Histone methyltransferases and demethylases reversibly modulate histone lysine methylation, which is considered a key epigenetic mark associated with gene regulation. Recently, aberrant regulation of gene expression by histone methylation modifiers has emerged as an important mechanism for tumorigenesis. However, it remains largely unknown how histone methyltransferases and demethylases co-regulate transcriptional profiles for cancer cell characteristics. Here, we show that in breast cancer cells, the histone H3 lysine 27 (H3K27) demethylase UTX (also known as KDM6A) positively regulates gene expression programs associated with cell proliferation and invasion. The majority of UTX-controlled genes, including a cohort of oncogenes and pro-metastatic genes, are co-regulated by the H3K4 methyltransferase mixed lineage leukemia 4 (MLL4, also called ALR, KMT2D, and MLL2). UTX interacted with a C-terminal region of MLL4. UTX knockdown resulted in significant decreases in the proliferation and invasiveness of breast cancer cells in vitro and in a mouse xenograft model. Such defective cellular characteristics of UTX-depleted cells were phenocopied by MLL4 knockdown cells. UTX-catalyzed demethylation of trimethylated H3K27 and MLL4-mediated trimethylation at H3K4 occurred inter-dependently at co-target genes of UTX and MLL4. Clinically, high levels of UTX or MLL4 were associated with poor prognosis in breast cancer patients. Taken together, these findings uncover that coordinated regulation of gene expression programs by a histone methyltransferase and a histone demethylase is coupled to the proliferation and invasion of breast cancer cells.

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

CtIP interacts with a group of tumor suppressor proteins including RB (retinoblastoma protein), BRCA1, Ikaros, and CtBP, which regulate cell cycle progression through transcriptional repression as well as chromatin remodeling. However, how CtIP exerts its biological function in cell cycle progression remains elusive. To address this issue, we generated an inactivated Ctip allele in mice by inserting a neo gene into exon 5. The corresponding Ctip ؊/؊ embryos died at embryonic day 4.0 (E4.0), and the blastocysts failed to enter S phase but accumulated in G 1 , leading to a slightly elevated cell death. Mouse NIH 3T3 cells depleted of Ctip were arrested at G 1 with the concomitant increase in hypophosphorylated Rb and Cdk inhibitors, p21. However, depletion of Ctip failed to arrest Rb ؊/؊ mouse embryonic fibroblasts (MEF) or human osteosarcoma Saos-2 cells at G 1 , suggesting that this arrest is RB dependent. Importantly, the life span of Ctip ؉/؊ heterozygotes was shortened by the development of multiple types of tumors, predominantly, large lymphomas. The wild-type Ctip allele and protein remained detectable in these tumors, suggesting that haploid insufficiency of Ctip leads to tumorigenesis. Taken together, this finding uncovers a novel G 1 /S regulation in that CtIP counteracts Rbmediated G 1 restraint. Deregulation of this function leads to a defect in early embryogenesis and contributes, in part, to tumor formation.

BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen genes, including angiopoietin-1 (ANG1), a secreted angiogenic factor, are corepressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3D culture. BRCA1, CtIP, and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in the ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells that form a capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization, and overexpressed ANG1. These results suggest that, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment.

Cumulative evidence indicates that breast cancer-associated gene 1 (BRCA1) participates in DNA damage repair and cell-cycle checkpoint control, serving as a tumor susceptibility gene to maintain the global genomic stability. However, whether BRCA1 has a direct role in cell proliferation and differentiation, two key biological functions in tumorigenesis, remains unclear. Here we demonstrate BRCA1 mediates differentiation of mammary epithelial cell (MEC) for acinus formation by using the in vitro 3D culture system. Reduction of BRCA1 in MEC by RNA interference impairs the acinus formation but enhances proliferation. Such aberrations can be rescued by expression of wild-type BRCA1 as well as a mutant at the RAD50-binding domain but not at the C-terminal BRCT domain, suggesting that the C-terminal BRCT domain has a critical role in these processes. Consistently, depletion of BRCA1 up-regulates the gene expression for proliferation but down-regulates that for differentiation. Moreover, application of the medium conditioned by differentiating normal MEC can reverse the phenotypes of differentiation-defective breast cancer cells bearing reduced BRCA1 functions. Our observation implies BRCA1 is involved in secretion of certain paracrine͞autocrine factors that induce MEC differentiation in response to extracellular matrix signals, providing, in part, an explanation for the etiological basis of either sporadic or familial breast cancer due to the loss or reduction of BRCA1.breast cancer ͉ tumor suppressor ͉ 3D culture ͉ matrix gel M utations in the breast cancer susceptibility gene breast cancer-associated gene 1 (BRCA1) account for up to half of hereditary breast cancer cases (1, 2) and almost all hereditary breast and ovarian cancer cases (3). Also, decreased BRCA1 expression is often found during sporadic breast cancer progression (4). Despite that a tissue-specific role of BRCA1 in breast and ovary is speculated (3), the cumulated evidence primarily converges on its universal functions, DNA damage repair, cell-cycle checkpoint control, and transcriptional regulation, to maintain genomic stability (3).The BRCA1 protein encompasses distinctive modules to interact with various proteins of diverse functions (5, 6). The N terminus possesses a RING finger domain, which dimerizes with BARD1 to exhibit ubiquitin ligase activity (5). The central region possesses two nuclear localization signals (5) and interacts with the DNA damage repair complex RAD50͞MRE11͞NBS1, transcription repressor ZBRK1, and BRCA2 (3, 6, 7). The C terminus possesses two tandem repeats of the BRCT motif, which is commonly found in DNA repair proteins (5) and interacts with CtIP, HDAC, and BACH1 (6,8,9). Loss of BRCA1 function leads to genomic instability, which diverges into two consequences (10). One is to trigger cell cycle arrest and apoptosis through activation of p53 (10). Alternatively, BRCA1 deficiency perturbs the chromosomal integrity (11) and increases the mutation rate of other genes (10). Breast tumors from the BRCA1 germ-line mutation carrie...

SUMMARY Epigenetic mechanisms regulating lineage differentiation of mammary stem cells (MaSCs) remain poorly understood. Pygo2 is a histone methylation reader and a context-dependent Wnt/β-catenin co-activator. Here we provide evidence for Pygo2’s function in suppressing luminal/alveolar differentiation of MaSC-enriched basal cells. We show that Pygo2-deficient MaSC/basal cells exhibit partial molecular resemblance to luminal cells such as elevated Notch signaling and reduced mammary repopulating capability upon transplantation. Inhibition of Notch signaling suppresses basal-level and Pygo2 deficiency-induced luminal/alveolar differentiation of MaSC/basal cells, whereas activation of Wnt/β-catenin signaling suppresses luminal/alveolar differentiation and Notch3 expression in a Pygo2-dependent manner. We show that Notch3 is a direct target of Pygo2, and that Pygo2 is required for β-catenin binding and maintenance of a poised/repressed chromatin state at the Notch3 locus in MaSC/basal cells. Together, our data support a model where Pygo2-mediated chromatin regulation connects Wnt signaling and Notch signaling to restrict the luminal/alveolar differentiation competence of MaSC/basal cells.