Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling

Abstract

Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes’ regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.

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Fig. 1: Genomic locus proteomics of hTERT.
Fig. 2: Genomic locus proteomic analysis of MYC promoter.

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Acknowledgements

We are grateful to S. Shipman and G. Church (Harvard University, Cambridge, MA, USA) for the tetracycline-inducible/puroR plasmid, and to A. Edge (Mass Eye and Ear, Boston, MA, USA) for the HUWE1 plasmids. We thank A. Ting, J. Doench, N. Udeshi, A. Regev, P. Thakore, B. Hamilton, and E. Kvedaraite for useful discussion; E.M. Perez, J. Ray, and R. Issner for technical assistance; and M. Papanastasiou, E. Kuhn, and J. Abelin for critical assessment of the manuscript. This work was funded by the NCI CPTAC PCC (grant 1U24CA210986-01 to S.A.C.), the Broad Institute (SPARC grant to S.A.M.), the Pediatric Scientist Development Program (to B.T.K.), and the March of Dimes Birth Defects Foundation (to B.T.K.).

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Contributions

S.A.M. and J.W. conceived the idea. S.A.M., J.W., and S.A.C. conceived the experimental setup and designed the research. S.A.M. created the constructs and stable lines, and performed labeling, western blotting, enrichments, mass spectrometry, and ChIP-qPCR for the c-MYC lines. J.W. performed ChIP-qPCR for all hTERT-related experiments. R.P. and S.A.M. performed the computational and statistical analyses. B.T.K. and S.A.M. performed the off-target analyses. S.A.M., J.W., R.P., B.T.K., F.Z., and S.A.C. interpreted the data. S.A.M., J.W., and S.A.C. wrote the manuscript.

Corresponding authors

Correspondence to Samuel A. Myers or Steven A. Carr.

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A patent application related to this work has been filed by The Broad Institute.

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Integrated supplementary information

Supplementary Figure 1 Workflow for the quantitative proteomics aspect of GLoPro.

Workflow for the quantitative proteomics aspect of GLoPro. Each individual sgRNA-293T-Caspex line is independently affinity labeled, lysed, enriched for biotinylated proteins by streptavidin-coated beads, digested, and TMT labeled. After mixing, the peptides are analyzed by LC-MS/MS, where the isobarically-labeled peptides from each condition are co-isolated (MS1), co-fragmented for peptide sequencing (MS2), and the relative quantitation of the TMT reporter ions are measured (MS2 inset). Subsequent data analysis compares the TMT reporter ions for each sgRNA line to the non-spatially constrained no-guide control line (grey) to identify reproducibly enriched proteins.

Supplementary Figure 2 Diagram of Caspex plasmid.

Diagram of Caspex plasmid. NLS, nuclear localization sequence; 3xFLAG, triple FLAG epitope tag; V5, V5 epitope tag; T2A, T2A self-cleaving peptide; GFP, Green fluorescent protein; TRE, Tetracycline response element; rtTA, reverse tetracycline-controlled transactivator; puror; puromycin acetyltransferase, ITRs, inverted terminal repeats. We designed GLoPro to have inducible expression to prevent constant CASPEX association with the locus of interest, potentially disrupting gene expression. Thus, the inducible expression and selection cassette is currently too large for viral transduction. Ongoing work in our laboratory has found that co-transfecting the piggybac transposase aids the generation of stable Caspex lines in cell culture models with poor transfection efficiency (data not shown). Thus, in its current form, Caspex can only be used in electroporation- or cationic lipid-based transfectable cells.

Supplementary Figure 3 CASPEX labeling validation at hTERT.

A) UCSC Genome Browser representation (hg19) of the TERT promoter, including genomic coordinates, and the location of sgRNA sites relative to the TSS. qPCR probes are numbered. B) ChIP-qPCR against biotin (blue boxes) and FLAG (green boxes) in hTERT-Caspex cells expressing either no sgRNA (far right) their respective sgRNA. ChIP probes refer to regions amplified and detected by qPCR. The primer pair used for the no-guide control spanned the respective targeted locus. The location of the sgRNA in each ChIP-qPCR is highlighted in red.

Supplementary Figure 4 On- and off-target CASPEX binding analysis.

On- and off-target CASPEX binding analysis for sgRNAs targeting hTERT. Anti-FLAG ChIP was performed in each sgRNA-expressing 293T-Caspex cell line and was probed for the predicted binding locus or the top predicted off-target locus. Each fold-enrichment was calculated via the delta Ct method 48 between the ChIP product and the input chromatin material from full ChIP replicates, measurement triplicates. A) Cumulative enrichment of tiled, on-target CASPEX binding at hTERT (green) compared to top predicated off-target binding sites for each individual sgRNA target (white). Cumulative refers to sgRNAs that reside within 2 kb of each other. No two off-targets sites (locus position labeled in grey) reside within 100 Mb from one another (Supplemental Table 1). Only the sgRNA-expressing 293T-Caspex lines used for proteomic analysis was used to calculate the cumulative on-target enrichment. B) Individual ChIP fold enrichment values for on- (grey) and the top predicted off-target CASPEX binding sites (white) for all sgRNA-expressing 293T-Caspex lines. The numbers above the bar graph indicates the fold difference in fold-enrichment between on-target binding to off-target CASPEX enrichment.

Supplementary Figure 5 Anti-Flag and anti-biotin western blots of hTERT Caspex lines.

Anti-FLAG and anti-biotin Western blots of hTERT Caspex lines treated for 12 hours with 0.5 ug/mL dox or vehicle, and labeled via Caspex-mediated biotinylation. Top panel shows anti-FLAG signals for cells treated with dox or vehicle. Middle panel shows anti-biotin signal from cells exposed to labeling protocols with or without dox treatment. Endogenous biotinylated proteins (stars) are used as the loading control. Bottom panel is a merge of both signals. Protein molecular weight ladder (left) separates two no-guide conditions from the TERT Caspex lines.

Supplementary Figure 6 Correlation analysis of protein enrichment from hTERT 293T-Caspex cells.

Multi-scatter plots for log2 fold enrichment values of proteins quantified, and the corresponding Pearson correlation coefficients between all pair-wise hTERT-293T-Caspex cells comparisons using the no sgRNA control line as the denominator (n = 5 independent sgRNA lines).

Supplementary Figure 7 Negative relationship between background protein abundance and enrichment at hTERT.

Scatterplot representing the abundance level of each protein (grey dots) from the no guide control (x-axis) and the statistical significance of its enrichment over background (y-axis). Blue line is the linear regression between the two variables (Pearson’s r = -0.23). The dotted line identifies the adj. p value cut-off of 0.05 for statistical enrichment at hTERT. Red dots indicate histone and RNAP subunits, green indicate a subset of proteins significantly enriched at hTERT. Significantly enriched proteins have, on average, statistically significant lower abundance than non-significantly enriched proteins (inset, Welch Two Sample t-test, p-val = 2.4e-16).

Supplementary Figure 8 CASPEX labeling validation at MYC.

A) UCSC Genome Browser representation (hg19) of the MYC promoter, including genomic coordinates, and the location of sgRNA sites relative to the TSS. B) ChIP-qPCR against CASPEX (FLAG epitope) in MYC-Caspex cells expressing their respective sgRNAs. ChIP probes either span the region targeted by the respective sgRNA or a non-overlapping region approximately 500 bp on either side of the sgRNA target site. Caspex cells expressing no sgRNA was used as the negative control.

Supplementary Figure 9 On- and off-target CASPEX binding analysis for sgRNAs targeting the MYC promoter.

On- and off-target CASPEX binding analysis for sgRNAs targeting MYC promoter. Anti-FLAG ChIP was performed in each sgRNA-expressing 293T-Caspex cell line and was probed for the predicted binding locus or the top predicted off-target locus. Each fold-enrichment was calculated via the delta Ct method 48 between the ChIP product and the input chromatin material from full ChIP replicates, measurement triplicates. A) Cumulative enrichment of tiled, on-target CASPEX binding at MYC (green) compared to top predicated off-target binding sites for each individual sgRNA target (white). Cumulative refers to sgRNAs that reside within 2 kb of each other. No two off-targets sites (locus position labeled in grey) reside on the same chromosome as one another (Supplemental Table 1). All MYC targeting sgRNA-expressing 293T-Caspex lines were used for proteomic analysis so all five were used to calculate the cumulative on-target enrichment. B) Individual ChIP fold enrichment values for on- (grey) and the top predicted off-target CASPEX binding sites (white) for all sgRNA-expressing 293T-Caspex lines. The numbers above the bar graph indicates the fold difference in fold-enrichment between on-target binding to off-target CASPEX enrichment.

Supplementary Figure 10 Pathway analysis of proteins associated with MYC.

Significantly enriched gene sets from proteins identified to associate with the MYC promoter by GLoPro. Only gene sets with an adjusted p-value of less than 0.01 are shown. MYC_ACTIVE_PATHWAY is highlighted in red and discussed in the text.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10

Reporting Summary

Supplementary Table 1

Oligonucleotides used in this study

Supplementary Table 2

hTERT GLoPro results

Supplementary Table 3

MYC GLoPro results

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Myers, S.A., Wright, J., Peckner, R. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat Methods 15, 437–439 (2018). https://doi.org/10.1038/s41592-018-0007-1

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