Suz12 or Eed deletion results in marked delay of mammary ductal outgrowth and perinatal lethality

Mammary gland development proceeds through distinct phases that include puberty and cycles of pregnancy, lactation, and involution. Suz12 expression, like Ezh2 [16,17], was detected at all stages of mammary gland development (S1A Fig) but was particularly high during puberty (4–6 week old mice). To examine the role of Suz12 in the mammary gland, we crossed mice bearing floxed Suz12 alleles with Cre transgenic mice that express Cre under control of the mouse mammary tumor virus promoter (MMTVcre). Genotyping of offspring revealed that two-thirds of MMTVcre^T/+Suz12^fl/f mice did not survive to weaning (S1 Data), and examination of newborn pups revealed abnormal lung development (S1B Fig), likely due to activity of MMTVcre in this tissue [19], and consistent with the reported role for PRC2/Ezh2 in lung [20].

MMTVcre^T/+Suz12^f/f mice that survived beyond birth appeared normal and did not differ from wild-type (Wt) mice with respect to bodyweight (S1C Fig). Examination of whole mounts and histological sections of mammary glands from MMTVcre^T/+Suz12^f/f mice during puberty revealed a heterogeneous phenotype. Some glands were indistinguishable from Wt or heterozygous littermates, while others comprised a small ductal tree, characteristic of the rudimentary ductal tree found in newborn mice (n = 4) (Fig 1A and S1D Fig). These small but otherwise normal mammary glands indicate that gene deletion leads to severe impairment of ductal growth during puberty [21,22]. Notably, Suz12 mRNA expression in MMTVcre^T/+Suz12^f/f and MMTVcre^T/+Suz12^f/+ glands was found to be comparable (S1E Fig), demonstrating that these ductal structures were derived from epithelial cells in which Suz12 had not been deleted. Moreover, protein expression of Ezh2, Suz12, and H3K27me3 was retained in sections from 5–6 week old MMTVcre^T/+Suz12^f/f mammary glands, indicative of strong selection against Suz12-deleted cells and retention of a functional PRC2 complex in the mammary epithelium (Fig 1B). Cell fate was also retained as assessed by immunostaining for estrogen receptor (ER), progesterone receptor (PR), and forkhead box A1 (Foxa1) (S1F Fig).

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Fig 1. Deletion of Suz12 selects for mammary outgrowths composed of cells that retain PRC2 function.

Representative images of whole mounts, HE stained sections (A), and immunohistochemical staining for Suz12, Ezh2, and H3K27me3 (B) in mammary glands from 6 week old MMTVcre^T/+Suz12^f/f and control mice. Arrows in whole mounts indicate the leading edge of the mammary epithelium. Isotype-control stained sections are shown in the inset. Scale bars: 4 mm (whole mounts, HE) and 50 μm (immunohistochemistry). Ezh2, Enhancer of Zeste homolog 2; HE, hematoxylin–eosin; H3K27me3, histone 3 lysine 27 trimethylation; PRC2, Polycomb repressive complex 2; Suz12, Suppressor of Zeste 12 protein homolog.

https://doi.org/10.1371/journal.pbio.2004986.g001

We next determined whether deletion of the second core subunit of canonical PRC2 member Eed produced a similar phenotype to deletion of Suz12. As seen with Suz12 deletion, conditional deletion of Eed with MMTVcre resulted in perinatal lethality of mice (S1 Data), and surviving mice appeared normal, except for a pronounced absence or delay in growth of the ductal tree (S2A Fig). Similar to Suz12 deletion, Eed mRNA expression was found to be indistinguishable between mammary epithelium from MMTVcre^T/+Eed^f/f and Wt mice (S2B Fig). In addition, Eed, Ezh2, and H3K27me3 proteins were detected by immunofluorescence (IF) and immunohistochemistry (IHC) at comparable levels to those in Wt mammary glands (S2C and S2D Fig). Taken together, these results suggest that canonical PRC2 complexes are critical for the proliferation and/or survival of MECs, and cells deleted for PRC2 function cannot contribute to the developing mammary gland.

Suz12 is required for the integrity of PRC2 and histone H3K27me2/me3 in MECs

To investigate which MEC subsets were affected by Suz12 loss, we acutely deleted Suz12 using the inducible and conditional Rosa26-creERT2 (R26creERT2) mouse model. R26creERT2^KI/+Suz12^f/f mice injected with tamoxifen die within 2 weeks because of hematopoietic failure (personal communication, S. Lee to E. Michalak), thus precluding the use of this model for in vivo studies. Basal/MaSC-enriched (basal, CD29^hiCD24⁺), committed luminal progenitor (CD29^loCD24⁺CD14⁺), and mature luminal (CD29^loCD24⁺CD14⁻) cell subsets were sorted from R26creERT2^KI/+Suz12^f/f and control mice and subjected to in vitro colony-forming assays to determine the effect of Suz12 deletion on the activity of mammary progenitor cells following induction by 4-hydroxytamoxifen (4OHT). Consistent with expression of Suz12 in all mammary epithelial subsets (Fig 2A), we observed fewer colonies in 2D colony forming assays on irradiated feeder cells (Fig 2B) upon induction of Suz12 deletion. In contrast to the hematopoietic system, in which loss of one allele of Suz12 [14] or Eed [23] enhances the activity of stem cells, deletion of one Suz12 allele did not affect the clonogenic activity of progenitor cells (Fig 2B). Unsorted MECs efficiently deleted Suz12 and yielded sufficient protein for western blot analysis (Fig 2C). As expected, Ezh2 protein was completely lost upon Suz12 deletion, while Ezh2 mRNA levels were not reduced (S3A Fig), suggesting rapid Ezh2 protein degradation leads to the absence of functional PRC2 upon Suz12 deletion. This is supported by the loss of H3K27me2 and H3K27me3 that accompanies Suz12 deletion (Fig 2C). Conversely, expression of the active H3K27me1 mark [4] did not change. No change in cleaved poly ADP ribose polymerase (PARP) was detected, suggesting that Suz12-deleted MECs do not undergo appreciable apoptosis. However, we observed increased expression of cyclin-dependent kinase p16 and p19 alternate reading frame (p19Arf), consistent with a known role for Ezh2/PRC2 in regulating cdkn2a gene expression (S3B Fig). These data suggest that Suz12 exerts an essential function in the mammary gland through maintenance of functional tri- as well as dimethylation of target loci.

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Fig 2. Suz12 is essential for MaSC and progenitor cell activity and integrity of the PRC2 complex.

(A) Western blot analysis of PRC2 proteins and histone marks in basal/MaSC-enriched (Basal, CD29^hiCD24⁺), committed luminal progenitor (CD29^loCD24⁺CD14⁺), and mature luminal cells (CD29^loCD24⁺CD14⁻) populations sorted from 10 week old C57BL/6 mice. Molecular mass in KDa of the protein ladder are shown on the left-hand side. (B) Representative images of Giemsa-stained colonies (left) and quantification of colonies (right) grown from sorted epithelial populations from R26creERT2^KI/+Suz12^f/f mice and littermate controls in the presence or absence of 4OHT. Basal, luminal progenitor, and mature luminal cell populations (defined in A) were sorted from R26creERT2^KI/+Suz12^f/f mice and control genotypes and 100 cells plated onto irradiated 3T3s. Colony formation was assessed 1 week following addition of 4OHT to induce Suz12 deletion. Mean ± S.E.M. (n = 3–5 independent mice per genotype performed in duplicate). ** P < 0.01 for KI/+ f/f compared with all other genotypes (one-way ANOVA for multiple comparisons). Scale bars: 2 mm. (C) Western blot analysis of protein expression in MECs from R26creERT2^KI/+Suz12^f/f mice and the indicated control genotypes following addition of 4OHT to induce Suz12 deletion on day 2. Cells were cultured for 1 week prior to preparation of protein lysates. Molecular mass in KDa of the protein ladder are shown on the left. Individual quantitative observations can be found in S6 Data. 4OHT, 4-hyrdoxytamoxifen; Ezh2, Enhancer of Zeste homolog 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3K27me1, histone 3 lysine 27 monomethylation; H3K27me2, histone 3 lysine 27 dimethylation; H3K27me3, histone 3 lysine 27 trimethylation; MaSC, mammary stem cell; MEC, mammary epithelial cell; PRC2, Polycomb repressive complex 2; Suz12, Suppressor of Zeste 12 protein homolog.

https://doi.org/10.1371/journal.pbio.2004986.g002

Suz12 deletion diminishes mammary organoid proliferation

To develop a system more amenable to molecular studies, we employed a 3D mammary organoid system, in which organoids grown from single cells in defined medium recapitulate many features of mammary tissue architecture and function [18]. Single basal or luminal progenitor cells sorted from R26creERT2^KI/+Suz12^f/f mice were plated, and deletion of Suz12 was induced by addition of 4OHT on day 1. Suz12 loss resulted in diminished numbers and smaller basal- and luminal progenitor–derived organoids over a 2 week culture period. Notably, PCR analysis of the resulting organoids revealed that selection against cre-mediated excision of both floxed alleles of Suz12 had occurred (S3C Fig). To circumvent this issue, small organoids were allowed to form before induction of deletion. Indeed, addition of 4OHT on day 4 resulted in smaller cystic-like organoids (Fig 3A and 3B) composed of Suz12-deleted cells (Fig 3C and S3D and S3E Fig) that had markedly reduced levels of H3K27me3 (S3E Fig). There were no notable differences in cleaved caspase 3 (CC3) in 2 week old Suz12-deleted organoids, but rather, a striking decrease in proliferative (Ki67⁺) cells was evident (Fig 3D). Consistent with the idea that cells lacking Suz12 are nonproliferative, repassaging resulted in the expansion of rare cells that had escaped Suz12 deletion (S3F and S3G Fig). Together, these data suggest that loss of Suz12 reduces the fitness and proliferation of mammary organoids.

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Fig 3. Suz12 loss perturbs the growth of mammary organoids.

(A) Representative best-focus Z stack and enlarged inset images of 2 week old organoids grown from single basal or luminal progenitor cells from R26creERT2^KI/+Suz12^f/f mice. Organoids were left untreated or treated with 4OHT on day 4 of culture. Scale bars: 200 μm and 40 μm (inset). (B) Quantification of organoid proliferation in 14–16 day old cultures from R26creERT2^KI/+Suz12^f/f mice or control mice described in (A), as measured by the cell titer-glow cell viability assay. Values were normalized to the average of the conditions per experiment. Mean ± S.E.M. (basal, n = 4–5 experiments; luminal, n = 6–7 experiments). *** P < 0.0001 (t test). (C) Image of genotyping PCR performed on 2 week old organoids described in (A) to distinguish the Wt, floxed (flox), and recombined (del) Suz12 alleles in basal- or luminal-derived organoids derived from 2 independent R26creERT2^KI/+Suz12^f/f mice. Controls included no DNA (left) and Suz12 Wt DNA (right). The size in bp of the DNA ladder is shown on the left-hand side. (D) Immunohistochemical staining for CC3 and Ki67 on 2 week old organoids from R26creERT2^KI/+Suz12^f/f mice or control mice, treated with 4OHT on day 4 of culture. Arrowheads indicate CC3-positive cells. Scale bars: 400 μm. Individual quantitative observations can be found in S6 Data. 4OHT, 4-hydroxytamoxifen; CC3, cleaved caspase 3; Suz12, Suppressor of Zeste 12 protein homolog; Wt, wild-type.

https://doi.org/10.1371/journal.pbio.2004986.g003

Suz12 loss results in changes to chromatin accessibility and gene transcription

To determine potential target genes that are dependent on Suz12 and therefore canonical PRC2 function, we performed RNA sequencing (RNA-seq) analysis of Suz12 Wt or Suz12-deleted basal- and luminal-derived mammary organoids grown from R26creERT2^KI/+Suz12^f/f mice. Suz12 was significantly down-regulated in both populations (Fig 4A, S4A Fig and S2 Data), and deletion of exon 5 was confirmed at the targeted locus (S4B Fig). In both basal- and luminal-derived organoids, the majority (>86%) of differentially expressed (DE) genes were up-regulated rather than down-regulated upon Suz12 deletion, consistent with the repressive role of PRC2 (Fig 4A, S4A and S4C Fig). A large overlap was apparent between up- and down-regulated DE genes (S4C Fig), as well as a strong correlation with gene expression changes (S4D Fig) in Suz12-deleted basal- versus luminal-derived organoids. This is consistent with a global effect of Suz12 deletion on PRC2 stability in MECs. Gene ontology (GO) enrichment analysis found that common DE down-regulated genes were significantly enriched for metabolic processes and H3K27 methylation, while common DE up-regulated genes were enriched for development and morphogenesis GO terms (S4C Fig).

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Fig 4. Suz12 deletion in basal-derived organoids results in changes in chromatin accessibility and de-repression of transcriptional programs.

(A) MD plot showing log2-fold expression changes versus average log2-expression identified by RNA-seq in Suz12-deleted basal-derived organoids compared with Wt organoids. Up- and down-regulated genes with changes significantly greater than 1.5-fold are highlighted in red and blue, respectively (Treat-FDR < 0.05). Suz12 expression is indicated. (B) MD plot showing windows identified by 150 bp tiling as significantly DA by ATAC-seq analysis of Suz12-deleted basal-derived organoids, compared with Wt average log-expression. Windows that were significantly up- or down-regulated are shown in red or blue, respectively. (C) A plot showing GC nucleotide enrichment in genomic regions of ATAC-seq reads of Suz12-deleted basal-derived organoids, compared with Wt. Shown is the average of the genome and functional DNA elements in the mouse genome. (D) MACS Peaks calling analysis of Suz12-deleted basal-derived organoids (KO), compared with Wt. (E) Box and whisker plot of DA genes found to be associated with windows found in (B) by ATAC-seq analysis of Wt and Suz12-deleted basal-derived organoids (KO), and their corresponding expression by RNA-seq analysis. Whiskers represent the 5%–95% intervals. *** P < 0.0001 (unpaired t test, Welch’s correction). (F) Venn diagram showing the overlap in up-regulated DE genes identified by RNA-seq and DA genes by ATAC-seq in Suz12-deleted basal-derived organoids. Individual quantitative observations can be found in S6 Data. ATAC-seq, assay for transposase-accessible chromatin using sequencing; DA, differentially accessible; DE, differentially expressed; FDR, false discovery rate; KO, knockout; MACS, model-based analysis of ChIP-seq; MD, mean-difference; RNA-seq, RNA-sequencing; Wt, wild-type.

https://doi.org/10.1371/journal.pbio.2004986.g004

To explore whether the changes in gene expression accompanying loss of Suz12 reflect alterations in chromatin compaction associated with loss of repressive H3K27me2/me3 marks, we performed global mapping of chromatin accessibility using ATAC-seq. Differential accessibility analysis identified 2,767 windows, of which 2,415 (87.3%) were more accessible upon Suz12 deletion (Fig 4B and S3 Data). Further, 1,377 of these windows overlapped with the transcription start site or gene body of 766 independent genes in Suz12-deleted basal organoids (referred to as differentially accessible [DA] genes from hereon). Nevertheless, the frequency of reads mapping to nucleosome-free regions versus mononucleosomes was not significantly altered. We observed only minor changes to insert length, suggesting no gross changes in nucleosome occupancy (S5A and S5B Fig), and a comparison of GC enrichment indicated a minor increased frequency of highly GC-rich regions, usually found at regulatory elements within promoters (Fig 4C). Moreover, model-based analysis of ChIP-seq (MACS) peaks analysis showed a small number of unique peaks upon Suz12 deletion (Fig 4D). Next, we compared the DA windows identified by ATAC-seq with gene expression from their associated gene to determine if changes in chromatin compaction elicited by Suz12 deletion were sufficient to induce changes in gene transcription. A strong correlation was evident between open chromatin and transcriptional up-regulation in Suz12-deleted basal-derived organoids (Fig 4E). However, analysis of Wt organoids did not reveal a correlation between genes assigned from ATAC-seq analysis and changes in their expression. Almost one-third (28%) of genes that became more accessible upon Suz12 deletion were significantly up-regulated by RNA-seq (Fig 4F). These 218 common genes included Cdkn2a (S6A Fig and S4 Data), a known target of Ezh2-mediated repression that encodes p16 and p19Arf, and transcription factors (TFs) involved in diverse lineage commitment, including Foxd1 and homeobox TFs (S6B and S6C Fig), and accordingly, represented sequence-specific DNA-binding GO terms (S5 Data). Notably, confocal imaging of organoids confirmed the persistence of basal and luminal cells in Suz12-deleted organoids (S6D Fig), suggesting lineage fidelity was maintained.

A similar ATAC-seq analysis was applied to Suz12-deleted luminal-derived organoids (S7A and S7B Fig), which revealed an increase in reads associated with nucleosomes (S5A and S5B Fig). These longer fragments were enriched in promoter-flanking and transcribed regions [24] consistent with increased expression of DE genes. Despite detecting many unique DA peaks (S7C Fig), particularly associated with promoters and intragenic/gene bodies (S7D Fig), the corresponding windows were less strongly associated with DE genes found by RNA-seq (S7E and S7F Fig).

Comparison of the Suz12-deleted gene expression signatures derived for both basal and luminal organoids showed that they were most similar to claudin-low and normal-like breast tumors (S7G Fig), similar to that observed for the basal/MaSC cell signature [25]. Likewise, the gene expression profile of Ezh2-deleted MECs was most concordant with claudin-low breast tumors [16]. The striking similarity between gene expression signatures of organoids lacking key PRC2 complex genes and claudin-low breast tumors—which exhibit a metaplastic, clinically aggressive phenotype in patients—suggests that PRC2 may play a role in tumor phenotype and behavior.

Ethics statement

All animal experiments were conducted using mice bred at and maintained in our animal facility according to the Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee guidelines, approval number 2017.002.

Mice

Suz12^f/f [34], Eed^f/f [35], Ezh2^f/f [36], MMTV-cre (line A) [37], and R26creERT2 [38] gene-targeted mice have been described previously. CD4cre-Eed^f/f T lymphocytes [39] were a gift from R. Allan. MMTV-cre mice were maintained as a pure strain on an FVB/N background, R26creERT2 mice were on a C57BL/6 background, and Ezh2, Eed, and Suz12 conditional knockout mice were on either a C57BL/6 or mixed FVB/N and C57BL/6 background. For analysis of postnatal day 1 lungs, adult female mice were subjected to timed pregnancies and injected with 0.2 mg progesterone on days 17 and 18 of pregnancy to delay parturition. At 19.5 days of pregnancy, pups were recovered by caesarian section and monitored for breathing for 1 hour with constant stimulation before collection of lungs into Bouin’s solution.

Mammary cell preparation and cell sorting

Primary mouse MEC cultures were isolated from both inguinal and/or thoracic mammary glands and prepared as described [17]. MEC suspensions and flow cytometry were performed as previously described [40]. Antibodies against mouse antigens were purchased from Biolegend and included CD24-PB (#101820), CD31 (#102410), CD45 (#103112), and Ter119 (#116212) conjugated to APC, CD29–FITC (#102206), and CD14-PE (#123309). Cells were sorted on a FACSAria or FACSDiva (BD PharMingen) and manually counted prior to plating on irradiated fibroblast feeders as described [40]. After 7 days, colonies were fixed and stained with Giemsa and manually counted. To induce R26creERT2-mediated deletion, 0.1 μM 4OHT was added to culture medium for 20 hours on day 1 of culture.

Organoid culture

Organoids were cultured as described previously [18] in 8 μl BME (Cultrex) drops (70 basal and 60 luminal progenitor cells per drop) on nontreated 24 well plates and covered by Advanced DMEM/F12 supplemented with growth factors excluding Wtn3a or FGF2. Rock inhibitor (Y-27632) was added to culture medium for the first 4 days, and the medium was refreshed every 2–3 days. To induce R26creERT2-mediated deletion, 0.1 μM 4OHT was added to culture medium for 16–20 hours on day 1 or day 4 of culture. Organoids were photographed using best focus projection images on the Nikon TiE System software after 12–14 days in culture. Prior to proliferation measurements, RNA-seq, or ATAC-seq analysis, 12–16 day old organoids were dissociated into single cells using TrypLE express (Thermo Fisher Scientific). Cell suspensions were mixed 1:1 with CellTiter-Glo Luminescent Cell Viability Assay (Promega) prior to detection of luminescence. Genomic DNA was extracted from organoids using DNeasy Blood and Tissue kit (Qiagen) and PCR used to distinguish the Wt, floxed, and recombined (deleted, referred to as “del”) Suz12 alleles as described [34].

Histology and whole mounting

Mice were injected with BrdU Cell Labeling Reagent (0.5 mg/10 g body weight, Amersham Biosciences) 1.5 hours prior to collection. For histology, tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Sections (5 μm) were stained with hematoxylin–eosin (HE). For whole-mount analysis, mammary glands were harvested and fixed in Carnoy’s solution (6:3:1 of 100% ethanol, chloroform, and glacial acetic acid) and stained with Carmine alum. The extent of ductal outgrowth was measured on inguinal whole mounts as the distance from the center of the lymph node to the leading edge of the ductal mass.

Immunostaining

IHC and IF were performed as described [16]. Paraffin-embedded sections (5 μm) were dewaxed in xylene and rehydrated through an alcohol series, blocked with 3% hydrogen peroxide, and subjected to antigen retrieval by boiling in 10 mM citrate buffer pH 6.0 for 30 seconds using a DAKO pressure cooker. Immunostaining was performed using the streptavidin-biotin peroxidase detection system as per the manufacturer’s instructions (ABC reagent, Vector Laboratories) and 3,3-diaminobenzidine was used as substrate (DAKO). In all cases, an isotype-matched control IgG was used as a negative control. The following antibodies were used: anti-BrdU (Bio Rad OBT0030), anti-Ezh2 (BD Biosciences #612667), anti-ERα (Santa Cruz sc-543), anti-PR (Santa Cruz sc-538), anti-Foxa1 (Abcam Ab23738), anti-H3K27me3 (Millipore #07–449), anti-Suz12 (Diagenode pAB-029-050), anti-Eed (R&D Systems AF5827-SP), anti-CC3 (Cell Signaling #9664L), and anti-Ki67 (Cell Signaling #12202S). Secondary antibodies were biotin-conjugated anti-rabbit IgG, anti-rat IgG and anti-mouse IgG (Vector Laboratories), anti-mouse alexa-488 (Invitrogen), and anti-rabbit alexa-647 (Invitrogen), and DAPI was used to detect nuclei (Thermo-Fischer Scientific). As described in [18], 3D imaging of organoids was performed on a SP8 Confocal microscope after staining with anti-cytokeratin 14 (Thermo-Fisher Scientific) and anti-cytokeratin 8/18 (TROMA-I, DSHB) antibodies.

Western blot analysis

Lysates were prepared in RIPA buffer [16], and western blotting was performed as described [17]. The following antibodies were used for western blot analysis: anti-Ezh2 (BD Biosciences #612666), anti-Suz12 (Cell Signaling 3737S), anti-Eed (Millipore 05–1320), anti-H3K27me1 (Millipore 07–448), anti-H3K27me2 (Millipore 07–452), anti-H3K27me3 (Millipore 07–449), anti-Histone 3 (Millipore 07–690), anti-β-actin (Sigma A5441), anti-Gapdh (Sigma G8795), anti-ERα (Millipore 07–690), anti-Ezh1 (Millipore 07–690), anti-PARP (Cell Signaling #9542), anti-p16 (Santa Cruz M-156), and anti-p19 (Rockland Immunochemicals #200-501-891). Secondary antibodies included HRP-conjugated anti-rabbit and anti-mouse (Southern Biotech), both 1:10,000.

ATAC-seq analysis

ATAC-seq was performed as described [24] with the following adaptations. Organoid cultures were dissociated, and 50,000 single cells were lysed and nuclei collected at 500 g for 10 minutes in lysis buffer containing 0.1% NP-40. Pelleted nuclei were tagmented with Nextera Tn5 Transposase (TDE1, Illumina FC-121-1030) for 20 minutes at 22 °C and 30 minutes at 37 °C. Transposed DNA was purified using Qiagen MinElute kit (28204) and fragments PCR amplified as described [24]. ATAC libraries were sequenced on a NextSeq using a 150H kit with 75 bp paired-end reads, and total reads were collated from 2 runs to obtain approximately 60 × 10⁶ reads per sample. Sequencing runs were pooled for each replicate and sample, and adapters were trimmed with Trim Galore! (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped to the mm10 mouse genome using bowtie2 [41], allowing for fragments <2,000 bases in length. Mitochondrial and duplicate reads were removed using Picard-Tools (http://broadinstitute.github.io/picard/), and bam files were run through macs2 (doi: 10.1186/gb-2008-9-9-r137) for peak calling using these parameters: “—nomodel—shift—75—extsize 150—qvalue 0.05.” For DA analysis, bam files were loaded in SeqMonk (v1.37.1, https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/), and probes were created using a tiling approach with 150 bp windows end to end across the genome. Raw counts were then processed through the differential gene expression pipeline within edgeR [42], and DA regions were called with an exact test and FDR < 0.05. Reads were quantified by reads per million and log2 transformed for visualizing graphically. For each gene, the 5 Kb region upstream of intron 1 was used in combination with the gene body to define the TSSs plus gene body. GC enrichment was quantified on mapped bam files using DeepTools [43] computeGCBias with an effective genome size of 2,150,570,000. These data have been deposited in the Gene Expression Omnibus (accession code GSE116431).

RNA-seq analysis

Total RNA was extracted from basal- or luminal-derived organoids grown from single sorted luminal or basal populations from the mammary glands of R26creERT2/Suz12^f/f female mice using the RNeasy Mini kit (Qiagen). Two biological replicates were prepared of the basal-derived organoids and 3 biological replicates of luminal-derived. RNA-seq was carried out on an Illumina Nextseq 500 to produce 20–65 million 80 bp reads per sample. Read pairs were mapped to the mouse genome (mm10) using the subread aligner [44] implemented in the Rsubread software package. Read counts for Entrez Genes were obtained using featureCounts [45] and its inbuilt mm10 annotation. Gene information was downloaded from the NCBI on 1 February 2017. Statistical analysis used the limma [46] and edgeR [42] software packages. Genes with at least 0.5 read counts per million (cpm) in at least 2 samples were considered to be expressed and were kept in the analysis. Immunoglobulin receptor segments, ribosomal genes, predicted and pseudogenes, and obsolete Entrez IDs were filtered out. Trimmed mean of M-values (TMM) scale normalization [47] was applied, and read counts were transformed to log2-cpm with a prior count of 3. Linear models were used to test for expression differences between 4OHT treated versus untreated samples from luminal cells and from basal cells. Each organoid sample was treated as a random block, allowing for correlation between repeats [48]. Differential expression was assessed using the Treat method [49], computing empirical Bayes moderated t statistics relative to a fold change threshold of 1.5, and allowing for an abundance trend in the standard errors and for robust estimation of the Bayesian hyperparameters [50]. The Benjamini and Hochberg method was used to control the FDR. These data have been deposited in the Gene Expression Omnibus (accession code GSE116431).

Expression profiles for breast cancer subtypes

Microarray expression profiles of breast tumors were downloaded from Gene Expression Omnibus series GSE18229 [51]. Probe intensities were normexp background corrected with offset 50 [52] and loess normalized using the limma package. Mouse Entrez Gene IDs were mapped to human using HUGO Gene Nomenclature Committee orthology predictions downloaded November 2016. Suz12-deficient expression signatures were computed for each tumor using a previously described method [25]. Briefly, the sum of products of RNA-seq log2-fold-changes with microarray log2-normalized intensities was computed for all genes DE in the Suz12 deficient basal- or luminal-derived organoids.

Quantitative reverse-transcriptase PCR (qRT-PCR) analysis

For qRT-PCR analysis, total RNA from MECs was reverse-transcribed using Superscript III (Invitrogen), and cDNA were analyzed on a LightCycler 480 (Roche). Input cDNA concentrations were normalized to GAPDH. Product accumulation was evaluated using the comparative Ct method (2^−ΔΔCT). Primer sequences were: Ezh2 For: 5′-ACATCCCTTCCATGCAACACC-3′; Ezh2 Rev: 5′-TCCCTCCAGATGCTGGTAACA-3′; Eed For: 5′-GTGAACAGCCTCAAGGAAGAT-3′; Eed Rev: 5′- ATAAGGTTACTCTGTGCTTC-3′; Gapdh For: 5′-TGACATCAAGAAGGTGGTGAAG; Gapdh Rev: AAGGTGGAAGAGTGGGAGTTGC-3′.