Kousuke Mouri, Hannah B. Dewey, Rodrigo Castro, Daniel Berenzy, Susan Kales et al.
Both upregulation and downregulation by cis-regulatory elements help modulate precise gene expression. However, our understanding of repressive elements is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, at genome-wide scale using a modified massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST (REST motif-intrinsic value [m-value]), above which cofactors colocalize and silence transcription. We identified 1,500 human variants that alter RE1 silencing and found that their effect sizes are predictable when they overlap with REST-binding sites above the m-value. Additionally, we demonstrate that non-canonical REST-binding motifs exhibit silencer function only if they precisely align half sites with specific spacer lengths. Our results show mechanistic insights into RE1, which allow us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization of transcription-factor-binding sites.
Kousuke Mouri, Hannah B. Dewey, Rodrigo Castro, Daniel Berenzy, Susan Kales, Ryan Tewhey. Whole-genome functional characterization of RE1 silencers using a modified massively parallel reporter assay. 2022-12-16. https://doi.org/10.1016/j.xgen.2022.100234
The whole-genome RE1 library paired 200-bp human REST-binding-site sequences and controls with five benchmark E elements in SK-N-SH. The ES plasmid library was assayed with four biological replicates and normalized to plasmid DNA.
A modified MPRAduo library tested native, swapped, scrambled, and spacer-edited noncanonical REST half-site arrangements in K562, with positive and negative controls. The assay measured reporter activity across the deposited analysis-level result set.
A benchmark MPRAduo pool tested 21 activating elements and candidate silencer/control elements in GM12878, including single E/S libraries and the silencer–enhancer (SE) duo alignment. Reporter RNA was normalized to plasmid DNA across four biological replicates.
The whole-genome RE1 library paired 200-bp human REST-binding-site sequences and controls with five benchmark E elements in HepG2. The ES plasmid library was assayed with four biological replicates and normalized to plasmid DNA.
The whole-genome RE1 library paired 200-bp human REST-binding-site sequences and controls with five benchmark E elements in GM12878. The ES plasmid library was assayed with four biological replicates and normalized to plasmid DNA.
The whole-genome RE1 library paired 200-bp human REST-binding-site sequences and controls with five benchmark E elements in K562. The ES plasmid library was assayed with four biological replicates and normalized to plasmid DNA.
A benchmark MPRAduo pool tested 21 activating elements and candidate silencer/control elements in GM12878, including single E/S libraries and the enhancer–silencer (ES) duo alignment. Reporter RNA was normalized to plasmid DNA across four biological replicates.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 27 files (ZIP)duoREST_benchmark_GM12878_results.txt.gzduoREST_RESTscreen_AF_GM12878_emVAR.out.gzduoREST_RESTscreen_AF_HepG2_emVAR.out.gzduoREST_RESTscreen_AF_K562_emVAR.out.gzduoREST_RESTscreen_AF_SKNSH_emVAR.out.gzduoREST_RESTscreen_derived_GM12878_results.run1.txt.gzduoREST_RESTscreen_derived_HepG2_results.run1.txt.gzduoREST_RESTscreen_derived_K562_results.run1.txt.gzduoREST_RESTscreen_derived_SKNSH_results.run1.txt.gzGSE196171_family.soft.gzGSE196171_nonCan_K562_results.run1.txt.gzGSE196171_RAW.tarGSM5861606_MPRAduo_counts_benchmark_run1.txt.gzGSM5861607_MPRAduo_counts_benchmark_run2.txt.gzGSM5861611_MPRAduo_counts_WGREST_GM12878.txt.gzGSM5861612_MPRAduo_counts_WGREST_HepG2.txt.gzGSM5861613_MPRAduo_counts_WGREST_K562.txt.gzGSM5861614_MPRAduo_counts_WGREST_SKNSH.txt.gzmmc15_allelic_skew_results.tsvmmc15_allelic_skew_results.zipmmc3_sequences_tested.tsvmmc3_sequences_tested.zipmmc5_benchmark_results.xlsxmmc7_WGREST_results.tsvmmc7_WGREST_results.zipmmc9_predicted_RE1_motif_scores.txtsource_notes.txt