Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma.

Polycomb-group (PcG) proteins, such as BMI-1 and EZH2, form multimeric gene-repressing complexes involved in axial patterning, hematopoiesis, and cell cycle regulation. In addition, BMI-1 is involved in experimental lymphomagenesis. Little is known about its role in human lymphomagenesis. Here, BMI-1 and EZH2 expression patterns are analyzed in a variety of B-cell non-Hodgkin lymphomas (B-NHLs), including small lymphocytic lymphoma, follicular lymphoma, large B-cell lymphoma, mantle-cell lymphoma, and Burkitt lymphoma. In contrast to the mutually exclusive pattern of BMI-1 and EZH2 in reactive follicles, the neoplastic cells in B-NHLs of intermediate- and high-grade malignancy showed strong coexpression of BMI-1 and EZH2. This pattern overlapped with the expression of Mib-1/Ki-67, a marker for proliferation. Neoplastic cells in B-NHL of low-grade malignancy were either BMI-1(low)/EZH2(+) (neoplastic centroblasts) or BMI-1(low)EZH2(-) (neoplastic centrocytes). These observations show that low-, intermediate-, and high grade B-NHLs are associated with increased coexpression of the BMI-1 and EZH2 PcG proteins, whose normal expression pattern is mutually exclusive. This expression pattern is probably caused by a failure to down-regulate BMI-1 in dividing neoplastic cells, because BMI-1 expression is absent from normal dividing B cells. These observations are in agreement with findings in studies of Bmi-1 transgenic mice. The extent of BMI-1/EZH2 coexpression correlated with clinical grade and the presence of Mib-1/Ki-67 expression, suggesting that the irregular expression of BMI-1 and EZH2 is an early event in the formation of B-NHL. This points to a role for abnormal PcG expression in human lymphomagenesis. (Blood. 2001;97:3896-3901)

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation.

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.

The polycomb group protein EED interacts with YY1, and both proteins induce neural tissue in Xenopus embryos.

Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction.

Coexpression of BMI-1 and EZH2 polycomb group genes in Reed-Sternberg cells of Hodgkin's disease.

The human BMI-1 and EZH2 polycomb group (PcG) proteins are constituents of two distinct complexes of PcG proteins with gene regulatory activity. PcG proteins ensure correct embryonic development by suppressing homeobox genes, and they also contribute to regulation of lymphopoiesis. The two PcG complexes are thought to regulate different target genes and probably have different tissue distributions. Altered expression of PcG genes is linked to transformation in cell lines and induction of tumors in mutant mice, but the role of PcG genes in human cancers is relatively unexplored. Using antisera specific for human PcG proteins, we used immunohistochemistry and immunofluorescence to detect BMI-1 and EZH2 PcG proteins in Reed-Sternberg cells of Hodgkin's disease (HRS). The expression patterns were compared to those in follicular lymphocytes of the lymph node, the normal counterparts of HRS cells. In the germinal center, expression of BMI-1 is restricted to resting Mib-1/Ki-67(-) centrocytes, whereas EZH2 expression is associated with dividing Mib-1/Ki-67(+) centroblasts. By contrast, HRS cells coexpress BMI-1, EZH2, and Mib-1/Ki-67. Because HRS cells are thought to originate from germinal center lymphocytes, these observations suggests that Hodgkin's disease is associated with coexpression of BMI-1 and EZH2 in HRS cells.

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers.

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.

RING1 interacts with multiple Polycomb-group proteins and displays tumorigenic activity.

Polycomb-group (PcG) proteins form large multimeric protein complexes that are involved in maintaining the transcriptionally repressive state of genes. Previously, we reported that RING1 interacts with vertebrate Polycomb (Pc) homologs and is associated with or is part of a human PcG complex. However, very little is known about the role of RING1 as a component of the PcG complex. Here we undertake a detailed characterization of RING1 protein-protein interactions. By using directed two-hybrid and in vitro protein-protein analyses, we demonstrate that RING1, besides interacting with the human Pc homolog HPC2, can also interact with itself and with the vertebrate PcG protein BMI1. Distinct domains in the RING1 protein are involved in the self-association and in the interaction with BMI1. Further, we find that the BMI1 protein can also interact with itself. To better understand the role of RING1 in regulating gene expression, we overexpressed the protein in mammalian cells and analyzed differences in gene expression levels. This analysis shows that overexpression of RING1 strongly represses En-2, a mammalian homolog of the well-characterized Drosophila PcG target gene engrailed. Furthermore, RING1 overexpression results in enhanced expression of the proto-oncogenes c-jun and c-fos. The changes in expression levels of these proto-oncogenes are accompanied by cellular transformation, as judged by anchorage-independent growth and the induction of tumors in athymic mice. Our data demonstrate that RING1 interacts with multiple human PcG proteins, indicating an important role for RING1 in the PcG complex. Further, deregulation of RING1 expression leads to oncogenic transformation by deregulation of the expression levels of certain oncogenes.

C-Terminal binding protein is a transcriptional repressor that interacts with a specific class of vertebrate Polycomb proteins.

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, we performed two-hybrid screens with Xenopus Pc (XPc) and human Pc 2 (HPC2). We find that the C-terminal binding protein (CtBP) interacts with XPc and HPC2, that CtBP and HPC2 coimmunoprecipitate, and that CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, we find that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, we show that CtBP is a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP.

The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain.

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins- RING1, BMI1, and hPc2-we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

Molecular analysis of the androgen-receptor gene in a family with receptor-positive partial androgen insensitivity: an unusual type of intronic mutation.

In the coding part and the intron-exon boundaries of the androgen-receptor gene of a patient with partial androgen insensitivity, no mutation was found. The androgen receptor of this patient displayed normal ligand-binding parameters and migrated as a 110-112-kD doublet on SDS-PAGE in the absence of hormone. However, after culturing of the patient's genital skin fibroblasts in the presence of hormone, the slower-migrating 114-kD protein, which reflects hormone-dependent phosphorylation, was hardly detectable. Furthermore, receptor protein was undetectable in the nuclear fraction of the fibroblasts, after treatment with hormone, which is indicative of defective DNA binding. By sequencing part of intron 2, a T-->A mutation was found 11 bp upstream of exon 3. In our screening of 102 chromosomes from unrelated individuals, this base-pair substitution was not found, indicating that it was not a polymorphism. mRNA analysis revealed that splicing involved a cryptic splice site, located 71/70 bp upstream of exon 3, resulting in generation of mRNA with an insert of 69 nucleotides. In addition, a small amount of a transcript with a deleted exon 3 and a very low level of wild-type transcript were detected. Translation of the extended transcript resulted in an androgen-receptor protein with 23 amino acid residues inserted between the two zinc clusters, displaying defective DNA binding and defective transcription activation.

Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain.

In Drosophila, the products of the Polycomb group (Pc-G) of genes act as chromatin-associated multimeric protein complexes that repress expression of homeotic genes. Vertebrate Pc-G homologues have been identified, but the nature of the complexes they form and the mechanisms of their action are largely unknown. The Polycomb homologue M33 is implicated in mesoderm patterning in the mouse and here we show that it acts as a transcriptional repressor in transiently transfected cells. Furthermore, we have identified two murine proteins, Ring1A and Ring1B, that interact directly with the repressor domain of M33. Ring1A and Ring1B display blocks of similarity throughout their sequences, including an N-terminal RING finger domain. However, the interaction with M33 occurs through a region at the C-terminus. Ring1A represses transcription through sequences not involved in M33 binding. Ring1A protein co-localizes in nuclear domains with M33 and other Pc-G homologues, such as Bmi1. The expression of Ring1A at early stages of development is restricted to the neural tube, whereas M33 is expressed ubiquitously. Within the neural tube, Ring1A RNA is located at the rhombomere boundaries of the hindbrain. Taken together, these data suggest that Ring1A may contribute to a tissue-specific function of Pc-G-protein complexes during mammalian development.

Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis.

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic.

In Drosophila melanogaster, the Polycomb-group (PcG) genes have been identified as repressors of gene expression. They are part of a cellular memory system that is responsible for the stable transmission of gene activity to progeny cells. PcG proteins form a large multimeric, chromatin-associated protein complex, but the identity of its components is largely unknown. Here, we identify two human proteins, HPH1 and HPH2, that are associated with the vertebrate PcG protein BMI1. HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and with BMI1. They also colocalize with BMI1 in interphase nuclei of U-2 OS human osteosarcoma and SW480 human colorectal adenocarcinoma cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic (Ph), and we, therefore, have named the novel proteins HPH1 and HPH2. HPH1, HPH2, and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. Two-hybrid analysis shows that homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts only with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. These data demonstrate the involvement of homology domains I and II in protein-protein interactions and indicate that HPH1 and HPH2 are able to heterodimerize.

MPc2, a new murine homolog of the Drosophila polycomb protein is a member of the mouse polycomb transcriptional repressor complex.

The evolutionarily conserved polycomb and trithorax-group genes are required to maintain stable expression patterns of homeotic genes and other target genes throughout development. Here, we report the cloning and characterization of a novel mouse polycomb homolog, MPc2, in addition to the previously described M33 polycomb gene. Co-immunoprecipitations and subnuclear co-localization studies show that MPc2 interacts with the mouse polycomb-group oncoprotein Bmi1 and is a new member of the mouse polycomb multiprotein complex. Gal4DB-MPc2 or -M33 fusion proteins mediate a five- to tenfold repression of stably integrated reporter constructs carrying GAL4 binding sites, demonstrating that these proteins are transcriptional repressors. The MPc2 gene is localized on chromosome 11, in close proximity to the classical mouse mutations tail short (Ts) and rabo torcido (Rbt). Ts and Rbt hemizygous mice display anemia and transformations of the axial skeleton reminiscent of phenotypes observed in mice with mutated polycomb or trithorax-group genes, suggesting that MPc2 is a candidate gene for Ts and Rbt.