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Histone Demethylase KDM6A Controls the Mammary Luminal Lineage through Enzyme-Independent Mechanisms Kyung Hyun Yoo,a Sumin Oh,a,b Keunsoo Kang,c Chaochen Wang,a,d Gertraud W. Robinson,a Kai Ge,d Lothar Hennighausena Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAa; Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Chungnam, Republic of Koreab; Department of Microbiology, Dankook University, Cheonan, Chungnam, Republic of Koreac; Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAd

Establishment of the mammary luminal cell lineage is controlled primarily by hormones and through specific transcription factors (TFs). Previous studies have linked histone methyltransferases to the differentiation of mammary epithelium, thus opening the possibility of biological significance of counteracting demethylases. We have now demonstrated an essential role for the H3K27me3 demethylase KDM6A in generating a balanced alveolar compartment. Deletion of Kdm6a in the mammary luminal cell lineage led to a paucity of luminal cells and an excessive expansion of basal cells, both in vivo and in vitro. The inability to form structurally normal ducts and alveoli during pregnancy resulted in lactation failure. Mutant luminal cells did not exhibit their distinctive transcription factor pattern and displayed basal characteristics. The genomic H3K27me3 landscape was unaltered in mutant tissue, and support for a demethylase-independent mechanism came from mice expressing a catalytically inactive KDM6A. Mammary tissue developed normally in these mice. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments demonstrated KDM6A binding to putative enhancers enriched for key mammary TFs and H3K27ac. This study demonstrated for the first time that the mammary luminal lineage relies on KDM6A to ensure a transcription program leading to differentiated alveoli. Failure to fully implement this program results in structurally and functionally impaired mammary tissue.

T

he establishment and maintenance of differentiation programs can be controlled by the status of specific histone modifications that ensure the precise execution of genetic programs. Notably, the histone methyltransferases EZH1 and EZH2, which catalyze the establishment of H3K27me3 repressive marks, and their counterparts, the H3K27me3 demethylases KDM6A (UTX) and KDM6B (JMJD3), are thought to constitute such programs. EZH1 and EZH2 have been extensively investigated using loss-offunction mouse genetics, and historically, their frequently redundant functions have been associated with the enzymatic activities of these proteins. However, more recent studies have also provided evidence of enzyme-independent functions (1–3) in addition to enzyme-dependent functions (4). Loss-of-function studies have also revealed specific roles for KDM6A, most notably in embryonic development (5); in cardiac development, where it is recruited to cardiac-specific enhancers (6); and in hematopoietic disorders (7). While in some settings KDM6A is the preponderant demethylase, in other cases, compensation by KDM6B has been observed. As with EZH2, enzyme-independent functions of KDM6A have been reported (8–10). The Kdm6a gene is located on the X chromosome, and conclusions on enzyme-independent functions were primarily drawn from studies with male cells containing a homolog on the Y chromosome, Kdm6c, which has no apparent enzymatic activity. However, in vitro enzymatic activity of KDM6C has been described (11). The sole purpose of mammary tissue is the production of milk to nourish the young. Mammary alveoli, the site of milk synthesis, are seasonal organs that develop during pregnancy under the influence of prolactin, secrete milk during lactation, and undergo apoptosis during involution upon weaning the pups (12). Prolactin controls the establishment of alveoli and their differentiation through the transcription factor (TF) STAT5 (13–17), which is

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key to the activation of mammary cell-specific genetic programs. In addition, ELF5 and GATA3 play key roles in the biology of luminal cells (18–22), and SOX9 appears to control the luminal lineage (23). Alveoli consist of two distinct cell types, luminal milk-secreting cells and basal or myoepithelial cells, both of which appear to originate from a common keratin 5 (K5)positive alveolar progenitor (24, 25). While genetic programs controlling luminal cell fate and differentiation have been well studied, less is known about the mechanisms that control the balance between basal and luminal cells. Among the signals that determine cell fates in mammary cell lineages, the Notch pathway likely plays a prominent role (26, 27), as the deletion of RBP-J, encoding the common downstream partner of all Notch intracellular domains (ICDs), and Pofut1, encoding a fucosyltransferase essential for the activity of Notch receptors, resulted in a vast expansion of basal cells characterized by the presence of cytokeratins K5 and K14 and smooth-muscle actin (SMA) (27). In vitro studies have revealed key roles for Notch signaling in

Received 17 February 2016 Returned for modification 4 March 2016 Accepted 8 May 2016 Accepted manuscript posted online 23 May 2016 Citation Yoo KH, Oh S, Kang K, Wang C, Robinson GW, Ge K, Hennighausen L. 2016. Histone demethylase KDM6A controls the mammary luminal lineage through enzyme-independent mechanisms. Mol Cell Biol 36:2108 –2120. doi:10.1128/MCB.00089-16. Address correspondence to Lothar Hennighausen, [email protected]. K.H.Y. and S.O. contributed equally to this work. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /MCB.00089-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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mammary stem cells (MaSCs) and luminal cell commitment (26), and the Notch–RBP-J pathway regulates alveoli by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. TRP63 is a definitive marker of basal cells, and its ablation resulted in impaired alveolar expansion and function during pregnancy (28), which was attributed to a loss of paracrine signaling by Ngr that activated STAT5A in luminal cells via the epidermal growth factor receptor (EGFR). Members of the family of inhibitors of differentiation (ID) also contribute to stem cell activity and differentiation choices between basal and luminal cells. ID4 is exclusively expressed in basal cells and suppresses luminal differentiation in an in vitro system (29). Overexpression of ID1 in mammary tissue of transgenic mice results in the preferential expansion of basal cells and ductal hyperplasia (30). Loss of LBH, a transcriptional cofactor highly expressed in basal cells and the MaSC population, results in delayed tissue expansion and increased luminal differentiation at the expense of basal cells (31). LBH positively induces Trp63 expression. In a quest to explore the importance of histone methyltransferases and demethylases in the establishment, expansion, and differentiation of mammary cell lineages during pregnancy, we used mouse genetics and initially inactivated the histone methyltransferase-encoding genes Ezh1 and Ezh2 in mammary stem cells. Deletion of Ezh2 did not adversely affect the genome-wide H3K27me3 landscape of alveolar cells but led to their accelerated differentiation during pregnancy (32). Mechanistically, EZH2 binds to target genes and thus controls the genomic access of EZH1, RNA polymerase II (Pol II), and STAT5 (32). Since genes key to mammary development and differentiation are bound by EZH2 but not decorated by H3K27me3 marks (32), we propose the possibility that active demethylation of these loci is an essential step in these programs. KDM6A and KDM6B are the two demethylases known to regulate H3K27me3 status, and they perform unique and redundant functions (33, 34). To interrogate the in vivo significance of KDM6A, we generated mice lacking its gene in mammary epithelium. Moreover, since enzymatic-independent functions of KDM6A have been reported (9, 10), we also analyzed mice expressing a catalytically inactive version. MATERIALS AND METHODS Mice. Kdm6af/f (10) and mouse mammary tumor virus (MMTV)-Cre transgenic mice (line A) with a mixed background (17) were used to generate mice lacking KDM6A in mammary stem cells (KDM6A knockout [KO]). All animals were handled and housed in accordance with the guidelines of the NIH, and the experiments were approved by the Animal Care and Use Committee (ACUC) of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). All the samples that were used for histological analysis, fluorescence-activated cell sorter (FACS) analysis, colony formation assay, RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) were randomly selected, but the experiments were not performed in a blind manner. Histological analysis and immunostaining. Whole mounts of mammary tissues from female virgin mice and from mice at day 13 of pregnancy (p13), p18, and day 1 of lactation (L1) were fixed in Carnoy’s mix (60% ethanol, 30% chloroform, and 10% glacial acetic acid), hydrated, and stained with carmine alum. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) by standard methods. For immunofluorescence staining, primary antibodies (anti-Ki-67 [Abcam; ab166677], anti␣-SMA [Sigma; A2547], anti-E-cadherin [BD Biosciences; 610182], anti-cleaved caspase 3 [Cell Signaling; 9661S], anti-keratin 5 [antiK5][Covance; PRB-160p], and anti-keratin 14 [anti-K14][Covance; PRB155p]) were incubated overnight at 4°C.

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MEC isolation and FACS analysis. Mammary epithelial cells (MECs) were isolated from 12-week-old female virgin mice using a standard protocol, and nonepithelial cells were removed using the EasySep mouse mammary stem cell enrichment kit (Stemcell Technology; 19757). MECs were stained with biotinylated anti-CD24 –fluorescein isothiocyanate (FITC) (BD Biosciences; 553261), anti-CD49f–R-phycoerythrin (R-PE) (BD Biosciences; 555736), and 7-amino-actinomycin D (7AAD) (BD Biosciences; 51-68981E) and were sorted into basal (CD24⫹ CD49fhi) and luminal (CD24hi CD49flo) cell populations after FACS analysis was performed, as previously described (35). qPCR. Genomic DNAs (gDNAs) were extracted from basal and luminal cells obtained from FACS sorting (Applied Biosystems; 11815-00). Twenty-five nanograms of gDNA was applied for quantitative PCR (qPCR) using the following primers: exon 24 of Kdm6a, forward, 5=-CAT CAAGAAATAAACTTCTGTTCAGT-3=, and reverse: 5=-AAAACACCCC AGTAGCCTTCAG-3=; 5= region of Kdm6a as a control, forward, 5=-CTC ATTCACAGCGAGTTCCA-3=, and reverse, 5=-TTTCAGCCACAGCAG TCATC-3=. Colony formation assay. MECs from 12-week-old nonparous female mice were plated overnight on ultra-low-attachment plates to obtain aggregates; 200 aggregates were cultured in GFR-Matrigel (BD no. 354230) with EGF medium (Dulbecco’s modified Eagle’s medium [DMEM]–F-12, 1% penicillin-streptomycin, 1⫻ ITS-X, 10 ng/ml EGF, and 0.0004% heparin) for 6 days. The medium was changed every 48 h. Colonies were fixed and stained for immunofluorescence as described previously (35). RNA-seq analysis. Distributions of mammary epithelial cells were investigated in three individual mice using FACS analysis. Basal or luminal cells from the three mice were combined. Total RNA was extracted from the combined cells, and RNA-seq was performed and analyzed as previously described (36). A Star aligner was used to map RNA-seq data to the mouse reference genome mm9 (37). Gene expression levels were calculated with Cufflinks (38). Plotly (https://plot.ly/) was used to generate the volcano blot. ChIP-seq. Frozen stored mammary tissues from female virgin and p13 mice were broken into powder with a mortar and pestle. Samples were processed as previously described (32). The following antibodies were used for immunoprecipitation: anti-KDM6A (Sigma; HPA002111) and anti-H3K4me1 (Abcam; ab-8895). Libraries for next-generation sequencing (NGS) were prepared and sequenced with HiSeq 2000 (Illumina). Active Motif was used to conduct ChIP-seq with H3K27me3 antibody (Millipore; 07-449) using pooled tissues from three WT and KDM6A KO mice at day 13 of pregnancy. ChIP-seq analysis. ChIP-seq data were mapped to mouse reference genome mm9 using Bowtie2. The total number of reads was normalized to 1.0 ⫻ 107 using HOMER (http://homer.salk.edu/homer/). Java TreeView (http://jtreeview.sourceforge.net/) was used to generate heat maps. There are two biological replicates for KDM6A ChIP-seq, and the correlation of replicates was calculated using deepTool with default parameters and Spearman correlation. KDM6A ChIP-seq data from the wild type (WT) and mutant were highly reproducible (Spearman’s correlation coefficients, 0.93 for the wild type and 0.78 for the mutant). Transplantation of embryonic mammary tissues. Embryonic mammary tissues of KDM6A knock-in (KI) mice (see below) were transplanted into the cleared fat pads of nu/nu (athymic) mice. Four weeks after transplantation, the nu/nu mice were bred and checked for a plug to obtain tissues at p13 and L1. Mammary tissues were harvested and prepared for histological analysis. Accession numbers. All the RNA-seq and ChIP-seq data sets have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE77586. GR, NFIB, and H3K27ac ChIP-seq data in mammary tissues at p13 were obtained from GSE74826. STAT5A and H3K27me3 ChIP-seq with p13 mammary tissues were deposited under accession number GSE70440. H3K4me3 ChIP-seq data were obtained from GSE48685.

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FIG 1 Impaired development of mammary epithelium in the absence of Kdm6a. (A) Western blot analysis of KDM6A during mammary development. Mammary tissues from nonparous (virgin) and pregnant (p) mice at days 6, 11, and 16 were analyzed. (B) Kdm6a mRNA levels in basal and luminal cell populations. Basal, 8.5 FPKM; luminal, 24.6 FPKM). (C) Kdm6a deletion in basal and luminal cell populations as determined by qPCR. The error bars indicate standard errors of the means (SEM) (***, P ⬍ 0.0001). (D) Mammary tissue whole mounts from 8-week-old (8w) nonparous mice, at p13, and at L1. The tissue was stained with carmine alum. Arrows, alveoli; arrowheads, main ducts. (E) Representative image of H&E-stained sections from WT and KDM6A KO tissues during mammary gland development. Arrows, luminal cells; arrowheads, myoepithelial cells; asterisk, milk globules in lumen.

RESULTS

Aberrant mammary epithelium forms in the absence of Kdm6a. KDM6A was detected in mammary tissue from nonparous mice and throughout pregnancy, albeit at lower levels (Fig. 1A). Since this study focused on the epithelial compartment of mammary tissue, we first determined Kdm6a levels in basal and luminal mammary cells that had been isolated by FACS. Kdm6a expression in the luminal compartment was 3-fold higher than in basal cells (25 fragments per kilobase per million [FPKM] versus 8 FPKM) (Fig. 1B). Next, we explored the biological functions of KDM6A in mammary epithelium of mice carrying two floxed Kdm6a alleles (10) and an MMTV-Cre transgene (17) that is active in mammary stem cells and also the luminal cell lineage (39). These mice are referred to as KDM6A KO mice. To validate that Kdm6a was absent from both the basal and luminal cell lineages, loss of Kdm6a in mutant cells was confirmed by RNA-seq (see Table S1 in the supplemental material). As both basal and luminal epithelial cells are descendants of mammary stem cells, we analyzed the degree of Kdm6a deletion (Fig. 1C). While more than 90% deletion was observed in luminal cells, the deletion efficiency was only 25% in

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the enriched basal cell population (Fig. 1C). This was a great surprise, as the MMTV-Cre line had floxed alleles reliably deleted in mammary stem cells in a large number of previously published studies. It is not clear whether transgene regulation of this line has changed over time and Cre expression is now preferentially linked to the luminal lineage or whether the particular floxed allele is resistant to deletion in mammary stem cells. Regardless, the efficient deletion of the Kdm6a gene in the luminal lineage permitted us to study the role of KDM6A in developing luminal cells during pregnancy and lactation. Next, we investigated the functional roles of KDM6A in mammary tissue at different developmental stages, i.e., nonparous mice and at day 13 of pregnancy (p13) and day 1 of lactation (L1). Whole-mount analysis revealed retarded ductal outgrowth during puberty in 8-week-old mice (Fig. 1D, top). However, complete ductal penetration of fat pads was observed in older mice, suggesting that retarded ductal outgrowth is a transient, time-restricted defect that can be easily overcome in adult mice. While the integrity of mammary ducts in KDM6A KO mice appeared overtly normal, as visualized in whole-mount analyses, H&E-stained sec-

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FIG 2 Deletion of Kdm6a prior to but not during differentiation affects mammary development. Histological analysis of mammary tissue from mice carrying two Kdm6a floxed alleles and either the MMTV-Cre transgene or the WapCre transgene. While the MMTV-Cre transgene results in deletion of floxed alleles in the mammary luminal lineage (KDM6A KO), the Wap-Cre transgene is active only in differentiating epithelial cells during late pregnancy (KDM6A WC). Tissue was harvested at day 18 of pregnancy. (Left) Low magnification. (Right) High magnification. Arrows, alveolus units; arrowheads, ducts.

tions revealed an aberrant arrangement of luminal and myoepithelial cells (Fig. 1E, top). While in wild-type tissue a single layer of luminal cells was surrounded by well-organized myoepithelial cells, disorganized multilayered cells were found in mutant tissue. Mammary alveoli sprout during pregnancy, preferentially from the tips of ductal, in particular tertiary, branches. Whole-mount analyses at p13 and L1 revealed aberrant development in mutant tissue, with alveoli budding from the main ducts (Fig. 1D, center and bottom). Histological sections not only verified the presence of alveoli originating from ducts but also established that alveoli had formed inside ducts with collapsed borders between myoepithelial and luminal cell layers (Fig. 1E, center and bottom). Dis-

placed alveoli, including those within ducts, expanded during pregnancy, with lumens filled with milk globules at parturition, demonstrating that Kdm6a is not required for alveolar differentiation (Fig. 1E, bottom). The histological defects were equivalent in the three biological replicates of each genotype. Even though mutant alveolar epithelium can overtly produce milk, mutant mice failed to nourish offspring. Based on histological investigations, structural blockage of ducts appears to be a primary reason for lactation failure. Alternatively, an abnormal composition of alveoli might disrupt their structural integrity. To determine whether the observed defects were due to the loss of Kdm6a from epithelium prior to differentiation during pregnancy, we independently deleted Kdm6a using a Wap-Cre transgene that is active in differentiated alveolar epithelium (17). Mammary development and histological appearance were normal, and dams were able to raise their litters (Fig. 2), suggesting a requirement for Kdm6a in the mammary lineage prior to and possibly during its expansion and differentiation. Mutant luminal cells display a basal shift. Normal development of ducts and alveoli requires the presence of distinct basal and luminal progenitors (40, 41), suggesting the possibility that aberrant development in mutant tissue was the result of altered or unbalanced epithelial progenitor cell populations. This was further investigated by isolation and molecular profiling of epithelial cell populations. Upon removal of hematopoietic and endothelial cells from freshly isolated cell suspensions obtained from nonparous mice, epithelial cell subpopulations were analyzed using FACS. The lineage-negative (Lin⫺) population was separated into four distinct subpopulations using antibodies against CD49f (␣6-integrin) and CD29 (24). While wild-type luminal and basal cells were distinctly segregated, mutant cells displayed an overlapping pattern (Fig. 3A). A discrete shift of luminal cells blurred their separation from basal cells. Based on these surface markers, the mutant luminal population was reduced (Fig. 3B). In contrast, an increase in MaSCs and basal cells was observed. Notably, 13% of the cells displayed a CD49f CD24 pattern that placed them into a category between basal and luminal cells in KDM6A KO mice (Fig. 3B, Inter). The shift of mutant luminal cells suggested a paucity of luminal characteristics and possibly the presence of a more basal character. This possibility was investigated through RNA-seq analyses of FACS-sorted basal and luminal cell populations (Fig. 3A and 4; see Table S1 in the supplemental material). For RNA-seq analyses, sorted cells from three individual mice were combined. First,

FIG 3 Loss of Kdm6a fails to separate basal and luminal subsets. (A) Flow cytometry analysis of MECs isolated from mammary tissue from 12-week-old nonparous WT and KDM6A KO mice. Luminal, CD24hi CD49flo; myoepithelial, CD24lo CD49fhi; stem cell enriched, CD24mid CD49fhi. The asterisk indicates intermediate cells, which do not clearly belong to the basal or luminal subset. Region A (pink), including the basal subset, and region B (aquamarine), presenting the luminal subset, were used for sorting. RNA-seq was performed with FACS-sorted cells in these regions from three individuals. (B) Percentages of four cell populations in MECs. Three individual mice from each genotype were analyzed. The error bars indicate standard deviations.

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FIG 4 KDM6A is necessary to maintain luminal cell character. (A) Numbers of genes enriched in basal and luminal cells and expressed in both (Common). Basal and luminal enriched gene sets were identified as those altered over 2-fold between basal and luminal cell populations in the wild type. (B) Percentages of differentially expressed genes in common, basal, or luminal enriched gene sets in the absence of KDM6A. (C) Hierarchical clustering of global gene expression levels in basal and luminal subsets from WT and KDM6A KO mice. The y axis shows distances based on Jensen-Shannon distances between tissue types (38). (D) Volcano blot showing genes with a ⬎2-fold increase or decrease in KDM6A KO cells. (E) GO analysis of differentially regulated genes in KDM6A KO. The GO terms were selected by association with the mammary gland (P value ⬍ 0.05; ⫺log10 P value ⬎ 1.3). Red, basal cells; green, luminal cells.

genes over 2-fold differentially expressed between wild-type basal and luminal cells were identified from a total of 17,034 genes expressed over 5 FPKM in wild-type mice (Fig. 4A). While expression of approximately 1,000 genes was enriched in the basal cell population and expression of ⬃4,070 genes was enriched in the luminal cell population, 11,960 genes were expressed in common in both cell populations. Out of the genes enriched in basal cells, the expression of 30% of the genes was increased or decreased over 2-fold in mutant cells (Fig. 4B). However, since deletion of Kdm6a in basal cells is incomplete, we propose that these changes are of an indirect nature and are possibly induced by juxtaposed mutant luminal cells. In contrast to basal cells, expression of approximately 80% of the luminal enriched genes was reduced in mutant cells. Only 17% of the genes equally expressed in basal and luminal cells were regulated in the absence of Kdm6a. In addition, hierarchical clustering demonstrated that the gene expression profile of mutant luminal cells was similar to that of wild-type basal cells (Fig. 4C). Most notably, expression of genes linked to luminal cell

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fate, such as those encoding the transcription factors STAT5A (4-fold), STAT3 (5-fold), GATA3 (3-fold), ID2 (3-fold), SOX9 (7-fold), and the progesterone receptor (5-fold), was reduced in mutant luminal cells (Fig. 4D; see Table S1 in the supplemental material). The prolactin-induced JAK2/STAT5 pathway is particularly critical for the establishment of a luminal cell lineage, and expression of PrlR and Jak2 was reduced more than 11-fold and 3-fold, respectively. Luminal cells are characterized by the presence of keratin 8 (K8) and keratin 18, and levels of the respective mRNAs were reduced 11-fold in mutant cells. Thus, the mutant luminal cell population fails to attain molecular characteristics and retains, or acquires, basal features. Gene ontology (GO) analysis validated that genes downregulated in luminal cells are associated with glandular development and cytokine signaling (Fig. 4E). Loss of Kdm6a from cancer stem cells has recently been associated with the induction of epithelial-mesenchymal transition (EMT)related genes, such as Snai1 (42). However, we have not observed induction of EMT-associated genes in mutant luminal cells.

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FIG 5 Loss of Kdm6a results in excessive establishment of basal cells. (A) Images of mammospheres taken at 0, 2, 4, and 6 days after placing primary MECs from WT and KDM6A KO mice in a three-dimensional (3D) culture system. (B) Immunofluorescence staining for Ki-67 in mammospheres at day 3. Green, E-cadherin; red, Ki-67; blue, DAPI (4=,6-diamidino-2-phenylindole). Scale bar, 20 ␮m. (C) Immunofluorescence staining for activated caspase 3 in mammospheres at day 6. The arrows indicate cells positive for activated caspase 3 in the lumens of hollow mammospheres (from WT cells). The arrowheads indicate mislocated cells stained with ␣-SMA in solid mammospheres (from KDM6A KO cells). Green, ␣-SMA; red, activated caspase 3; blue, DAPI. (D) Confocal images of K5 in mammospheres at days 1, 3, and 5. The arrows indicate K5-positive cells inside solid mammospheres (from KDM6A KO cells). Green, E-cadherin; red, K5; blue, DAPI. (E) Representative confocal images of K14, K6, and K8 in mammospheres from WT and KDM6A KO cells. The images of K14 were taken at day 3, and the images of K6 and K8 were taken at day 5. Green, E-cadherin; red, K14 (top), K6 (middle), and K8 (bottom); blue, DAPI. The arrows indicate mislocated cells stained with K14 and K6.

Aberrant basal cell proliferation and localization in mammospheres. The paucity of luminal cells, together with their failure to acquire a defining transcriptome profile, suggested that the stem cell or progenitor compartments might be affected by the absence of KDM6A. Such a notion would be in agreement with the basal characteristics of mutant luminal cells and the fact that

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normal MaSCs possess basal cell characteristics (43). To address the possibility of an altered stem cell compartment, we investigated the ability of primary mammary cells to form mammospheres (Fig. 5). Cell suspensions were prepared from wild-type and KDM6A KO mammary tissue from nonparous mice and cultured in vitro. Although both wild-type and mutant cells yielded

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mammospheres, their structures were distinctly different. As expected, mammospheres derived from wild-type cells were hollow, but those formed by mutant cells had a dense appearance without a lumen (Fig. 5A). While hollow mammospheres appear to be derived from luminal progenitors, solid mammospheres are thought to originate from basal-type progenitors (44), further supporting the idea that loss of KDM6A results in enrichment of basal progenitors. The lack of lumens in mutant mammospheres is likely the result of two mechanisms, excessive proliferation of epithelial cells and a paucity of cell death. To investigate proliferation and apoptosis of epithelial cells in mutant mammospheres, immunostaining for Ki-67 and activated caspase 3 was performed (Fig. 5B and C). Multilayered Ki-67-positive cells were observed in mutant mammospheres at day 3 and lack of activated caspase 3 staining at day 6. Notably, while wild-type mammospheres were surrounded by a single layer of ␣-SMA-positive cells, basal cell-like cells were observed inside mutant mammospheres (Fig. 5C). In addition, localization of the basal marker K5 was investigated during mammosphere formation (Fig. 5D). While in wild-type mammospheres only the single layer of basal cells was K5 positive, K5positive cells had infiltrated mutant mammospheres. In addition, expression of the basal marker K14, keratin 6 (K6) as a marker for luminal progenitors, and the luminal marker K8 was investigated (Fig. 5E). K14- and K6-positive cells were observed inside mutant mammospheres. Moreover, K8-positive cells were detected in an irregular layer of epithelial cells in the absence of KDM6A. These data suggest that loss of KDM6A induced enhanced proliferation and diminished apoptosis of epithelial cells, as well as mislocalization of basal cell-like cells. Genome-wide binding of KDM6A. ChIP-seq experiments identified genome-wide KDM6A occupancy in mammary tissue. A total of approximately 75,000 sites were occupied by KDM6A in wild-type mammary tissue. ChIP-seq experiments on KDM6A KO tissue revealed the presence of approximately 35,000 peaks with counterparts in wild-type tissue (Fig. 6A). Since these binding peaks were independent of the KDM6A status; they are either nonspecific in nature or the result of stromal cells or basal cells in which the Kdm6a gene is intact. Since 35,000 sites identified in the KDM6A ChIP-seq fell into this category, they were discarded from further analyses. Approximately 40,000 KDM6A binding sites were seen in wild-type tissue but not in mutant tissue and were therefore considered to be specific. Approximately 25,000 sites were located within promoter regions (within 700 bp upstream or downstream of the transcription start site [TSS]), with the presence of H3K4me3, and also putative enhancer regions, as they coincided with H3K4me1 and H3K27ac marks (Fig. 6B). Thirty-four percent of specific KDM6A binding sites were associated with active enhancer regions (Fig. 6B), suggesting their involvement in regulating gene expression in luminal cells. Coverage plots showed that specific KDM6A binding at active enhancers and promoters was greatly diminished in the absence of KDM6A (Fig. 6C). Genes guiding the luminal lineage are under KDM6A control. RNA-seq data demonstrated greatly reduced expression of genes encoding transcription factors and signaling molecules that are key to the progression and differentiation of the luminal lineage (Fig. 4D). Next, to explore the functional significance of KDM6A binding, we investigated gene expression levels associated with active promoters and enhancers that do or do not bind KDM6A (Fig. 6D). Approximately 75,900 active promoter and

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enhancer sites were discriminated and annotated to 14,091 genes that are expressed at over 5 FPKM. Of these genes, 6,655 were bound by KDM6A and 7,436 were not. Genes associated with KDM6A binding at active promoter and enhancer sites were significantly regulated only in luminal cells. Notably, 52% of the genes enriched in luminal cells were specifically recognized by KDM6A at active promoters and enhancers, in contrast to 24% enriched in basal cells (Fig. 6E; see Table S2 in the supplemental material). To determine whether additional transcription factors coincide with KDM6A binding, we searched for and identified binding motifs for transcription factors highly relevant to the mammary lineage, including ELF5, NFIB, and STAT5 (Fig. 6F). Next, we investigated cobinding of several key mammary factors and KDM6A with established ChIP-seq data in mammary tissue (Fig. 6G). Cobinding of these transcription factors was found at active enhancers of genes that were under positive control by KDM6A (Fig. 6H). Notably, a putative enhancer of the Sox9 gene is bound by KDM6A and several transcription factors, including STAT5 and NFIB, along with H3K27ac marks. Also genes encoding structural proteins characteristic of luminal cells, such as K18 and K8, are under KDM6A control. The putative enhancer of the Krt8 gene is also bound by KDM6A and mammary enriched transcription factors (Fig. 6H). In vivo binding of these factors is further validated by a mirror gap in the H3K27ac profile (Fig. 6H). These results suggest that KDM6A binding at enhancer regions regulates genes associated with the luminal lineage through cobinding with other mammary enriched transcription factors. Genetic validation would need to come from mice lacking specific enhancer binding sites. Integrity of the H3K27me3 status in the absence of KDM6A. KDM6A is considered the preponderant H3K27me3 demethylase, suggesting that its absence in mammary epithelium would yield an altered H3K27me3 landscape. To test this hypothesis, MECs were isolated from day 13 pregnant mice, and the H3K27me3 status was analyzed by Western blotting and ChIP-seq experiments (Fig. 7A). The Western blots revealed that overall H3K27me3 levels in MECs were not affected by the loss of KDM6A (Fig. 7A). H3K27me3 genome-wide occupancy at TSSs was investigated in wild-type and mutant mammary tissues (Fig. 7B), and no distinguishable differences were observed. In addition, ChIP-seq experiments determined equivalent H3K27me3 coverage at approximately 23,700 loci in WT and mutant tissue (Fig. 7C). Of note, the basal cell compartment, which accounts for approximately onethird of the mammary epithelium, displayed incomplete Cre-mediated loss of Kdm6a, which could contribute to some H3K27me3 marks. However, since the degrees of H3K27me3 were equivalent in WT and mutant tissues, we propose that KDM6A is not the guiding demethylase in mammary epithelium and that KDM6B (JMJD3) possibly compensates for its loss. Notably, KDM6A is not required to activate mammary cell-specific genes, and their respective loci are devoid of any H3K27me3 marks regardless of the status of KDM6A. Next, we investigated whether mammary cell-specific KDM6A binding sites coincided with H3K27me3 marks, and H3K27me3 occupancy was investigated at mammary cell-specific KDM6A binding sites (Fig. 7D). Only approximately 1% of mammary cellspecific KDM6A binding sites coincided with H3K27me3 marks (Fig. 7E). Loss of KDM6A did not alter the H3K27me3 status of genes with low H3K27me3 occupancy, such as Krt80 (Fig. 7F),

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FIG 6 KDM6A occupancy coincides with other transcription factors. (A) Percentages of specific peaks representing genuine KDM6A binding. (B) Percentages of specific KDM6A binding in active promoters and enhancers. Active promoter, H3K4me3 marks within 700 bp upstream or downstream of the TSS; silent promoter, no H3K4me3 within 700 bp upstream or downstream of the TSS; active enhancer, H3K4me1 and H3K27ac marks; silent enhancer, no H3K27ac marks but H3K4me1 marks. (C) Coverage plot of KDM6A enrichment within 1 kb upstream or downstream of specific KDM6A binding sites at active promoter and enhancer regions. Asterisks indicate the reduction of KDM6A binding at active promoters and enhancers. (D) Box plots showing the biological significance of KDM6A binding at active promoters and enhancers. ***, P ⬍ 2.2E⫺16. Median, middle bars inside the boxes; interquartile ranges (IQR), 50% of the data; whiskers, 1.5 times the IQR. (E) Numbers of genes with KDM6A binding at active promoter or enhancer regions in luminal and basal cells. These genes are among the genes enriched in the two cell types. (F) Transcription factor binding motifs enriched at specific KDM6A binding sites in active enhancer regions. (G) Coverage plot of KDM6A and mammary enriched TFs, including STAT5, NFIB, and ELF5, with H3K27ac marks. (H) Snapshots of sites recognized by KDM6A and other TFs. Asterisks indicate the absence of KDM6A binding at active enhancers. Different ChIP-seq tracks were labeled in different colors as indicated. Numbers displayed the scale of each ChIP-seq track.

which is specifically expressed in luminal cells. Likewise, loss of KDM6A did not yield elevated H3K27me3 levels at genes highly enriched for H3K27me3, such as the Hoxa locus (Fig. 7F). These results demonstrate that global and gene-specific H3K27me3 patterns are likely independent of KDM6A. Demethylase-independent function of KDM6A in mammary epithelium. Since the presence of KDM6A was not required for a balanced H3K27me3 landscape in mammary tissue, its function in mammary development might be independent of its demeth-

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ylase activity. This was investigated in mice carrying point mutations in the Kdm6a gene (KDM6A KI) that yielded an enzymedead protein (10). Mammary anlagen from all genotypes was transplanted into cleared fat pads from adult recipients, and the mammary tissue was analyzed at p13 and immediately following delivery (Fig. 8B). The histological appearances of KDM6A KI and wild-type tissue were indistinguishable. To determine whether enzyme-dead KDM6A KI adversely affects luminal characteristics, mammary tissue from pregnant mice and at parturition was

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FIG 7 H3K27me3 levels are independent of KDM6A. (A) Western blot for H3K27me3 in MECs isolated from mammary tissue at p13 from WT and KDM6A KO (KO) mice. Histone extracts were prepared from MECs and mouse embryonic fibroblasts (MEFs) from WT (PC, positive control) and EZH2 KO (NC, negative control) mice to confirm the position of the H3K27me3 band. (B) Coverage plot of H3K27me3 levels at TSSs in WT and KDM6A KO tissues. (C) Heat map of global H3K27me3-enriched sites within 10 kb upstream or downstream of the binding site in WT and KDM6A KO tissues. (D) Heat map of KDM6A and H3K27me3 enrichment within 1 kb upstream or downstream of the binding site at specific KDM6A binding sites. (E) Venn diagram showing the numbers of overlapping binding sites between H3K27me3 and KDM6A. (F) Genome browser snapshots of KDM6A and H3K27me3 enrichment at the Hoxa and Krt80 loci.

stained for markers characteristic of basal and luminal cells (Fig. 8C). While ducts and alveoli were characterized by a single layer of luminal cells surrounded by a single layer of basal cells in wild-type and KDM6A KI tissues, extensive expansion of basal cells was observed in the absence of KDM6A. In addition, within mutant alveoli, basal cell-like cells were interspersed with luminal cells, something not seen in wild-type and KDM6A KI tissues. This provides evidence that the demethylase activity is dispensable for KDM6A function in mammary epithelium. Aberrant expansion of basal cell-like cells does not induce basal tumors. Since the excessive expansion of the basal compartment during pregnancy occurs in the absence of KDM6A, we asked whether this was a transient feature or would be retained after weaning, possibly resulting in basal tumors. This question was initially addressed in mammary tissue that had gone through

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a full pregnancy and lactation followed by 3 weeks of involution and remodeling (Fig. 9A). Complete remodeling occurred in both wild-type and KDM6A KO mammary tissue and was accompanied by the loss of the entire alveolar compartment. Thus, the massive expansion of the basal compartment during pregnancy is temporarily restricted and completely remodeled once pregnancy-dependent hormonal stimuli cease. Since the mammary epithelium transiently proliferates and differentiates with each estrous cycle (45), we tested whether the continuous cyclical hormonal stimulation would result in a permanent accumulation of the basal epithelium in the absence of KDM6A and possibly in basal tumors. To investigate this, mammary tissue from 14-month-old female mice was analyzed (Fig. 9B). Whole-mount evaluation did not reveal any differences between wild-type and mutant mammary ducts. These data indicated that loss of KDM6A was relevant to luminal cell differentiation.

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FIG 9 Hormone dependency expansion of basal development. (A) Representative H&E-stained sections obtained from mammary tissue at day 21 of involution. (Top) Low magnification. (Bottom) High magnification. (B) Representative sections of whole-mount mammary glands from 14-month-old nonparous mice.

FIG 8 Normal mammary development in the presence of an enzymatically inactive KDM6A. (A) Diagram of enzyme-dead KI and KO in the Kdm6a gene. The KI allele carries two mutations (H1146A and E1148A) in exon 24 of the Kdm6a gene, and the resulting protein has no enzymatic activity (10). (B) Histological analysis of WT, KDM6A KI, and KDM6A KO mammary tissue at p13 (top) and L1 (bottom). Arrowheads, ducts; arrows, alveoli; asterisks, milk globules. (C) Immunofluorescence staining for K14 in mammary tissues from nonparous mice and at p13 and L1. The asterisks indicate disorganized luminal layers in ducts. The arrows show disorganized myoepithelial layers surrounding luminal cells. Green, E-cadherin; red, K14; blue, DAPI.

DISCUSSION

In vitro systems have provided evidence of the importance of KDM6 and its demethylase activity in establishing gene expression programs specifying lineage commitment (46). However, its in vivo roles, in particular, the specific contribution of the enzymatic activity, are less clear and possibly more nuanced. While enzymatically active KDM6A is encoded on the X chromosome, the gene encoding an apparently enzyme-dead paralogue, KDM6C, is located on the Y chromosome. While Kdm6a homozygous mutant females display severe and lethal developmental defects, males are only mildly affected (8–10, 47). In light of emerging evidence of in vitro demethylase activity of KDM6C (11), the in vivo importance of KDM6A demethylase-dependent and -independent functions needs to be revisited. We have now investigated the impact of KDM6A in the establishment of mammary tissue during pregnancy using mice lacking Kdm6a, preferentially in the luminal cell lineage, and in mice ex-

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pressing a catalytically inactive KDM6A (10). The presence of KDM6A is critical for creating a genuine luminal genetic program, and in its absence, mammary luminal epithelium is coerced to retain basal characters and structurally abnormal ducts, and nonfunctional alveoli are formed. KDM6A functions independently of the enzymatic activity, and mammary development is normal in the presence of a catalytically inactive KDM6A. ChIP-seq data demonstrated that KDM6A binds, in concert with other mammary enriched transcription factors, to enhancers of key genes controlling the luminal lineage and regulates their expression. KDM6A plays an essential role in somatic cell reprogramming and muscle regeneration in a demethylase activity-dependent manner (4, 48). In contrast, various lineage establishments require KDM6A protein but not its demethylase activity of KDM6A, including embryonic stem cell differentiation, Caenorhabditis elegans development, and early embryonic development (10, 49, 50). The role of KDM6A in regulating gene expression, aside from demethylating histones, is still not understood. KDM6A is an exclusive subunit in the MLL3/MLL4 complex, a histone modifier essential for shaping lineage-specific enhancers during cell differentiation (1, 51). Our data also indicated that KDM6A worked on enhancers in luminal cells. It is possible that KDM6A depletion impaired MLL3/MLL4 complex recruitment onto these enhancers, resulting in failed enhancer establishment. On the other hand, KDM6A has been reported to interact with a Brg1-containing SWI/SNF remodeler complex (6, 52). KDM6A may facilitate uncovering of chromatin by the remodeler complex when enhancer activation is needed. Without KDM6A, cofactors

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were no longer able to bind undisclosed enhancers during cell differentiation. However, further studies are needed to reveal the mechanisms in detail. The balanced expansion of the basal and luminal lineages is disrupted upon deletion of Kdm6a from the mammary luminal epithelium and results in the disproportionate expansion of the basal compartment and a paucity of genuine luminal cells. A shift in, or even the paucity of, cell populations in the absence of KDM6A has also been observed in other systems, especially in the T cell compartment (53, 54). Of note, in these cases, the H3K27me3 demethylase activity of KDM6A appears to be required for its function. T follicular helper cells are particularly sensitive to the presence of KDM6A, which asserts its function through its enzymatic activity (55). Cardiac development is severely impaired in the absence of KDM6A and partially dependent on its enzymatic activity (5, 6). Since genome-wide and gene-specific H3K27me3 occupation was not adversely affected by the absence of KDM6A, we propose that KDM6A is not the defining demethylase in mammary tissue. Experiments with mammary tissue lacking both KDM6A and KDM6B are needed to determine the degree of redundancy between the two demethylases. Of note, methylation and demethylation of H3K27 might not be major developmental drivers in mammary epithelial cell lineages, as key genes linked to epithelial development and differentiation are completely devoid of H3K27me3 marks, both in wild-type cells and in cells lacking EZH1/2 (32) and KDM6A, as shown in this study. Rather, we propose, based on ChIP-seq experiments, that KDM6A is a cofactor of mammary enriched transcription factors binding to enhancers of key genes, such as Sox9, required for the complete establishment of the luminal cell lineage. The most conspicuous feature of mammary epithelium lacking KDM6A was the striking expansion of basal cells, especially during pregnancy. This aberrant developmental process is under hormonal control and distinctly reversible, as these structures are being remodeled and vanish in the involution process. Thus, the absence of KDM6A from mammary luminal cells does not result in oncogenic transformation of mammary epithelium. In contrast, in T cell acute lymphoblastic leukemia (T-ALL), KDM6A is considered a tumor suppressor (33, 34). While in bona fide mammary epithelium KDM6A controls the balance of the two lineages, it appears to have different roles in breast cancer. Experimental reduction of KDM6A can result in reduced growth (56), and also in EMT-mediated breast cancer stem cell properties (42). In the context of specific breast cancer cell lines, KDM6A acts as a negative regulator of genes key to repressing EMT. In contrast to the biology of mammary luminal cells, the expansion of basal cells is less well understood. The transcriptional coactivator LBH is required for the establishment of the basal lineage (31), and Brca1 haploinsufficiency biases luminal cells toward basal cells (57). Intriguingly, experimental loss of Notch signaling results in expansion of basal cells (27) reminiscent of that observed in the absence of KDM6A. Expression of the Notch genes is greatly reduced in the luminal cell population in the absence KDM6A, suggesting a link between these two key signaling pathways. This study highlights the essential requirement for KDM6A in establishing a balanced population of basal and luminal mammary cells. Specifically, KDM6A is an integral part of enhancers that activate luminal-cellspecific genetic programs, including transcription factors and differentiation markers.

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ACKNOWLEDGMENTS We thank Harold Smith from the NIDDK Genomics Core for reliable and fast NGS. We thank Chul Min Yang for discussions and analysis of RNAseq data for the study. This work was funded by the IRP of the NIDDK/NIH. K.H.Y. designed the project, conducted mouse work, histology, RNAseq, and ChIP-seq, analyzed data, prepared figures, and wrote the manuscript. S.O. analyzed NGS data and prepared the relevant figures. K.K. analyzed NGS data. K.G. and C.W. provided Kdm6a mutant mice (floxed and KI) and edited the manuscript. G.W.R. conducted mammary transplants and histology experiments. L.H. designed the project, analyzed data, and wrote the manuscript. We declare no conflict of interest.

FUNDING INFORMATION This work, including the efforts of Kyung Hyun Yoo, Sumin Oh, Keunsoo Kang, Chaochen Wang, Gertraud Wasner Robinson, Kai Ge, and Lothar Hennighausen, was funded by the Intramural Research Program (IRP) of the National Institutes of Health.

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