific manner.

A map of direct TF–DNA interactions in the human genome

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF–DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF–DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF–DNA interactions. Our work culminated with predicted interactions covering >2% of the human genome, obtained by uniformly processing 1983 ChIP-seq peak data sets from the ReMap database for 232 unique TFs. The predictions were a posteriori assessed using protein binding microarray and ChIP-exo data, and were predominantly found in high quality ChIP-seq peaks. The set of predicted direct TF–DNA interactions suggested that high-occupancy target regions are likely not derived from direct binding of the TFs to the DNA. Our predictions derived co-binding TFs supported by protein-protein interaction data and defined cis-regulatory modules enriched for disease- and trait-associated SNPs. We provide this collection of direct TF–DNA interactions and cis-regulatory modules through the UniBind web-interface (http://unibind.uio.no).

A map of direct TF-DNA interactions in the human genome

ABSTRACTChromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF-DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF-DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF-DNA interactions. Our work culminated with predicted interactions covering >4% of the human genome, obtained by uniformly processing 1,983 ChIP-seq peak data sets from the ReMap database for 232 unique TFs. The predictions were a posteriori assessed using protein binding microarray and ChIP-exo data, and were predominantly found in high quality ChIP-seq peaks. The set of predicted direct TF-DNA interactions suggested that high-occupancy target regions are likely not derived from direct binding of the TFs to the DNA. Our predictions derived co-binding TFs supported by protein-protein interaction data and defined cis-regulatory modules enriched for disease- and trait-associated SNPs. Finally, we provide this collection of direct TF-DNA interactions and cis-regulatory modules in the human genome through the UniBind web-interface (http://unibind.uio.no).