Epigenomic disorder and partial EMT impair luminal progenitor integrity in Brca1-associated breast tumorigenesis
In breast cancer related to the BRCA1 mutation, luminal progenitor cells are believed to be the cells of origin, yet how these cells transform into invasive cancer cells remain poorly understood. Here, we combine single-cell epigenomic and transcriptomic data to reconstitute sequences of events in luminal cells that lead to tumorigenesis. Upon deletion of Trp53 and Brca1, we find that luminal progenitors display an extensive epigenomic disorder associated with a loss of cell identity. These cells then progress to tumor formation through a partial epithelial-to-mesenchymal transition, orchestrated by Snail and the timely activation of immunosuppressive and FGF signaling with their microenvironment. In human samples, pre-tumoral changes can be detected in early stage, basal-like tumors, which rarely recur, as well as in normal-like mammary glands of BRCA1 mutation carriers who have had cancer. Our study fills critical gaps in our understanding of BRCA1-driven tumorigenesis, opening perspectives for the early monitoring of individuals with high cancer risk. Overall design: snCUT&Tag was adapted from  Cusanovic et al. 2021. All washes were made with 500 µL unless otherwise stated and all centrifugations were done using a swinging bucket centrifuge at 1300g for 4 min at 4°C for nuclei preparation, or 600g for 8 min at 4°C for the subsequent steps. Nuclei were extracted from 1-2 million cells for 10 min on ice in 6 mL ice-cold NE1 buffer (20 mM HEPES pH7.2, KCl 10 mM, spermidine 0.5 mM, glycerol 20%, BSA 1%, NP-40 0.1%, digitonin 0.01%, proteases inhibitor 1x). Nuclei were filtered with a 30 uM cell strainer, washed in 6 mL PBS + BSA 1% and resuspended in Dig-Wash buffer (20 mM HEPES pH7.2, NaCl 150 mM, spermidine 0.5 mM, BSA 1%, NP-40 0.01%, digitonin 0.01%, proteases inhibitor 1x), checked under microscope and counted with 4,6-diamidino-2-phenylindole (DAPI) staining. 50,000 to 100,000 nuclei were resuspended in 50uL antibody buffer (EDTA 2 mM, 20 mM HEPES pH7.2, NaCl 150 mM, spermidine 0.5 mM, BSA 1%, NP-40 0.01%, digitonin 0.01%, proteases inhibitor 1x) with 1:50 antibody (Anti-H3K4me1 #5326 D1A9, Cell Signaling) and incubated overnight at 4°C with rotation. Next day, nuclei were washed with Dig-Wash buffer and resuspended in 100uL Dig-300 buffer (20 mM HEPES pH7.2, NaCl 300 mM, spermidine 0.5 mM, BSA 1%, NP-40 0.01%, digitonin 0.01) with the proteinA-Tn5 fusion (Diagenode, #C01070001, 1:250) and incubated for 1 h at room temperature with rotation. Then, nuclei were washed three times with Dig-300 buffer, resuspended in 300 µL Tag-Buffer (20 mM HEPES pH7.2, NaCl 300mM, spermidine 0.5mM, BSA 1%, NP-40 0.01%, digitonin 0.01%, MgCl2 10 mM) and incubated for 1 h at 37°C. Tagmentation was stopped by addition of one volume of 1x Diluted Nuclei Buffer (DNB, 10X Genomics) supplemented with 2% BSA, 12.5 mM EDTA. The nuclei were then centrifuged at 1300g, 4 min at 4°C and washed twice with 200 µL 1x DNB supplemented with 2% BSA. The nuclei were resuspended in 10-70 µl of DNB + 2% BSA. If the sample did not show nuclei aggregates, nuclei were loaded on a 10x Chromium system using 10x Chromium Single Cell ATAC–Seq kit v2 (10X Genomics) as described16. Final library amplification with 15 PCR cycles was performed according to the Chromium Single Cell ATAC Library kit manual. snCUT&Tag libraries were sequenced on a NovaSeq 6000 (Illumina) in PE50 mode.