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

Abstract

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.

Figure 1.

Validation of PCRP mAbs in ChIP-exo. (A) Comparison of ChIP-exo data at cognate versus noncognate motifs. ChIP-exo heatmap, composite, and DNA-sequence four-color plots were generated for NRF1, USF1, YY1, and IgG ChIP-exo data sets against the complete matrix of bound motifs from Supplemental Figure 2. The 5′ end of aligned sequence reads for each set of experiments was plotted relative to the distance from the cognate motif for each indicated target. Reads are strand-separated (blue, motif strand; red, opposite strand) and total-tag-normalized across samples. Rows are linked across samples and sorted based on their combined average rank-order in a 100-bp bin around each motif midpoint. High levels of background result in a more uniform distribution of reads across the window (as seen with the IgG control). (B) Enrichment comparisons of four unique HSF1 hybridoma clones (HSF1-1A10, HSF1-1A8, HSF1-1C1, HSF1-1D11). ChIP-exo heatmap, composite, and DNA-sequence four-color plots are shown for the indicated number and type of bound motifs for the indicated antibody hybridoma clones (A,B) or interaction partners (C) tested in K562 cells. The 5′ end of aligned sequence reads for each set of experiments was plotted against the distance from the cognate motif, present in the union of all called peaks between the data sets for each indicated target. Reads are strand-separated (blue, motif strand; red, opposite strand) and total-tag-normalized across samples. Rows are linked across samples and sorted based on their combined average rank-order in a 100-bp bin around each motif midpoint.

Figure 2.

Examining specificity of PCRP mAbs. (A) Heatmaps and composite plots displaying the global loss of NRF1-3D4 and NRF1-3H1 ChIP-seq signal after NRF1 RNAi. HCT116 cells were treated with nontargeting (sh Control) or two different NRF1-directed shRNAs (shRNA 1 and shRNA 2). Rows are linked across samples and sorted in descending order by mean score per region. (B) Western blot analysis of NRF1 knockdown by two different shRNAs (SH1 and SH2) or a nontargeting shRNA (NonT). HCT116 cells were infected with the indicated shRNAs and selected with puromycin (2 μg/mL). Total cell extracts were prepared for SDS-PAGE and immunoblotting against NRF1 and tubulin beta as the loading control. NRF1 knockdown efficiency (upper band in top panel) was quantified after normalizing with tubulin beta levels using ImageJ, and the normalized values shown. (C) Motif enrichment analysis of ChIP-exo. Cartoons depict models for binding via the cognate motif of the target ssTF or noncognate binding. Box plots of TPM expression values of target ssTFs associated to antibodies stratified by AUROC value. Results from analysis of 100 putative ssTF binding motifs within each ChIP-exo data set with more than 500 peaks (259 data sets in total). We assigned to each ChIP-exo data set the PWM with the highest AUROC (“top motif”) and quantified its centering as the mean distance of the PWM match from the peak's summit. In the scatter plot, each point represents the enrichment/centering of the top motif in one of the 259 putative TF ChIP-exo data sets. Colors indicate the expression level (RNA-seq TPM value; unavailable values are shown in gray) (The ENCODE Project Consortium 2012) of the gene specific for the antibody used in the ChIP assay. Point sizes indicate the number of ChIP-exo peaks in the data set. Top motifs with AUROC > 0.6 (dashed line) and TPM values from duplicate RNA-seq experiments are indicated. (D) Results from enrichment analysis of 100 TF binding motifs within each of 19 ChIP-seq data sets. Points are formatted as in C.

Figure 3.

Cell type comparison of antibody performance. (A,B) ChIP-exo heatmap, composite, and DNA-sequence four-color plots are shown for the indicated number of bound motifs for the indicated targets, in the indicated cell types. The 5′ end of aligned sequence reads for each set of experiments was plotted against the distance from the cognate motif, present in the union of all called peaks among the data sets for each indicated target. Reads are strand-separated (blue, motif strand; red, opposite strand) and total-tag-normalized across samples. Rows are linked across samples and sorted based on their combined average in a 100-bp bin around each motif midpoint.

Figure 4.

Application of ChIP-exo in human tissue using PCRP mAbs. ChIP-exo heatmap, composite, and DNA sequence four-color plots are shown for the indicated number and type of bound motifs for the indicated targets, in the indicated organ types (the liver includes two donors). The 5′ end of aligned sequence reads for each set of experiments was plotted against the distance from the cognate motif, present in the union of all called peaks between the data sets for each indicated target. Reads are strand-separated (blue, motif strand; red, opposite strand) and total-tag-normalized across samples. Rows are linked across samples and sorted based on their combined average in a 100-bp bin around each motif midpoint.

Figure 5.

PCRP mAb assayed by CUT&RUN. (A,B) CUT&RUN heatmap, composite, and DNA-sequence four-color plots are shown relative to the motifs defined and sorted in Figure 2. The 5′ end of aligned sequence reads is plotted. Reads are strand-separated (blue, motif strand; red, opposite strand) for ChIP-exo and combined (black) for CUT&RUN. Reads are aligned as above (Fig. 1A), and individual data set results are available in Supplemental Table 5.

Figure 6.

TF binding motifs derived from PBM experiments performed using anti-GST or PCRP antibodies. Each full-length TF was assayed with its PCRP mAb(s) and compared against its corresponding motif derived from an anti-GST PBM experiment and anticipated motif from the UniPROBE or Cis-BP databases (Weirauch et al. 2014; Hume et al. 2015). The TFs from UniPROBE or Cis-BP were assayed as extended DNA-binding domains. For the display of sequence motifs, probability matrices were trimmed from left and right until two consecutive positions with information content of 0.3 or greater were encountered, and logos were generated from the resulting trimmed matrices using enoLOGOS (Workman et al. 2005).