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A P Otte

Publications and source records attributed to A P Otte.

At least 19 recordsLinked to original sources
PubMed2001

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)

Adolescent↗
PubMed2001

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.

CD4-Positive T-Lymphocytes↗
PubMed2001

Differential expression of human Polycomb group proteins in various tissues and cell types.

Polycomb group proteins are involved in the maintenance of cellular identity. As multimeric complexes they repress cell type-specific sets of target genes. One model predicts that the composition of Polycomb group complexes determines the specificity for their target genes. To study this hypothesis, we analyzed the expression of Polycomb group genes in various human tissues using Northern blotting and immunohistochemistry. We found that Polycomb group expression varies greatly among tissues and even among specific cell types within a particular tissue. Variations in mRNA expression ranged from expression of all analyzed Polycomb group genes in the heart and testis to no detectable Polycomb group expression at all in bone marrow. Furthermore, each Polycomb group gene was expressed in a different number of tissues. RING1 was expressed in practically all tissues, while HPH1 was expressed in only a few tissues. Also within one tissue the level of Polycomb group expression varied greatly. Cell type-specific Polycomb group expression patterns were observed in thyroid, pancreas, and kidney. Finally, in various developmental stages of fetal kidney, different Polycomb group expression patterns were observed. We conclude that Polycomb group expression can vary depending on the tissue, cell type, and development stage. Polycomb group complexes can only be composed of the Polycomb group proteins that are expressed. This implies that with cell type-specific Polycomb group expression patterns, cell type-specific Polycomb group complexes exist. The fact that there are cell type-specific Polycomb group targets and cell type-specific Polycomb group complexes fits well with the hypothesis that the composition of Polycomb group complexes may determine their target specificity. J. Cell. Biochem. Suppl. 36: 129-143, 2001.

Blotting, Northern↗
PubMed2001

Polycomb-group genes as regulators of mammalian lymphopoiesis.

Polycomb proteins form DNA-binding protein complexes with gene-suppressing activity. They maintain cell identity but, also, contribute to the regulation of cell proliferation. Mice with mutated Polycomb-group genes exhibit various hematological disorders, ranging from the loss of mature B and T cells to development of lymphomas. Lymphopoiesis in humans is associated with characteristic expression patterns of Polycomb-group genes in defined lymphocyte populations. Collectively, these results indicate that Polycomb-group genes encode novel gene regulators involved in the differentiation of lymphocytes. The underlying mechanism is related, most probably, to gene silencing by chromatin modification, and might affect proliferative behavior and account for the irreversibility of lineage choice.

Animals↗
PubMed2001

The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma.

Polycomb group (PcG) proteins are involved in the stable transmittance of the repressive state of their gene targets throughout the cell cycle. Mis-expression of PcG proteins can lead to proliferative defects and tumorigenesis. There are two separate multimeric PcG protein complexes: an EED-EZH2-containing complex and a BMI1-RING1-containing complex. In the normal human follicle mantle, both PcG complexes have mutually exclusive expression patterns. BMI1-RING1 is expressed, but EZH2-EED is not. Here, we studied the expression of both complexes in six cases of mantle cell lymphoma (MCL), which is derived from the follicle mantle. MCL cells can be cultured in vitro and stimulated to proliferation. We found that resting MCL cells expressed BMI1-RING1, but not EZH2-EED, like normal mantle cells. Proliferating MCL cells, however, showed strongly enhanced expression of EZH2. Also, BMI1 and RING1 continued to be expressed in proliferating MCL. This is the first demonstration that EZH2 expression can be upregulated in fresh lymphoma cells. To test whether the enhanced EZH2 expression was causal for the increased proliferation in MCL, we overexpressed EZH2 in two different cell lines. In the B cell-derived Ramos cell line, EZH2 overexpression caused an increase in the proliferation rate. This suggests a possible causal effect between EZH2 upregulation and increased proliferation in haematopoietic cells.

Blotting, Western↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2000

Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization.

In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.

Amino Acid Sequence↗
PubMed2000

Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators.

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

Animals↗
PubMed2000

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.

B-Lymphocyte Subsets↗
PubMed2000

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.

Adolescent↗
PubMed1999

Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation.

Polycomb-group (PcG) proteins form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. Components of PcG complexes and their mutual interactions have been identified and analysed through extensive genetic and biochemical analyses. Molecular mechanisms underlying PcG-mediated repression of gene activity, however, have remained largely unknown. Previously we reported the existence of two distinct human PcG protein complexes. The EED/EZH protein complex contains the embryonic ectoderm development (EED) and enhancer of zeste 2 (EZH2; refs 9,10) PcG proteins. The HPC/HPH PcG complex contains the human polycomb 2 (HPC2; ref. 11), human polyhomeotic (HPH), BMI1 (ref. 13 ) and RING1 (refs 14, 15) proteins. Here we show that EED (refs 4, 5, 6, 7, 8) interacts, both in vitro and in vivo, with histone deacetylase (HDAC) proteins. This interaction is highly specific because the HDAC proteins do not interact with other vertebrate PcG proteins. We further find that histone deacetylation activity co-immunoprecipitates with the EED protein. Finally, the histone deacetylase inhibitor trichostatin A (ref. 17) relieves transcriptional repression mediated by EED, but not by HPC2, a human homologue of polycomb. Our data indicate that PcG-mediated repression of gene activity involves histone deacetylation. This mechanistic link between two distinct, global gene repression systems is accomplished through the interaction of HDAC proteins with a particular PcG protein, EED.

Acetylation↗
PubMed1999

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.

3T3 Cells↗
PubMed1999

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.

Alcohol Oxidoreductases↗
PubMed1998

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.

Cell Line↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

Ectopic expression of wnt-5a in human renal cell carcinoma cells suppresses in vitro growth and telomerase activity.

Loss of genes located on chromosome 3p has been reported in many different types of human cancers, including renal cell carcinoma. Previous studies using a nontumorigenic human renal cell carcinoma cell line (RCC23) established from a stage III nonpapillary carcinoma with a loss of heterozygosity on 3p showed that microcell hybrids containing an introduced intact chromosome 3 resulted in a more differentiated phenotype including restored cellular senescence and repression of telomerase activity. Human wnt-5a has been cloned and mapped to chromosome 3p14-21. We have stably transfected human wnt-5a into RCC23 cells which results in in vitro growth suppression and repression of telomerase activity in a manner similar to that found in microcell hybrids containing an introduced intact chromosome 3.

Carcinoma, Renal Cell↗
PubMed1997

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.

Amino Acid Sequence↗