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Results for “Polycomb Repressive Complex 1”

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PubMed2005

[Differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells].

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.

Cell Differentiation↗

PubMed2000

Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling.

Pc-G and trx-G genes are responsible for maintenance of transcriptional regulation and provide a cellular memory mechanism throughout development. Studies in fly, yeast, mouse, and human have implicated modulation of higher-order chromatin structure as an important component in this process. Specifically, connections between SWI/SNF complexes and trx-G genes have provided a mechanistic link between chromatin remodeling and transcriptional regulation. Here we discuss recent genetic and biochemical data that has shed light on the molecular mechanisms and pathways associated with Pc-G and trx-G function in developmental processes such as cell cycle control and hematopoiesis. genesis 26:189-197, 2000.

Animals↗

PubMed1998

Heritable chromatin states induced by the Polycomb and trithorax group genes.

In Drosophila the Polycomb group (PcG) and trithorax group (trxG) genes are required to maintain differential expression patterns of many important developmental regulatory genes. The PcG is responsible for heritable silencing throughout development. At target genes PcG response elements (PREs) attract PcG protein complexes and induce the formation of higher-order chromatin structures. We have mapped the distribution of Polycomb and other PcG members at various target genes by using an improved formaldehyde cross-linking and chromatin immunoprecipitation technique. We find that Polycomb spreads locally from PREs over several kilobases, thereby probably stabilizing the silencing complexes. Members of the trxG co-localize at PREs. GAGA factor was found to be constitutively bound to PREs independently of gene activity. PREs associated with active genes appear to have increased amounts of bound GAGA. We have developed a system capable of switching a PRE between the on/off modes. PREs and trxG-regulated elements are common chromosomal elements through which the proteins of the PcG/trxG exert their maintenance function on adjacent chromatin structures.

Animals↗

PubMed2001

Remembering silence.

Polycomb response elements (PREs) are regulatory switch elements that can direct the genes that they control to be either active or silenced. Once decided, this on or off state is maintained through subsequent cell divisions. We do not know how the switching works, or how it is copied to newly replicated chromosomes. Experiments that switch a silenced PRE to an active state have provided insights into both questions. A PRE switched experimentally can remember its previously silenced state and return to it after several cell divisions. In the most recent study of this phenomen on, the data show that several distinct variables affect the ability of PREs to "remember" and restore their previous state. The authors' interpretation of these results is discussed here.

Animals↗

PubMed2005

To SIR with Polycomb: linking silencing mechanisms.

Yeast SIR2, the most evolutionarily conserved deacetylase, plays an essential role in epigenetic silencing at the silent mating type loci and telomeres. SIR2 has been implicated in chromatin silencing and lifespan determination in several organisms. Discovery that Drosophila SIR2 is also involved in epigenetic silencing mediated by the Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase has wide implications. These findings suggest possible link of Polycomb system to diverse cellular processes including senescence.

Animals↗

PubMed1995

Transcriptional silencing of homeotic genes in Drosophila.

Homeotic genes are subject to transcriptional silencing, which prevents their expression in inappropriate body regions. Here, we shall focus on Drosophila, as little is known about this process in other organisms. Evidence is accumulating that silencing of Drosophila homeotic genes is conferred by two types of cis- regulatory sequences: initiation (SIL-1) and maintenance (SIL-M) elements. The former contain target sites for transient repressors with a highly localised distribution in the early embryo and the latter for constitutive repressors that are likely to be present in all cells. We discuss how SIL-1 elements may cooperate with SIL-M elements to promote formation of a silencing complex. We propose that this complex consists of specific non-histone proteins, the so-called Polycomb group proteins, and that it is anchored at SIL-M elements and at the promoter.

Animals↗

PubMed2005

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM).

BACKGROUND: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. METHODS AND RESULTS: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simulate, display, and discuss the effects of blurring and fluorophore heterogeneity on lifetime imaging in widefield and confocal configurations. CONCLUSION: Sectioning improves the quality of lifetime images of samples with multiple fluorophores or spatially varying Förster resonance energy transfer.

Animals↗

PubMed1991

Direct interaction of the Polycomb protein with Antennapedia regulatory sequences in polytene chromosomes of Drosophila melanogaster.

The Polycomb (Pc) gene is responsible for the elaboration and maintenance of the expression pattern of the homeotic genes during development of Drosophila. In mutant Pc- embryos, homeotic transcripts are ectopically expressed, leading to abdominal transformations in all segments. From this it was suggested that PC+ acts as a repressor of homeotic gene transcription. We have mapped the cis-acting control sequences of the homeotic Antennapedia (Antp) gene regulated by Pc. Using Antp P1 and P2 promoter fragments linked to the E. coli lacZ reporter gene we show different expression patterns of beta-galactosidase (beta-gal) in transformed Pc+ and Pc- embryos. In addition we are able to visualize by immunocytochemical techniques on polytene chromosomes the direct binding of the Pc protein to the transposed cis-regulatory promoter fragments. However, short Antp P1 promoter constructs which are--due to position effects--ectopically activated in salivary glands, do not reveal a Pc binding signal.

Animals↗

PubMed1992

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.

Animals↗

PubMed1993

Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.

Polycomb group genes are necessary for maintaining homeotic genes repressed in appropriate parts of the body plan. Some of these genes, e.g. Psc, Su(z)2 and E(z), are also modifiers of the zeste-white interaction. The products of Psc and Su(z)2 were immunohistochemically detected at 80-90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of zeste protein and two other Polycomb group proteins, Polycomb and polyhomeotic. The five proteins co-localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in another Polycomb group gene, E(z), the Su(z)2 and Psc products become dissociated from chromatin at non-permissive temperatures from most but not all sites, while the binding of the zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions. These results suggest that the binding of at least some Polycomb group proteins requires interactions with other members of the group and, although zeste can bind independently, its repressive effect on white involves the presence of at least some of the Polycomb group proteins.

Animals↗

PubMed1994

A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression.

Segmentation genes provide the signals for the activation and regulation of homeotic genes in Drosophila but cannot maintain the resulting pattern of expression because their activity ceases halfway through embryogenesis. Maintenance of the pattern is due to the Polycomb group of genes (Pc-G) and the trithorax group of genes (trx-G), responsible for the persistence of the active or repressed state of homeotic genes. We have identified a regulatory element in the Ubx gene that responds to Pc-G and trx-G genes. Transposons carrying this element create new binding sites for Pc-G products in the polytene chromosomes. This Pc-G maintenance element (PRE), establishes a repressive complex that keeps enhancers repressed in cells in which they were originally repressed and maintains this state through many cell divisions. The trx-G products stimulate the expression of enhancers in cells in which they were originally active. This mechanism is responsible for the correct regulation of imaginal disc enhancers, which lack themselves antero-posterior positional information. The PRE also causes severe variegation of the mini-white gene present in the transposon, a phenomenon very similar to heterochromatic position-effect variegation. The significance of this mechanism for homeotic gene regulation is discussed.

Animals↗

PubMed1995

Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.

The genes of the Polycomb-group (Pc-G) are responsible for maintaining the inactive expression state of homeotic genes. They act through specific cis-regulatory DNA elements termed PREs (Pc-G Response Elements). Multimeric complexes containing the Pc-G proteins are thought to induce heterochromatin-like structures, which stably and heritably inactivate transcription. We have tested the functional role of the FAB fragment, a PRE of the bithorax complex. We find that this element behaves as an orientation dependent silencer, capable of inducing mosaic gene expression on neighboring genes. Transgenic fly lines were constructed containing a PRE adjacent to a reporter gene inducible by the yeast GAL4 trans-activator. The competition between the activator and Pc-G-containing chromatin was visualized on polytene chromosomes using immunocytochemistry. The Pc-G protein Polycomb and GAL4 have mutually exclusive binding patterns, supporting the notion that Pc-G-induced chromatin structures can prevent activators from binding to their target sequences. However, this antagonistic function can be overcome by high doses of GAL4, even in the absence of DNA replication.

Animals↗

PubMed1995

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.

3T3 Cells↗

PubMed1995

Transcriptional silencing by the Polycomb protein in Drosophila embryos.

Polycomb group (Pc-G) proteins act to keep homeotic genes stably and heritably silenced during Drosophila development. Here, it is shown that Polycomb (Pc), one of the Pc-G proteins, acts as a transcriptional silencer in Drosophila embryos if tethered to reporter genes by the DNA binding domain of GAL4 (i.e. as a GAL-Pc fusion protein). The results suggest that silencing by GAL-Pc requires the C-terminal portion of Pc, but not the chromodomain. If a pulse of Gal-Pc is provided, synthetic reporter genes are repressed, though only transiently. In contrast, reporter genes containing homeotic gene sequences remain stably and heritably silenced in a Pc-G gene-dependent fashion, even when GAL-Pc is no longer present. This implies that GAL-Pc recruits Pc-G proteins to DNA and suggests that maintenance of silencing requires the anchoring of Pc-G proteins to specific cis-regulatory sequences present in homeotic genes. The extent of DNA over which the Pc-G machinery acts is quite selective, as silencing established on one enhancer does not necessarily 'spread' to a juxtaposed synthetic enhancer.

ATP-Binding Cassette Transporters↗

PubMed1995

The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.

trithorax is required to stably maintain homeotic gene expression patterns established during embryogenesis by the action of the transiently expressed products of the segmentation genes. The large trithorax proteins contain a number of highly conserved novel motifs, some of which have been hypothesized to interact directly with specific DNA sequences in their target genes. Using antibodies directed against trithorax proteins, we show that they are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. trithorax binding is detected at the sites of its known targets, the Bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature-sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. We localized one trithorax binding site within a portion of the large 5' regulatory region of the Ubx gene, to an interval which also contains binding sites for Polycomb group proteins. These results suggest that trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co-localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action.

Animals↗

PubMedDate not supplied

Molecular cloning and characterization of a RING-H2 finger protein, ANAPC11, the human homolog of yeast Apc11p.

Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases. The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed. This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids. The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes. In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte. The ANAPC11 gene is located at the human chromosome 17q25. ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected. Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

BACKGROUND: Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro. METHODS: We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro. RESULT: The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording. CONCLUSIONS: The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.

Action Potentials↗

PubMed2026

ZNF695 Promotes Colorectal Cancer Progression Through Transcriptional Activation of CBX8 and Subsequent Wnt/β-Catenin Signaling Activation.

In this investigation, we examined the functional mechanism of the transcription factor zinc finger protein 695 (ZNF695) and its target gene chromobox protein homolog 8 (CBX8) in colorectal cancer (CRC) migration and invasion. HCT-116 and LOVO cell lines were used to establish cell models with knocked-down ZNF695 and knocked-down or over-expressed CBX8. To comprehensively evaluate the functional contributions of ZNF695 and CBX8 to cellular phenotypes, we employed CCK-8, wound-healing, and Transwell assays to evaluate cell proliferation, migration, and invasion, respectively. To assess the impact of ZNF695 on tumor progression, we generated a xenograft model utilizing nude mice. A FLAG-ZNF695 expression plasmid was constructed, and ChIP-seq experiments were performed. By integrating mRNA sequencing data following ZNF695 knockdown with highly expressed genes in CRC from the TCGA database, CBX8 was identified as a putative downstream target of ZNF695. We employed a dual-luciferase reporter assay to validate the specific binding affinity of ZNF695 toward the CBX8 promoter region. To elucidate the specific biological cascades modulated by ZNF695 and CBX8, we conducted a comprehensive pathway enrichment analysis. Rescue experiments were conducted to determine whether the ZNF695/CBX8 regulatory axis upregulates the expression of the Wnt signaling pathway downstream targets, AXIN2 and CCND1. Both in vitro assays and in vivo models confirmed that silencing ZNF695 dramatically suppresses CRC cell proliferation, migration, and invasion, while concurrently impeding tumor progression. ChIP-seq coupled with dual-luciferase reporter assays substantiated the direct binding of ZNF695 to the CBX8 promoter. Furthermore, CBX8 depletion significantly attenuated the migratory and invasive phenotypes of CRC cells. Restoring CBX8 expression effectively rescued the migratory and invasive deficits in CRC cells induced by ZNF695 silencing. Re-expression of CBX8 in ZNF695-silenced cells restored Wnt/β-catenin signaling activity, accompanied by increased expression of AXIN2 and CCND1. ZNF695 promotes CRC progression by transcriptionally activating CBX8 and subsequently enhancing Wnt/β-catenin signaling, thereby promoting tumor cell proliferation, migration, and invasion.

Humans↗