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Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens

Citation

Sprouffske, Kathleen et al. (2021), Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens, Dryad, Dataset, https://doi.org/10.5061/dryad.t1g1jwt20

Abstract

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

Methods

ChIP-seq was performed as previously described (Galli, G.G., et al., Mol Cell, 2015. 60(2): p. 328-37). Briefly, cells were cross-linked in 1% formaldehyde for 10 minutes at room temperature after which the reaction was stopped by addition of 0.125M glycine. Cells were lysed and harvested in ChIP buffer (100 mM Tris at pH=8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) and the chromatin disrupted by sonication using a EpiShear sonicator (Active Motif) to obtain fragments of average 200-500 bp in size. Chromatin extracts were incubated for 16 hours with the following antibodies: ER (Cell Signaling, 13258), FOXA1 (Cell Signaling, 58613), GATA3 (Cell Signaling, 5852), CTCF (Cell Signaling, 2899), H3K27ac (Cell Signaling, 8173) and H3K4me1 (Cell Signaling, 5326). Immunoprecipitated complexes were recovered using Protein G Dynabeads (Invitrogen) and DNA was recovered by reverse-crosslinking and purified using SPRI Select beads (Beckman Coulter). Libraries for ChIP-sequencing were generated using Ovation® Ultralow Library System V2 (NuGEN) and barcodes were added using NEBNext Multiplex Oligos for Illumina (NEB, Index Primers Set 1) according to the manufacturer’s recommendation. All next-generation sequencing experiments were run on a HiSeq2500 (Illumina). Fastq files were aligned to a human reference genome (hg38) using bowtie2 v2.3.4.1 and sorted using samtools v1.8. Duplicates were marked and removed using Picard MarkDuplicates v2.18.7 (http://broadinstitute.github.io/picard), and low quality mapped reads (below 20) were removed using samtools. Samtools view was used to retain reads mapping to human chromosomes and to discard reads mapping to chrM for ATAC-seq samples. Deeptools was used to generate RPKM-normalized bigwig files.