Polycomb Group Protein BMI1 Research Articles

UBAP2L is a novel BMI1-interacting protein essential for hematopoietic stem cell activity

AbstractMultipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L.

Transcriptional repression of Sin3B by Bmi-1 prevents cellular senescence and is relieved by oncogene activation.

The Polycomb group protein Bmi-1 is an essential regulator of cellular senescence and is believed to function largely through the direct repression of the Ink4a/Arf locus. However, concurrent deletion of Ink4a/Arf does not fully rescue the defects detected in Bmi-1−/− mice, indicating that additional Bmi-1 targets remain to be identified. The expression of the chromatin associated Sin3B protein is stimulated by oncogenic stress, and is required for oncogene-induced senescence. Here we demonstrate that oncogenic stress leads to the dissociation of Bmi-1 from the Sin3B locus, resulting in increased Sin3B expression and subsequent entry into cellular senescence. Furthermore, Sin3B is required for the senescent phenotype and elevated levels of reactive oxygen species elicited upon Bmi-1 depletion. Altogether, these results identify Sin3B as a novel direct target of Bmi-1, and establish Bmi-1-driven repression of Sin3B as an essential regulator of cellular senescence.

Prognostic value of polycomb proteins EZH2, BMI1 and SUZ12 and histone modification H3K27me3 in colorectal cancer.

Numerous changes in epigenetic mechanisms have been described in various types of tumors. In search for new biomarkers, we investigated the expression of Polycomb-group (PcG) proteins EZH2, BMI1 and SUZ12 and associated histone modification H3K27me3 in colorectal cancer. Nuclear expression of PcG proteins and histone modification H3K27me3 were immunohistochemically (IHC) stained on a tissue microarray (TMA), including 247 tumor tissues and 47 normal tissues, and scored using the semi-automated Ariol system. Tumor tissues showed higher expression of EZH2 (p = 0.05) and H3K27me3 (p<0.001) as compared to their normal counterparts. Combined marker trend analyses indicated that an increase in the number of markers showing high expression was associated with better prognosis. High expression of all four markers in the combined marker analyses was correlated with the best patient survival and the longest recurrence-free survival, with overall survival (p = 0.01, HR 0.42(0.21–0.84)), disease-free survival (p = 0.007, HR 0.23(0.08–0.67) and local recurrence-free survival (p = 0.02, HR 0.30(0.11–0.84)). In conclusion, we found that expression of PcG proteins and H3K27me3 showed prognostic value in our study cohort. Better stratification of patients was obtained by combining the expression data of the investigated biomarkers as compared to the individual markers, underlining the importance of investigating multiple markers simultaneously.

Psip1/Ledgf p75 restrains Hox gene expression by recruiting both trithorax and polycomb group proteins.

Trithorax and polycomb group proteins are generally thought to antagonize one another. The trithorax family member MLL (myeloid/lymphoid or mixed-lineage leukemia) is presumed to activate Hox expression, counteracting polycomb-mediated repression. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, whose PWWP domain binds to H3K36me3, interacts with MLL and tethers MLL fusion proteins to HOXA9 in leukaemias. Here we show, unexpectedly, that Psip1/p75 regulates homeotic genes by recruiting not only MLL complexes, but also the polycomb group protein Bmi1. In Psip1−/− cells binding of Mll1/2, Bmi1 and the co-repressor Ctbp1 at Hox loci are all abrogated and Hoxa and Hoxd mRNA expression increased. Our data not only reveal a potential mechanism of action for Psip1 in the regulation of Hox genes but also suggest an unexpected interplay between proteins usually considered as transcriptional activators and repressors.

Down Regulation of miR200c Promotes Radiation‐Induced Thymic Lymphoma by Targeting BMI1

The miR-200c has recently been implicated in the epithelial to mesenchymal transition (EMT) process by directly target the EMT related transcriptional factors ZEB1 and ZEB2. The expression of this miRNA is inversely correlated with tumorgenecity and invasiveness in several human cancers. However, little is known about the expression and targets of the miR-200c in radiation carcinogenesis. Here in this study, using a split radiation induced thymic lymphoma (RITL) model in BALB/c mice, we found that miR-200c is down-regulated in RITL samples. Cell death and apoptosis in lymphoma cells was induced by miR-200c mimic while decreased by miR-200c inhibitor. Computational analysis found a putative target site of miR-200c in the 3'UTR of one of the polycomb group (PcG) protein BMI1 mRNA, which was verified by a luciferase reporter assay. Forced over-expression of miR-200c decreased the level of BMI1 protein and moreover, over-expression of BMI1 rescued the biological effects of miR-200c, indicating BMI1 is a direct mediator of miR-200c functions. Furthermore, the BMI1 expression level was up-regulated and inversely correlated with miR-200c in RITL samples. Finally, our data also indicates that Adenovirus over-expression of pre-miR-200c reduced tumorgenesis in vivo. Taken together, we conclude that down-regulated expression of miR-200c and up-regulation of its direct target BMI1 in radiation-induced thymic lymphoma, which may indicate a novel therapeutic method for RITL through induction of miR-200c or inhibition of BMI1.

Ubiquitination and Degradation of Ribonucleotide Reductase M1 by the Polycomb Group Proteins RNF2 and Bmi1 and Cellular Response to Gemcitabine

Ribonucleotide reductase M1 (RRM1) is required for mammalian deoxyribonucleotide (dNTP) metabolism. It is the primary target of the antimetabolite drug gemcitabine, which is among the most efficacious and most widely used cancer therapeutics. Gemcitabine directly binds to RRM1 and irreversibly inactivates ribonucleotide reductase. Intra-tumoral RRM1 levels are predictive of gemcitabine’s therapeutic efficacy. The mechanisms that regulate intracellular RRM1 levels are largely unknown. Here, we identified the E3 ubiquitin-protein ligases RNF2 and Bmi1 to associate with RRM1 with subsequent poly-ubiquitination at either position 48 or 63 of ubiquitin. The lysine residues 224 and 548 of RRM1 were identified as major ubiquitination sites. We show that ubiquitinated RRM1 undergoes proteasome-mediated degradation and that targeted post-transcriptional silencing of RNF2 and Bmi1 results in increased RRM1 levels and resistance to gemcitabine. Immunohistochemical analyses of 187 early-stage lung cancer tumor specimens revealed a statistically significant co-expression of RRM1 and Bmi1. We were unable to identify suitable reagents for in situ quantification of RNF2. Our findings suggest that Bmi1 and possibly RNF2 may be attractive biomarkers of gemcitabine resistance in the context of RRM1 expression. They also provide novel information for the rational design of gemcitabine-proteasome inhibitor combination therapies, which so far have been unsuccessful if given to patients without taking the molecular context into account.

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.

MicroRNA-141 regulates BMI1 expression and induces senescence in human diploid fibroblasts

Polycomb group protein BMI1 is an important regulator of senescence, aging, and cancer. On one hand, it is overexpressed in cancer cells and is required for self-renewal of stem cells. On the other hand, it is downregulated during senescence and aging. MicroRNAs have emerged as major regulators of almost every gene associated with cancer, aging, and related pathologies. At present, very little is known about the miRNAs that regulate the expression of BMI1. Here, we report that miR-141 posttranscriptionally downregulates BMI1 expression in human diploid fibroblasts (HDFs) via a miR-141 targeting sequence in the 3′ untranslated region of BMI1 mRNA. We also show that overexpression of miR-141 induces premature senescence in HDFs via targeting of BMI1 in normal but not in exogenous BMI1-overexpressing HDFs. Induction of premature senescence in HDFs was accompanied by upregulation of p16INK4a, an important downstream target of BMI1 and a major regulator of senescence. Our results suggest that miR-141-based therapies could be developed to treat pathologies where BMI1 is deregulated.

Ubap2l-Bmi-1-Rnf2 Define a Novel Polycomb Complex Essential For Self-Renewal Of Hematopoietic Stem Cells

The polycomb group protein Bmi-1 is a well known determinant of hematopoietic stem cell function. Bmi-1-/- mice display severe hematopoietic defects, including progressive loss of hematopoietic cells from the bone marrow. Bmi-1 is dispensable for hematopoietic stem cell specification, but essential for their maintenance, an effect attributable to its ability to promote HSC self-renewal. The mechanism by which Bmi-1 regulates this process is not completely understood. Bmi-1 has been shown to repress the INK4A/ARF locus encoding the cell cycle inhibitors p16ink4a and p19arf , to interact with the E4F1 protein and to regulate the DNA damage response pathway, however experimental manipulation of these proteins/pathways only partially rescues the hematopoietic defects of the Bmi-1-/-mice. It thus appears that the mechanism by which Bmi-1 regulates HSC self-renewal remains to be determined.Towards this goal, we purified Bmi-1 containing protein complexes from cellular extracts and identified Bmi-1 interaction partners by mass spectrometry. We observed that the protein Ubap2l, which has never been shown to associate with Bmi-1 and for which no link with polycomb group protein function has been described, was consistently found in Bmi-1-containing protein complexes. Immunoprecipitation experiments revealed that Ubap2l indirectly associates with Bmi-1 via an interaction with the polycomb group protein Rnf2. We then evaluated the possibility that Ubap2l might be involved in the regulation of HSC activity. We observed that Ubap2l transcripts are more abundant in primitive HSC populations compared to total BM. CFC assays performed with BM cells infected with Ubap2l shRNAs revealed that Ubap2l knockdown causes a modest and progressive loss of progenitor activity when cells are kept in culture, with multipotent and bipotent progenitors being substantially more affected than unipotent progenitors. We transplanted these cells in mice and observed a gradual decrease in the percentage of donor derived cells expressing Ubap2l shRNAs in the peripheral blood of the recipient mice, with the most striking effect observed 16 weeks post-transplantation in the BM. Bmi-1 has been shown to regulate the proliferative capacity of both progenitor and stem cells, and its deletion in BM cells is known to dramatically reduce the reconstitution activity of these cells at early time points following transplantation. In contrast, Ubap2l appears to preferentially regulate LTR-HSC activity. We tested the effects of Ubap2l silencing on leukemic cells in vivo and observed that a reduction of Ubap2l levels in these cells had an important impact on their ability to reconstitute recipient mice, suggesting that Ubap2l also plays a role in leukemic stem cell activity. We determined if the mechanism by which Ubap2l regulates HSC activity is related to Bmi-1 function by simultaneously introducing Bmi-1 cDNA and Ubap2l shRNAs in BM cells and found that Bmi-1 is able to rescue the long-term reconstitution defect caused by Ubap2l downregulation in these cells. We observed that Ubap2l silencing does not significantly affect the expression of the known Bmi-1 targets p16ink4a and p19arf, implying that Ubap2l regulates HSC activity via a Bmi-1-dependent mechanism that does not involve repression of the INK4A/ARF locus. One explanation for the two Bmi-1 dependent mechanisms at play in the regulation of HSC activity could be that Bmi-1 is part of two separate protein complexes, each regulating different aspects of hematopoietic cell function. To test this hypothesis, we fractionated cellular extracts and were indeed able to resolve two distinct Bmi-1 containing protein complexes, distinguishable by the presence of Ubap2l.Based on the results we obtained, we propose a model in which two different Bmi-1 containing protein complexes regulate hematopoietic stem cell function. An Ubap2l-independent complex, which is most likely involved in the repression of the INK4A/ARF locus, and could be responsible for the effects of Bmi-1 on multipotent progenitors and STR-HSCs, and an Ubap2l-dependent complex, which operates via a yet to be defined mechanism unrelated to p16Ink4a and p19Arf, and would account for the effects of Bmi-1 on LTR-HSC activity. These results position Ubap2l as a key regulator of LTR-HSC activity and unveil a novel protein complex mediating the effects of Bmi-1 on LTR-HSCs. Disclosures:No relevant conflicts of interest to declare.

BMI1 Promotes Erythropoiesis Through Regulating Ribosome Biogenesis

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome of childhood manifested as macrocytic anemia with insufficient erythroid precursors in the bone marrow. Within the decade following the demonstration that mutations in the ribosomal protein gene RPS19 can lead to DBA, this disease has become a paradigm for an emerging group of pathologies (ribosomopathies) linked to defects in ribosome biogenesis. Mutations in ribosomal protein genes impair ribosome biogenesis, resulting in activation of the p53 tumor suppressor pathway, cell cycle arrest and defective erythropoiesis. While mutations in ribosomal protein genes have been found in 50-60% of DBA patients, genetic abnormalities in the remaining patients are largely unknown. Despite improvements in our understanding of the pathophysiology of DBA, the molecular basis for selective impairment of the erythroid lineage in this disorder is not understood. In particular, how ribosome biogenesis is regulated in erythroid precursors remains elusive. Our laboratory has been investigating the role of Polycomb group protein Bmi1 in regulating hematopoietic stem cell (HSC) self-renewal and lineage commitment. Recently, we found that Bmi1 is a critical downstream target of AKT signaling and AKT-mediated phosphorylation of Bmi1 inhibits HSC self-renewal (Liu et al., Science Signaling, 2012). Upon more detailed analysis of the hematopoietic phenotype of the Bmi1 knockout mice, we have observed that these mice develop macrocytic anemia and show delayed recovery following phenylhydrazine (PHZ)-induced hemolytic anemia. This phenotype suggests defective erythropoiesis and we identified that loss of Bmi1 expression results in a block in erythroid differentiation and decreased erythroid colony formation. Gene expression profiling indicated that multiple ribosomal protein genes were downregulated in Bmi1 null erythroid precursors. Moreover, we discovered that the p53 pathway is activated in Bmi1 null erythroid progenitor cells and genetic inhibition of p53 activity rescued the erythroid defects in the Bmi1 deficient mice. Thus, Bmi1 null mice recapitulate many critical features of human DBA. Furthermore, we demonstrated that BMI1 plays a critical role in human erythropoiesis as knockdown of BMI1 in human CD34+ cells decreases ribosomal protein gene expression, activates the p53 pathway, and blocks erythroid differentiation. Importantly, we observed that BMI1 expression is downregulated in bone marrow cells from some DBA patients. Thus, BMI1 plays a critical role in regulating ribosome biogenesis in erythroid precursors and BMI1 deficiency may contribute to the pathogenesis of DBA. Understanding how BMI1 regulates ribosome biogenesis and erythroid development will provide novel insight into the processes by which BMI1 and ribosomopathies contribute to the pathogenesis of DBA and potentially new targets for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Nigericin selectively targets cancer stem cells in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is prevalent in southern China, northern Africa, and Alaska. The prognosis for NPC patients at early stage is good, while it is poor for patients at late stages. Cancer stem cells (CSCs) have been proposed to be associated with tumor initiation, relapse and metastasis, and the poor prognosis of NPC likely results from residual CSCs after therapy. Study on the therapy targeting CSCs in NPC remains poor, though it received intensive attentions in other cancers. Here, we used NPC cell lines with high and low proportion of CSCs as models to explore the effect of nigericin, an antibiotic, on CSCs. We found that nigericin could selectively target CSCs and sensitize CSCs in NPC to the widely used clinical drug cisplatin both in vitro and in vivo. Moreover, downregulation of the polycomb group protein Bmi-1 may contribute to the inhibitory effect of nigericin on CSCs. Furthermore, by using the in vitro NPC cell models, we found that nigericin could significantly decrease the migration and invasion abilities, which are known to be associated with CSCs. Taken together, our results suggest that nigericin can selectively target CSCs in NPC, which could be a candidate CSCs targeting drug for clinical evaluation.

In Vivo RNAi Screen for BMI1 Targets Identifies TGF-β/BMP-ER Stress Pathways as Key Regulators of Neural- and Malignant Glioma-Stem Cell Homeostasis

In mouse and human neural progenitor and glioblastoma "stem-like" cells, we identified key targets of the Polycomb-group protein BMI1 by combining ChIP-seq with in vivo RNAi screening. We discovered that Bmi1 is important in the cellular response to the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) and endoplasmic reticulum (ER) stress pathways, in part converging on the Atf3 transcriptional repressor. We show that Atf3 is a tumor-suppressor gene inactivated in human glioblastoma multiforme together with Cbx7 and a few other candidates. Acting downstream of the ER stress and BMP pathways, ATF3 binds to cell-type-specific accessible chromatin preloaded with AP1 and participates in the inhibition of critical oncogenic networks. Our data support the feasibility of combining ChIP-seq and RNAi screens in solid tumors and highlight multiple p16(INK4a)/p19(ARF)-independent functions for Bmi1 in development and cancer.

Abstract 1794: Cross-regulation of polycomb group protein BMI1 and WNT inhibitors.

Abstract Polycomb group (PcG) proteins are evolutionarily conserved gene silencers, which determine cell fate decisions during development. These proteins are often aberrantly expressed in cancer cells. In particular, BMI1 and EZH2 are known to be overexpressed in a number of human malignancies including breast and prostate cancers. In vitro models of cancer development strongly support oncogenic role of overexpressed BMI1 in cancer and metastasis. BMI1 is known to be required for self-renewal of neural, hematopoietic, intestinal and mammary stem cells. In addition, BMI1 is downregulated in senescent cells, and this downregulation may contribute to age-related pathologies. BMI1 is also suspected to play a role in cancer stem cell development and increase stem cell-ness of tumors to promote drug resistance. Despite its role in cancer, stem cell phenotype, development and aging, very little is known about the signaling pathways that regulate the expression of BMI1. Here we report that BMI1 activates WNT pathway and upregulates its target genes such as Cyclin D1 and c-Myc. Using promoter-reporter assays, quantitative RT-PCR and Western blot analyses, we show that BMI1 auto-activates its expression via an E-box present in its promoter. Because auto-activation of BMI1 depends on E-box sequences present in its promoter, we hypothesized that BMI1 may regulate expression of c-Myc directly or indirectly via activation of WNT pathway. Indeed, our data suggest that BMI1 upregulated expression of c-Myc via activation of WNT pathway. Further dissection of this regulation revealed that BMI1 represses expression of WNT inhibitors, in particular DKK family of proteins such as DKK1, DKK2 and DKK3. Interestingly, we also found that DKK1 inhibits expression of BMI1, which also depends on E-box present in BMI1 promoter. Thus BMI1 and WNT inhibitors such as DKK1 cross-regulate each other's expression. We speculate that such regulation might favor BMI1 overexpression in cancer cells via increased WNT activity and c-Myc expression. On the other hand, increased expression of WNT inhibitors such as DKK1 might favor BMI1 downregulation in senescent and aged tissues. Thus, a fine tuned positive feedback loop controls BMI1 expression via WNT signaling pathway, and this feedback loop may be relevant to the role of BMI1 in cancer, stem cells and senescence. Citation Format: Joon-Ho Cho, Manjari Dimri, Goberdhan P. Dimri. Cross-regulation of polycomb group protein BMI1 and WNT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1794. doi:10.1158/1538-7445.AM2013-1794

Abstract 1483: Bmi1 is required for Twist1-mediated breast cancer cell invasion.

Abstract Metastasis is the spread of cancer cells from the site of primary tumors to distant organs and the establishment of secondary tumors there. It is a multistep process, and the initial step is cancer cell invasion during which cancer cells move into tissues surrounding the tumor and the vasculature. Transcription factor Twist1 has been identified as a key regulator of metastasis in a murine breast tumor model as suppression of Twist1 expression in metastatic mammary carcinoma cells specifically inhibits their ability to metastasize, and overexpression of Twist1 induces cell motility and promotes an epithelial-mesenchymal transition (EMT). Previous studies have shown that Twist1 works together with polycomb group protein Bmi1 to promote EMT. Here we show that in breast cancer cells, Bmi1 acts downstream of Twist1 to promote the cancer cell invasion. Suppression of Bmi1 in breast cancer cells expressing high level Twist1 reduces cell motility, and ectopic expression of Bmi1 in Twist1-knockdown cells promote cell invasion. The results delineate a novel pathway that leads to cancer cell invasion and metastasis, which may benefit the development of novel therapeutic approaches to treat advanced breast cancer. Citation Format: Yu-Huey Lin, Guihua Jing, Junjun Liu. Bmi1 is required for Twist1-mediated breast cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2013-1483 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Nonredundant and locus-specific gene repression functions of PRC1 paralog family members in human hematopoietic stem/progenitor cells

AbstractThe Polycomb group (PcG) protein BMI1 is a key factor in regulating hematopoietic stem cell (HSC) and leukemic stem cell self-renewal and functions in the context of the Polycomb repressive complex 1 (PRC1). In humans, each of the 5 subunits of PRC1 has paralog family members of which many reside in PRC1 complexes, likely in a mutually exclusive manner, pointing toward a previously unanticipated complexity of Polycomb-mediated silencing. We used an RNA interference screening approach to test the functionality of these paralogs in human hematopoiesis. Our data demonstrate a lack of redundancy between various paralog family members, suggestive of functional diversification between PcG proteins. By using an in vivo biotinylation tagging approach followed by liquid chromatography-tandem mass spectrometry to identify PcG interaction partners, we confirmed the existence of multiple specific PRC1 complexes. We find that CBX2 is a nonredundant CBX paralog vital for HSC and progenitor function that directly regulates the expression of the cyclin-dependent kinase inhibitor p21, independently of BMI1 that dominantly controls expression of the INK4A/ARF locus. Taken together, our data show that different PRC1 paralog family members have nonredundant and locus-specific gene regulatory activities that are essential for human hematopoiesis.

Identification of regions in the HOX cluster that can confer repression in a Polycomb-dependent manner

Polycomb Group (PcG)-mediated repression is critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We have identified and characterized two new potential PcG-dependent regulatory elements within the human HOXB and HOXC clusters. Their repressive activities are similar to a previously identified element in the HOXD cluster. The PcG proteins BMI1 and SUZ12 are recruited to a reporter construct in mesenchymal stem cells and confer repression that was dependent upon PcG expression. In addition, JARID2 was observed to localize to these three elements. Interestingly, the requirement for JARID2 is variable at the different regions despite its localization. We conclude that distinct regions of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression and that JARID2 plays a role in the recruitment of PRC2.

A Positive Feedback Loop Regulates the Expression of Polycomb Group Protein BMI1 via WNT Signaling Pathway

Polycomb group protein BMI1 plays an important role in cellular homeostasis by maintaining a balance between proliferation and senescence. It is often overexpressed in cancer cells and is required for self-renewal of stem cells. At present, very little is known about the signaling pathways that regulate the expression of BMI1. Here, we report that BMI1 autoactivates its own promoter via an E-box present in its promoter. We show that BMI1 acts as an activator of the WNT pathway by repressing Dickkopf (DKK) family of WNT inhibitors. BMI1 mediated repression of DKK proteins; in particular, DKK1 led to up-regulation of WNT target c-Myc, which in turn further led to transcriptional autoactivation of BMI1. Thus, a positive feedback loop connected by the WNT signaling pathway regulates BMI1 expression. This positive feedback loop regulating BMI1 expression may be relevant to the role of BMI1 in promoting cancer and maintaining stem cell phenotype.

Expression of Polycomb Group Proteins EZH2 and Bmi-1 During Progression of Esophageal Squamous Intraepithelial Neoplasia

Expression of Polycomb Group Proteins EZH2 and Bmi-1 During Progression of Esophageal Squamous Intraepithelial Neoplasia

JMJD2A Promotes Cellular Transformation by Blocking Cellular Senescence through Transcriptional Repression of the Tumor Suppressor CHD5

Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5, a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Ras-mediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors.

Deacetylase inhibitors modulate proliferation and self-renewal properties of leukemic stem and progenitor cells

Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal. The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.