Elevated expression of FoxM1 in breast cancer correlates with an undifferentiated tumor phenotype and a negative clinical outcome. However, a role for FoxM1 in regulating mammary differentiation was not known. Here, we identify another function of FoxM1, the ability to act as a transcriptional repressor, which plays an important role in regulating the differentiation of luminal epithelial progenitors. Regeneration of mammary glands with elevated levels of FoxM1 leads to aberrant ductal morphology and expansion of the luminal progenitor pool. Conversely, knockdown of FoxM1 results in a shift toward the differentiated state. FoxM1 mediates these effects by repressing the key regulator of luminal differentiation, GATA-3. Through association with DNMT3b, FoxM1 promotes methylation of the GATA-3 promoter in an Rb-dependent manner. This study identifies FoxM1 as a critical regulator of mammary differentiation with significant implications for the development of aggressive breast cancers.

E2F/DP transcription factors regulate cell proliferation and apoptosis. Here, we investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because we show that this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. We attribute this defect to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, our results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation.

The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype.

Cancer is characterized by aberrant patterns of expression of multiple genes. These major shifts in gene expression are believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including histone modifications. However, it is unclear whether the binding of histone-modifying proteins to genomic regions and the placing of histone modifications efficiently discriminates corresponding genes from the rest of the genes in the human genome. We performed gene expression analysis of histone demethylases (HDMs) and histone methyltransferases (HMTs), their target genes and genes with relevant histone modifications in normal and tumor tissues. Surprisingly, this analysis revealed the existence of correlations in the expression levels of different HDMs and HMTs. The observed HDM/HMT gene expression signature was specific to particular normal and cancer cell types and highly correlated with target gene expression and the expression of genes with histone modifications. Notably, we observed that trimethylation at lysine 4 and lysine 27 separated preferentially expressed and underexpressed genes, which was strikingly different in cancer cells compared to normal cells. We conclude that changes in coordinated regulation of enzymes executing histone modifications may underlie global epigenetic changes occurring in cancer.

We examined whether differences in tumor DNA methylation were associated with more aggressive hormone receptor-negative breast cancer in an ethnically diverse group of patients in the Breast Cancer Care in Chicago (BCCC) study and using data from The Cancer Genome Atlas (TCGA).

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement.

Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters.

N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m6A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m6A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6A-mRNA levels and transcriptional changes of m6A-associated genes. These results build a direct link between NO and m6A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome.

Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol's inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.

Repair of the endothelial cell barrier after inflammatory injury is essential for tissue fluid homeostasis and normalizing leukocyte transmigration. However, the mechanisms of endothelial regeneration remain poorly understood. Here we show that the endothelial and hematopoietic developmental transcription factor Sox17 promotes endothelial regeneration in the endotoxemia model of endothelial injury. Genetic lineage tracing studies demonstrate that the native endothelium itself serves as the primary source of endothelial cells repopulating the vessel wall following injury. We identify Sox17 as a key regulator of endothelial cell regeneration using endothelial-specific deletion and overexpression of Sox17. Endotoxemia upregulates Hypoxia inducible factor 1α, which in turn transcriptionally activates Sox17 expression. We observe that Sox17 increases endothelial cell proliferation via upregulation of Cyclin E1. Furthermore, endothelial-specific upregulation of Sox17 in vivo enhances lung endothelial regeneration. We conclude that endotoxemia adaptively activates Sox17 expression to mediate Cyclin E1-dependent endothelial cell regeneration and restore vascular homeostasis.

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3). The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me3. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.