Profiling ERα and FOXA1 Binding in Breast Cancer

We used an immunotethered strategy for profiling the binding of the ERα and FOXA1 TF in human MCF7 breast cancer cells. MCF7 cells were estrogen-withdrawn for 72 hours before being plated and then treated with either ethanol (vehicle control) or 10⁻¹⁰ M E2 for 1 hour before cell collection. The CUT&RUN method uses an antibody to a specific chromatin epitope to tether protein A–MNase at chromosomal binding sites within permeabilized cells. The nuclease is activated by the addition of calcium and cleaves DNA around binding sites (10 (link)). Cleaved DNA is isolated and subjected to paired-end Illumina sequencing to map the distribution of the chromatin epitope genome-wide. We used a primary antibody to human ERα (ab3575, Abcam, Cambridge, MA) and human FOXA1 (ab170933) and protein A–MNase fusion (pA-MNase, a gift from S. Henikoff, Fred Hutchinson Cancer Research Center, Seattle WA) (10 (link)). CUT&RUN profiling with 5 × 10⁵ cells and library amplification with 13 cycles of PCR were performed as described (10 (link)). Libraries were sequenced for 10 million paired-end reads on the Illumina NovaSeq 6000 platform at the University of Colorado Denver Cancer Center Genomics Shared Resource. Paired-end reads were mapped to the GRch38 assembly of the human genome using Bowtie2 (58 (link)).