10.5061/DRYAD.T1G1JWT20
Sprouffske, Kathleen
0000-0001-7081-2598
Novartis (Switzerland)
Lopes, Rui
Novartis (Switzerland)
Sheng, Caibin
0000-0002-7706-1253
Novartis (Switzerland)
Uijttewaal, Esther C. H.
0000-0003-3892-2820
Novartis (Switzerland)
Wesdorp, Adriana Emma
0000-0001-6656-2151
Novartis (Switzerland)
Dahinden, Jan
Novartis (Switzerland)
Wengert, Simon
0000-0003-1271-1996
Novartis (Switzerland)
Diaz-Miyar, Juan
Novartis (Switzerland)
Yildiz, Umut
Novartis (Switzerland)
Bleu, Melusine
Novartis (Switzerland)
Apfel, Verena
Novartis (Switzerland)
Mermet-Meillon, Fanny
Novartis (Switzerland)
Krese, Rok
Novartis (Switzerland)
Eder, Mathias
Novartis (Switzerland)
Olsen, André Vidas
0000-0003-0845-4632
University of Copenhagen
Hoppe, Philipp
Novartis (Switzerland)
Knehr, Judith
Novartis (Switzerland)
Carbone, Walter
0000-0001-6150-8295
Novartis (Switzerland)
Cuttat, Rachel
0000-0001-6019-4837
Novartis (Switzerland)
Waldt, Annick
0000-0002-4670-1588
Novartis (Switzerland)
Altorfer, Marc
Novartis (Switzerland)
Naumann, Ulrike
0000-0001-7783-7675
Novartis (Switzerland)
Weischenfeldt, Joachim
University of Copenhagen
deWeck, Antoine
0000-0003-0887-6771
Novartis (Switzerland)
Kauffmann, Audrey
Novartis (Switzerland)
Roma, Guglielmo
0000-0002-8020-4219
Novartis (Switzerland)
Schübeler, Dirk
0000-0002-9229-2228
Friedrich Miescher Institute
Galli, Giorgio G.
Novartis (Switzerland)
Systematic dissection of transcriptional regulatory networks by
genome-scale and single-cell CRISPR screens
Dryad
dataset
2021
FOS: Biological sciences
breast cancer
MCF7 breast cancer
ChIP-seq
2021-09-30T00:00:00Z
2021-09-30T00:00:00Z
en
https://doi.org/10.1126/sciadv.abf5733
5411275536 bytes
3
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
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.