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Epigenetic alterations of chromatin structure affect chromatin accessibility and collaborate with genetic alterations in the development of cancer. Lysine demethylase 4B (KDM4B) has been identified as a JmjC domain‐containing epigenetic modifier that possesses histone demethylase activity. Although recent studies have demonstrated that KDM4B positively regulates the pathogenesis of multiple types of solid tumors, the tissue specificity and context dependency have not been fully elucidated. In this study, we investigated gene expression profiles established from clinical samples and found that KDM4B is elevated specifically in acute myeloid leukemia (AML) associated with chromosomal translocation 8;21 [t(8;21)], which results in a fusion of the AML1 and the eight‐twenty‐one (ETO) genes to generate a leukemia oncogene, AML1‐ETO fusion transcription factor. Short hairpin RNA‐mediated KDM4B silencing significantly reduced cell proliferation in t(8;21)‐positive AML cell lines. Meanwhile, KDM4B silencing suppressed the expression of AML1‐ETO‐inducible genes, and consistently perturbed chromatin accessibility of AML1‐ETO‐binding sites involving altered active enhancer marks and functional cis‐regulatory elements. Notably, transduction of murine KDM4B orthologue mutants followed by KDM4B silencing demonstrated a requirement of methylated‐histone binding modules for a proliferative surge. To address the role of KDM4B in leukemia development, we further generated and analyzed Kdm4b conditional knockout mice. As a result, Kdm4b deficiency attenuated clonogenic potential mediated by AML1‐ETO and delayed leukemia progression in vivo. Thus, our results highlight a tumor‐promoting role of KDM4B in AML associated with t(8;21).

Obesity increases the risk of metabolic disorders like diabetes mellitus and dyslipidemia. However, how metabolic status is sensed and regulates cellular behavior is unclear. Utx is an H3K27 demethylase that influences adipocyte function in vitro. To examine its role in vivo, we generated mice lacking Utx in adipocytes (UtxAKO). Although all UtxAKO mice grew normally on a normal chow diet (NCD), female UtxAKO mice on a high fat diet (HFD) showed striking reductions in body fat compared to control mice (Ctrl). Gene expression profiling of adipose tissues of HFD-fed UtxAKO female mice revealed decreased expression of rate-limiting enzymes of triacylglycerol synthesis but increased expression of those of cholesterol/steroid hormone synthesis. Moreover, these animals resisted adiposity induced by ovariectomy and exhibited increased estrogen in visceral adipose tissues. Thus, upon HFD feeding, Utx regulates lipid metabolism in adipose tissues by influencing the local hormonal microenvironment. Conversely, Utx deficiency skews lipid catabolism to enhance cholesterol/steroid hormone production and repress obesity.

Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.

Understanding the molecular mechanisms that drive adipogenesis is important in developing new treatments for obesity and diabetes. Epigenetic regulations determine the capacity of adipogenesis. In this study, we examined the role of a histone H3 lysine 27 demethylase, the ubiquitously transcribed tetratricopeptide repeat protein on the X chromosome (Utx), in the differentiation of mouse embryonic stem cells (mESCs) to adipocytes. Using gene trapping, we examined Utx-deficient male mESCs to determine whether loss of Utx would enhance or inhibit the differentiation of mESCs to adipocytes. Utx-deficient mESCs showed diminished potential to differentiate to adipocytes compared to that of controls. In contrast, Utx-deficient preadipocytes showed enhanced differentiation to adipocytes. Microarray analyses indicated that the β-catenin/c-Myc signaling pathway was differentially regulated in Utx-deficient cells during adipocyte differentiation. Therefore, our data suggest that Utx governs adipogenesis by regulating c-Myc in a differentiation stage-specific manner and that targeting the Utx signaling pathway could be beneficial for the treatment of obesity, diabetes, and congenital utx-deficiency disorders.

Cellular senescence prevents the aberrant proliferation of damaged cells. The transcription factor Bach1 binds to p53 to repress cellular senescence, but it is still unclear how the Bach1–p53 interaction is regulated. We found that the Bach1–p53 interaction was inhibited by oncogenic Ras, bleomycin, and hydrogen peroxide. Proteomics analysis of Bach1 complex revealed its interaction with p19(ARF), a tumor suppressor that competitively inhibited the Bach1–p53 interaction when overexpressed within cells. Reduction of MDM2 expression in wild‐type murine embryonic fibroblasts (MEFs) did not result in slower proliferation, showing that Bach1 plays a role in keeping the proliferation of MEFs independent of MDM2. Consistent with this interpretation, expression of p21 was highly induced in MEFs when both Bach1 and MDM2 were abrogated. The level of Bach1 protein was reduced on knockdown of p53. These results suggest that p53 activation involves its dissociation from Bach1, achieved in part by the competitive binding of p19(ARF) to Bach1. The p19(ARF)–Bach1 interaction constitutes a regulatory pathway of p53 in parallel with the p19(ARF)–MDM2 pathway. (Cancer Sci 2012; 103: 897–903)

Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.

Oxidative stress contributes to both aging and tumorigenesis. The transcription factor Bach1, a regulator of oxidative stress response, augments oxidative stress by repressing the expression of heme oxygenase-1 (HO-1) gene (Hmox1) and suppresses oxidative stress-induced cellular senescence by restricting the p53 transcriptional activity. Here we investigated the lifelong effects ofBach1deficiency on mice.Bach1-deficient mice showed longevity similar to wild-type mice. Although HO-1 was upregulated in the cells ofBach1-deficient animals, the levels of ROS inBach1-deficient HSCs were comparable to those in wild-type cells.Bach1−/−;p53−/−mice succumbed to spontaneous cancers as frequently asp53-deficient mice.Bach1deficiency significantly altered transcriptome in the liver of the young mice, which surprisingly became similar to that of wild-type mice during the course of aging. The transcriptome adaptation toBach1deficiency may reflect how oxidative stress response is tuned upon genetic and environmental perturbations. We concluded thatBach1deficiency and accompanying overexpression of HO-1 did not influence aging or p53 deficiency-driven tumorigenesis. Our results suggest that it is useful to target Bach1 for acute injury responses without inducing any apparent deteriorative effect.

Cellular senescence is induced in response to DNA damage, caused by genotoxic stresses, including oxidative stress, and serves as a barrier against malignant transformation. Tumor-suppressor protein p53 induces genes critical for implementing cellular senescence. However, the identities of p53 target genes and other regulators that achieve senescence under oxidative stress remain to be elucidated. Effector genes for oxidative stress-induced cellular senescence were sought, based on the fact that transcription factor Bach1 inhibits this response by impeding the transcriptional activity of p53. pRb became hypophosphorylated more rapidly in Bach1-deficient MEFs than in wild-type cells, suggesting that pRb activation was involved in their senescence. Bach1-deficient MEFs bypassed the senescence state when the expression of a subset of p53 target genes, including p21, Pai1, Noxa, and Perp, was simultaneously reduced by using RNAi. Combined knockdown of p21 and pRb resulted in vigorous re-proliferation. These results suggest that oxidative stress-induced cellular senescence is registered by multiple p53 target genes, which arrest proliferation redundantly, in part by activating pRb. Our elucidations contrast with previous reports describing monopolistic regulations of senescence by single p53 target genes.

The tumor suppressor p53 induces cellular senescence, an irreversible form of proliferation arrest, to inhibit carcinogenesis as well as aging of organs and a body. While the major cause of cellular senescence and aging is oxidative stress, little is known about how the p53 activity is regulated under such conditions. Bach1 inhibits expression of oxidative stress responsive genes by competing with Nrf2, the key activator of oxidative stress response. Bach1 inhibits p53-dependent cellular senescence induced by oxidative stress. Bach1 forms a protein complex with p53, is recruited to p53 target genes, and inhibits their expression. These findings provide completely novel insights into how the activity of p53 is regulated under oxidative stress. Since p53 is the critical tumor suppressor with huge clinical implications, the newly identified mechanism should open new research fields.

Apoptosis, transient cell cycle arrest, and cellular senescence mediated by the tumor suppressor protein p53 are the key strategies to suppress expansion of cells with mutations. Our group has reported that transcription factor Bach1 is a crucial negative regulator of oxidative stress-induced cellular senescence (Dohi et al., Nature Struct. Mol. Biol., 2008). Bach1 forms a complex that contains p53, histone deacetylase 1 (HDAC-1) and nuclear corepressor N-coR. Bach1 is recruited to a subset of p53 target genes and contributes to impeding p53 action by promoting histone deacetylation of p53 target genes. Therefore, Bach1 may play a role in cancer transformation by inhibiting p53 activity. We have explored the possibility of Bach1 being as a positive regulator for cancer. First, we compared the timing of immortalization of wild-type and Bach1-deficient mouse embryonic fibroblasts (MEFs). Immortalization is an important step in cellular transformation and tumorigenesis, conferring upon cells the ability to grow indefinitely by bypassing cellular senescence. Bach1-deficient MEFs entered into the p53-dependent senescence state more rapidly than wild-types. However, they became immortalized in a similar kinetics as wild-types when cultured by 3T3 protocol. Thus, while Bach1-deficient MEFs were kept in senescence state longer than wild-type cells, they ultimately bypassed senescence as wild-type cells did. Inactivation of tumor suppressor genes such as p53 or p19 was observed in both types of MEFs, indicating that the p19–p53 pathway was critical for the maintenance of senescence in these cells. Next, we compared transformation of immortalized wild-type and Bach1-deficient MEFs by the activated oncogene H-RasV12. We found that formation of transformed colonies on dish and in soft agar was markedly reduced in Bach1-deficient cells as compared with wild type cells. RasV12-transduced wild-type MEFs produced apparent tumors in all athymic nude mice when injected subcutaneously, whereas Bach1-deficient cells formed significantly smaller tumors at best. Based on these observations, we suggest that Bach1 plays important roles in freeing from contact inhibition and acquiring anchorage-independent growth. To understand how Bach1 contribute to the carcinogenesis, we carried out following experiments using RasV12-expressing immortalized MEFs. Reactive oxygen species (ROS) levels were very low in Bach1-deficients. Phospholylation of ERK1/2 and H2AX was markedly reduced in Bach1-deficients. These data suggest that some intra-cellular signal pathways or DNA-repairing pathways are blocked in Bach1-deficients. We are currently identifying Bach1-target genes which are involved in these processes. Based on these observations, we concluded that Bach1 is critical for transformation induced by RasV12. Bach1 is necessary for transformation of MEFs by not only impeding the function of p53 but also acting through other pathways for transformation. We will discuss about the mechanism of transformation supposed by Bach1. Citation Information: Cancer Res 2009;69(23 Suppl):A59.

The epithelial-mesenchymal transition (EMT) is a complex biological program that allows cancer cells to aquire motility and invasiveness. Transcriptional repressor Bach1 (BTB and CNC homology 1) belongs to cap'n'collar basic leucin zipper transcription factors family and represses transcription of stress-responsive genes such as heme oxygenase-1 (Hmox-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) by binding to the MARE (Maf recognition element) sequenceses in the regulatory regions of these genes. We have recently reported that Bach1 also recruites histon deacetylase-1 (HDAC1) to a subset of p53 target genes (Perp1, p21, Noxa, Puma, PAI-1) and contributes to the inhibition of p53-dependent cellular senescence (Dohi Y. et al. Nat Struct Mol Biol. 2008;15(12):1246–54). In the present study we addressed whether Bach1 regulated cells motility and invasiveness, contributing to EMT. First, we analyzed the phenotype of Bach1-deficient (Bach1−/−) mice. We found increased spontaneous motility of immortalized embryonic fibroblasts (MEF) derived from Bach1−/− mice in the wound-healing assay. To identify differential gene expression which might explain enhanced motility of Bach−/− MEFs, we compared gene expression profiles of Bach1−/− and wild type (WT) MEFs. We found that NF-кB essential modulator, NEMO (IKBKG) was highly expressed in Bach1−/− MEFs. Mouse genome database search revealed three MARE sites located in 10 kb upstream of the first exon of NEMO gene. We confirmed that Bach1 bound to these sites in ChIP and EMSA assays and also repressed transcription of the reporter gene construct containing MARE sequences from the NEMO promoter. DNA binding activity of NF-кB, measured by EMSA, and expression of NF-кB-dependent reporter construct were enhanced in Bach1−/− MEFs. To identify Bach1 target genes downstream of NF-кB, we compared mRNA expression profiles of the Bach1−/− and WT MEFs, treated with TNF-α. Genes encoding cytokines, chemokines and matrix metalloproteinases (IL-6, CXCL-1, CXCL-2, MMP2, MMP3, MT2-MMP) were up-regulated in Bach1−/− cells. Interestingly, IL-6 mRNA level changed earlier and more intensively than other factors; moreover, we detected higher level of IL-6 in the culture supernatants from Bach1−/− MEFs. Phoshorylation of STAT3, a target of IL-6 and a regulator of many MMPs and chemokines, was enhanced in Bach1−/− MEFs. RNAi targeting of both NEMO and IL-6 significantly redused motility of Bach1−/− MEFs indicating causative role of the both factors. Thus, Bach1 may regulate cells motility by fine-tuning the expression of chemokines and matrix metaloproteinases via cross-talk with the NF-кB signal transduction pathway in IL-6 dependent manner. Next, we addressed whether the lost of Bach1 in epithelia or stroma would activate EMT program in cancer cells. RNAi targeting of Bach1 in breast cancer cell line MCF-7 resulted in lower E-cadherin expression, reduced number of cell-to-cell, cell-to-substrate contacts and increased spontaneous motility. Co-culture with Bach1−/− but not WT MEFs induced transwell migration and basement membrane matrix invasion of MCF-7 cells. In conclusion, our data indicate that Bach1 regulates cell autonomous and non-autonomous induction of chemokines and extracellular matrix degrading enzymes, inhibiting motility, invasiveness and EMT. Citation Information: Cancer Res 2009;69(23 Suppl):B7.