Polycomb Group Gene Products Research Articles

Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8

Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of <i>Hoxb8</i>

3‐Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor‐initiating hepatocellular carcinoma cells

Recent advances in stem cell biology have identified tumor-initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self-renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self-renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor-initiating HCC cells remains unclear. In this study, we conducted loss-of-function assay of EZH2 using short-hairpin RNA and pharmacological inhibition of EZH2 by an S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep). Both EZH2-knockdown and DZNep treatment impaired cell growth and anchorage-independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)(+) tumor-initiating cells. Administration of 5-fluorouracil (5-FU) or DZNep suppressed the tumors by implanted HCC cells in non-obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5-FU predominantly reduced the number of EpCAM(+) cells and diminished the self-renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor-initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor-initiating HCC cells.

The polycomb group gene product Ezh2 regulates proliferation and differentiation of murine hepatic stem/progenitor cells

Polycomb group proteins initiate and maintain gene silencing through chromatin modifications and contribute to the maintenance of self-renewal in a variety of stem cells. Among polycomb repressive complexes (PRCs), PRC2 initiates gene silencing by methylating histone H3 lysine 27, and PRC1 maintains gene silencing through mono-ubiquitination of histone H2A lysine 119. We have previously shown that Bmi1, a core component of PRC1, tightly regulates the self-renewal of hepatic stem/progenitor cells. In this study, we conducted lentivirus-mediated knockdown of Ezh2 to characterise the function of Ezh2, a major component of PRC2, in hepatic stem/progenitor cells. Loss of Ezh2 function in embryonic murine hepatic stem/progenitor cells severely impaired proliferation and self-renewal capability. This effect was more prominent than that of Bmi1-knockdown and was partially abrogated by the deletion of both Ink4a and Arf, major targets of PRC1 and PRC2. Importantly, Ezh2-knockdown but not Bmi1-knockdown promoted the differentiation and terminal maturation of hepatocytes, followed by the up-regulation of several transcriptional regulators of hepatocyte differentiation. Our findings indicate that Ezh2 plays an essential role in the maintenance of both the proliferative and self-renewal capacity of hepatic stem/progenitor cells and the full execution of their differentiation.

Bmi1 regulates memory CD4 T cell survival via repression of the Noxa gene

The maintenance of memory T cells is central to the establishment of immunological memory, although molecular details of the process are poorly understood. In the absence of the polycomb group (PcG) gene Bmi1, the number of memory CD4+ T helper (Th)1/Th2 cells was reduced significantly. Enhanced cell death of Bmi1−/− memory Th2 cells was observed both in vivo and in vitro. Among various proapoptotic genes that are regulated by Bmi1, the expression of proapoptotic BH3-only protein Noxa was increased in Bmi1−/− effector Th1/Th2 cells. The generation of memory Th2 cells was restored by the deletion of Noxa, but not by Ink4a and Arf. Direct binding of Bmi1 to the Noxa gene locus was accompanied by histone H3-K27 methylation. The recruitment of other PcG gene products and Dnmt1 to the Noxa gene was highly dependent on the expression of Bmi1. In addition, Bmi1 was required for DNA CpG methylation of the Noxa gene. Moreover, memory Th2-dependent airway inflammation was attenuated substantially in the absence of Bmi1. Thus, Bmi1 controls memory CD4+ Th1/Th2 cell survival and function through the direct repression of the Noxa gene.

Bmi1 regulates memory Th2 cell survival via repression of the Noxa gene.

The maintenance of memory T cells is central to the establishment of immunological memory, although molecular details of the process are poorly understood. In the absence of the Polycomb group (PcG) gene Bmi1, the number of memory Th2 cells was reduced significantly. Enhanced cell death of Bmi1−/− memory Th2 cells was observed both in vivo and in vitro. Among various pro‐apoptotic genes that are regulated by Bmi1, the expression of pro‐apoptotic BH3 only protein Noxa was increased in Bmi1−/− effector CD4 T cells even in the absence of Ink4a and Arf. The generation of memory Th2 cells was restored by the deletion of Noxa but not by Ink4a and Arf. Direct binding of Bmi1 to the Noxa gene locus was accompanied by histone H3‐K27 methylation. The recruitment of other PcG gene products to the Noxa gene was highly dependent on the expression of Bmi1. In addition, DNA methylation appeared to control the recruitment of PcG gene products and the expression levels of Noxa. Moreover, memory Th2‐dependent airway inflammation was attenuated substantially in the absence of Bmi1. Thus, Bmi1 controls memory Th2 cell survival and function through the direct repression of the Noxa gene.

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains ofHoxb8

To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, we have focused on the binding patterns of PcG gene products to the flanking regions of the Hoxb8 gene in expressing and non-expressing tissues. In parallel, we followed the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4), and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. We show that developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene.

Regulation of hematopoietic stem cell self-renewal by a polycomb group gene product, Bmi-1

Regulation of hematopoietic stem cell self-renewal by a polycomb group gene product, Bmi-1

Involvement of winged eye encoding a chromatin-associated bromo-adjacent homology domain protein in disc specification

How organ identity is determined is a fundamental question in developmental biology. In Drosophila, field-specific selector genes, such as eyeless (ey) for eyes and vestigial (vg) for wings, participate in the determination of imaginal disc-specific identity. We performed gain-of-function screening and identified a gene named winged eye (wge), which encodes a bromo-adjacent homology domain protein that localizes at specific sites on chromosomes in a bromo-adjacent homology domain-dependent manner. Overexpression of wge-induced ectopic wings with antero-posterior and dorso-ventral axes in the eye field in a region-specific Hox gene-(Antennapedia) independent manner. Overexpression of wge was sufficient for ectopic expression of vg in eye discs. A context-dependent requirement of wge was demonstrated for vg expression in wing discs and for expression of eyes absent (eya), a control gene for eye development downstream of ey, in eye discs. In contrast to vg, however, overexpression of wge inhibited EY-mediated expression of eya. Consistent with colocalization on polytene chromosomes of WGE and Posterior sex combs (PSC), a Polycomb group gene product, we demonstrated an antagonistic genetic interaction between wge and Psc. These findings suggest that wge functions in the determination of disc-specific identity, downstream of Hox genes.

The polycomb group gene product Mel-18 interacts with cyclin D2 and modulates its activity

Considerable evidence supports the view that D-type cyclins play a role in G1-S progression. We found that cyclin D2 directly interacts with Mel-18, one of the polycomb group gene products in a yeast two hybrid screen. Further, we have determined the binding domains that are required for interaction between cyclin D2 and Mel-18. The proline/serine-rich domain (P/S domain) of Mel-18 is required to interact with cyclin D2, and the N-terminal region of cyclin D2 is necessary to interact with Mel-18. A co-localization study shows that cyclin D2 and Mel-18 interact within the nucleus. To determine whether Mel-18 affects cyclin D2 activity, we blocked Mel-18 expression using an anti-sense strand system in cyclin D2 over-expressing cells. The results indicate that cells with reduced Mel-18 expression levels show more proliferative activity than the controls. These findings are the first report that Mel-18 directly interacts with cyclin D2 and may inhibit cyclin D2 activity.

Regulation of Th2 Cell Differentiation by mel-18, a Mammalian Polycomb Group Gene

Polycomb group ( PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNγ was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.

Proliferative involvement of ENX-1, a putative human polycomb group gene, in haematopoietic cells.

Homeobox genes have important roles in haematopoiesis and are regulated in an activated state by the trithorax group (trxG) of genes. In a repressed state, they are regulated by the Polycomb group (PcG) of genes. ENX-1, a putative human PcG gene product, interacts with the proto-oncogene product Vav. We report an investigation of the role of ENX-1 in human haematopoiesis. CD34+ cells mobilized to peripheral blood strongly expressed ENX-1. When stimulated to proliferate, both T and B lymphocytes rapidly up-regulated ENX-1. ENX-1 was expressed in all cell lines of the various lineages examined. When HL-60 cells were differentiated to mature granulocytes with all-trans retinoic acid, ENX-1 was down-regulated. Moreover, ENX-1 antisense oligodeoxynucleotide suppressed DNA synthesis in HL-60 cells. Our data indicate that ENX-1 is involved in the proliferation of both normal and malignant haematopoietic cells.

The Role of mel-18, a Mammalian Polycomb Group Gene, during IL-7–Dependent Proliferation of Lymphocyte Precursors

mel-18 is a mammalian homolog of Drosophila melanogaster Polycomb group genes. Mice lacking the mel-18 gene show a posterior transformation of the axial skeleton, severe combined immunodeficiency, and a food-passing disturbance in the lower intestine due to hypertrophy of the smooth muscle layer. In this study, the severe combined immunodeficiency observed in mel-18 mutant mice is correlated with the impaired mitotic response of lymphocyte precursors upon interleukin-7 stimulation. Strikingly, the axial skeleton and lymphoid phenotypes are identical in both mel-18 and bmi-1 mutants, indicating that the Mel-18 and Bmi-1 gene products might act in the same genetic cascade. These results suggest that mammalian Polycomb group gene products are involved in cell cycle progression in the immune system.

A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton.

Segment identity in both invertebrates and vertebrates is conferred by spatially restricted distribution of homeotic gene products. In Drosophila, the expression of Homeobox genes during embryogenesis is initially induced by segmentation gene products and then maintained by Polycomb group and Trithorax group gene products. Polycomb group gene homologs are conserved in vertebrates. Murine mel-18 and closely related bmi-1 are homologous to posterior sex combs and suppressor two of zeste. Mel-18 protein mediates a transcriptional repression via direct binding to specific DNA sequences. To gain further insight into the function of Mel-18, we have inactivated the mel-18 locus by homologous recombination. Mice lacking mel-18 survive to birth and die around 4 weeks after birth after exhibiting strong growth retardation. Similar to the Drosophila posterior sex combs mutant, posterior transformations of the axial skeleton were reproducibly observed in mel-18 mutants. The homeotic transformations were correlated with ectopic expression of Homeobox cluster genes along the anteroposterior axis in the developing paraxial mesoderm. Surprisingly, mel-18-deficient phenotypes are reminiscent of bmi-1 mutants. These results indicate that the vertebrate Polycomb group genes mel-18 and bmi-1, like Drosophila Polycomb group gene products, might play a crucial role in maintaining the silent state of Homeobox gene expression during paraxial mesoderm development.

Mel-18, a Polycomb group-related mammalian gene, encodes a transcriptional negative regulator with tumor suppressive activity.

The mammalian mel-18/bmi-1 gene products share an amino acid sequence and a secondary structure, including a RING-finger motif, with the Drosophila Polycomb group (PcG) gene products Psc and Su(z)2, implying that they represent a gene family with related functions. As Drosophila PcG gene products are thought to function as transcriptional repressors by modifying chromatin structure, Mel-18/Bmi-1 might be expected to have similar activities. Here we have analyzed the function of mel-18 and found that Mel-18 acts as a transcriptional repressor via its target DNA sequence, 5'-GACTNGACT-3'. Interestingly, this binding sequence is found within regulatory or non-coding regions of various genes, including the c-myc, bcl-2 and Hox genes, suggesting diverse functions of mel-18 as the mammalian homolog of the PcG gene. We also demonstrate that mel-18 has tumor suppressor activity, in contrast to bmi-1, which has been defined as a proto-oncogene.

Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group of genes and encodes a chromatin protein required for negative regulation of homeotic genes and other loci, in particular the ph locus itself. We have studied the genetic control of ph transcription during development. Early ph expression is under the control of bicoid and engrailed as activators and of oskar as an inhibitor. The negative autoregulation of ph starts at the blastoderm stage and is partly mediated by a transvection effect. As the number of functional copies of ph increases in the same genome, a concomitant reduction of the transcription of each copy is observed. This regulation is ensured, likely at the chromatin level, positively by the trithorax group and negatively by the Polycomb group gene products like a homeotic gene, but it occurs in the same cells. We propose that an equilibrium between these two states of chromatin activity ensures an accurate level of ph transcription.

Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.

The polycomb group (Pc-G) genes are responsible for maintaining the repressed state of homeotic genes during development. It has been suggested that the Pc-G exerts its transcriptional control by regulating higher order chromatin structure. In particular, the finding of genetic and molecular similarities to components involved in heterochromatin formation, led to the proposal that homeotic genes are permanently repressed by mechanisms similar to those responsible for heterochromatin compaction. Because of synergistic effects, Pc-G gene products are thought to act in a multimeric complex. Using immunoprecipitation we show that two members of the Pc-G, Polycomb and polyhomeotic, are constituents of a soluble multimeric protein complex. Size fractionation indicates that a large portion of the two proteins are found in a distinct complex of molecular weight 2-5 x 10(6) Da. During embryogenesis the two proteins show the same spatial distribution. In addition, by double-immunofluorescence labelling we can demonstrate that Polycomb and polyhomeotic have exactly the same binding patterns on polytene chromosomes of larval salivary glands. We propose that some Pc-G proteins act in multimeric complexes to compact the chromatin of stably repressed genes like the homeotic regulators.