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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↗

Epigenetic regulation of mammalian genomic imprinting.

Imprinted genes play important roles in development, and most are clustered in large domains. Their allelic repression is regulated by 'imprinting control regions' (ICRs), which are methylated on one of the two parental alleles. Non-histone proteins and nearby sequence elements influence the establishment of this differential methylation during gametogenesis. DNA methylation, histone modifications, and also polycomb group proteins are important for the somatic maintenance of imprinting. The way ICRs regulate imprinting differs between domains. At some, the ICR constitutes an insulator that prevents promoter-enhancer interactions, when unmethylated. At other domains, non-coding RNAs could be involved, possibly by attracting chromatin-modifying complexes. The latter silencing mechanism has similarities with X-chromosome inactivation.

Animals↗

Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

3T3 Cells↗

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue.

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.

Amino Acid Sequence↗

Identification and characterization of polyhomeotic PREs and TREs.

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), which are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated. ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, we have functionally characterized PREs and TREs at the ph locus that function in transgenic constructs. We have identified a strong PRE and TRE in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. We have used chromatin immunoprecipitation experiments on cultured cells and embryos to show that Pc-G proteins are located in restricted regions, close to the ph promoters that overlap functionally defined PRE/TREs. Our data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation.

Animals↗

Identification of Polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced.

The Drosophila homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products. When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of the Polycomb and trithorax group loci.

ATP-Binding Cassette Transporters↗

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↗

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53.

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.

Burkitt Lymphoma↗

Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-oncogene.

The bmi-1 proto-oncogene has been implicated in B-cell lymphomagenesis in E mu-myc transgenic mice. Distinct domains of the Bmi-1 protein are highly conserved within the Drosophila protein Posterior Sex Combs, a member of the Polycomb group involved in maintaining stable repression of homeotic genes during development. We have inactivated the bmi-1 gene in the germ line of mice by homologous recombination in ES cells. Null mutant mice display three phenotypic alterations: (1) a progressive decrease in the number of hematopoietic cells and an impaired proliferative response of these cells to mitogens; (2) neurological abnormalities manifested by an ataxic gait and sporadic seizures; and (3) posterior transformation, in most cases along the complete anteroposterior axis of the skeleton. The observations indicate that Mbi-1 plays an important role in morphogenesis during embryonic development and in hematopoiesis throughout pre- and postnatal life. Furthermore, these data provide the first evidence of functional conservation of a mammalian Polycomb group homolog.

Animals↗

Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1.

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O(2) concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.

Cell Division↗

E3 ubiquitin ligase RNF2 interacts with the S6' proteasomal ATPase subunit and increases the ATP hydrolysis activity of S6'.

We reported previously that the human RNF2 (RING finger protein 2) protein is an E3 ubiquitin ligase that interacts with the human ubiquitin-conjugating enzyme Hip-2/hE2-25K. In the present study, we show that RNF2 interacts with S6' ATPase, a subunit of the proteasomal 19 S regulatory complex. S6' interacts with RNF2 through its N-terminal RING domain, and RNF2 interacts with S6' through its C-terminal region. Interestingly, the RNF2-S6' interaction increases the ATP hydrolysis activity of the S6' protein. Moreover, S6' ATPase activity is highly increased in the presence of ubiquitinated proteins. The present study suggests that the E3 ubiquitin ligase RNF2 might have a dual function: facilitating the ubiquitination of its target substrates and recruiting the substrates to the proteasome. Furthermore, ATP hydrolysis in the E3/proteasome complex might act as an important signal for the protein degradation pathway.

Adenosine Triphosphatases↗

Activation and repression activities of ash2 in Drosophila wing imaginal discs.

Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. In this study, we have used the patterning of veins and interveins in the wing as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). We show that ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. Our results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes; and (2) keeping kni in an off state in tissues beyond the L2 vein. We propose that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development.

Animals↗

Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation.

CTLA-4 is a negative regulator of T-cell activation, and its inhibitory effects can be accomplished either by competition with CD28 or by transmitting negative signals through its intracellular domain. To utilize the cytoplasmic domain of CTLA-4 to suppress allergic inflammation, we fused it to a novel protein-transduction domain in the human transcriptional factor Hph-1. Transduction efficiency was verified in vitro and in vivo after ocular, intranasal and intradermal administration. After transduction into T cells, the Hph-1-ctCTLA-4 fusion protein inhibited the production of interleukin (IL)-2, and downregulated CD69 and CD25. Intranasal administration of Hph-1-ctCTLA-4 resulted in markedly reduced infiltration of inflammatory cells, secretion of T helper type 2 (T(H)2) cytokines, serum IgE levels and airway hyper-responsiveness in a mouse model of allergic airway inflammation. These results indicated that Hph-1-ctCTLA-4 constitutes an effective immunosuppressive protein drug for potential use in the treatment of allergic asthma, via nasal administration.

Administration, Intranasal↗

Expression of the polycomb-group gene BMI1 is related to an unfavourable prognosis in primary nodal DLBCL.

BACKGROUND: Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is highly variable and poorly predictable. Microarray studies showed that patients with DLBCL with a germinal centre B cell-like (GCB) phenotype have a better prognosis than those with an activated B cell-like (ABC) phenotype. The BMI1 proto-oncogene was identified as one of the genes present in the signature of the ABC type of DLBCL, associated with a poor prognosis. OBJECTIVES: (1) To investigate, in primary nodal DLBCL, the expression of BMI1 and its association with clinical outcome and DLBCL signature; (2) to look for an association between BMI1 expression and the expression of its putative downstream targets p14ARF and p16INK4a. RESULTS: BMI1 expression was found to be associated with poor clinical outcome, but not clearly with an ABC-like phenotype of DLBCL. Expression of BMI1 was frequently, but not always, related to low levels of expression of p14ARF and p16INK4a. CONCLUSION: Expression of BMI1 is associated with an unfavourable clinical outcome of primary nodal DLBCL.

Adult↗

Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group, but not Polycomb group, genes.

Genomic imprinting is a form of epigenetic inheritance that is characterized by differential expression of a gene depending on its parental origin. The mini-X chromosome Dp(1;f)LJ9 in Drosophila shows this type of classical imprinting; when transmitted by the maternal parent genes on this chromosome are fully expressed, but when the chromosome is transmitted by the male parent at least three genes are subject to silencing, resulting in a variegated expression pattern. Chemical and environmental modifiers of position-effect variegation have been shown to alter the somatic maintenance of the imprint. To extend these observations, several mutations in chromatin-associated proteins were examined for their effect on imprinting on the Dp(1;f)LJ9 mini-X chromosome. Effects on establishment and maintenance were independently assessed by genetically associating the mutations in chromatin modifiers with the mini-X chromosome in either the parents, where the imprint is established, or the progeny, in which the imprint must be maintained. Nine Suppressor of variegation [ Su(var)] mutations, including alleles of the Su(var)2-5 gene, which encodes the well characterized heterochromatin-associated protein HP1, abolished maintenance but not the establishment of the imprint. Mutant alleles of two genes in the trithorax group ( trx-G), brahma and trithorax, showed a maternal-effect enhancement of the paternal imprint. Surprisingly, however, with the exception of an Enhancer of Polycomb [ E(Pc)] allele, none of the Polycomb-group ( Pc-G) mutations tested affected the imprint. Thus, the maintenance of this imprint relies on the wild-type products of Su(var) and trx-G, but not Pc-G, genes. Finally, none of the mutations tested affected the maintenance of the maternal imprint or the establishment of either the maternal or paternal imprint, suggesting that the maternal and paternal imprints depend on different molecular processes and that imprint establishment and maintenance are independently regulated.

Animals↗

Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: one of the early inducible clones encodes a novel protein sharing several highly homologous regions with a Drosophila polyhomeotic protein.

To elucidate regulatory mechanisms triggering early mammalian differentiation, 17 retinoic acid (RA)-inducible clones were isolated from 1.4 x 10(5) plaques of cDNA libraries prepared from mouse embryonal carcinoma F9 cells, using the differential plaque hybridization method. Partial nucleotide sequences of these clones demonstrated that ten clones correspond to known genes. Interestingly, only 2 of the 17 clones are among the previously documented up-regulated genes. Therefore, there are many more unidentified genes up-regulated in the course of RA-induced differentiation of F9 cells. As RNAs hybridizable with one of the seven unidentified clones were induced in F9 cells after 3 h of RA treatment, we chose this 'Rae-28' clone as being representative of developmentally up-regulated unidentified clones and its properties were characterized. We determined the Rae-28 cDNA sequence and deduced the RAE-28 protein structure. The deduced RAE-28 protein shared several motifs and highly homologous regions with a Drosophila polyhomeotic protein. As the Drosophila polyhomeotic gene is involved in regulating morphogenesis, the rae-28 gene may participate in regulating early mammalian development.

Amino Acid Sequence↗

RNF2 interacts with the linker region of the human P-glycoprotein.

The human P-glycoprotein (Pgp) is a drug-efflux pump responsible for innate or acquired multidrug resistance in many cancers. Pgp contains a unique approximately 75 amino acid long linker region in its middle, which is critically important for its drug transport and ATPase functions. To identify cellular proteins that bind to this linker region and modulate Pgp function, a yeast two-hybrid analysis was carried out. This procedure identified RNF2 (RING finger protein 2), an E3 ubiquitin ligase, as a prominent Pgp-interacting protein. Co-expression of RNF2 with Pgp in Sf9 insect cells resulted in decreased ATPase activity and proteolytic protection of the transporter protein. Immunoprecipitation experiments confirmed the physical interaction between these two proteins. Confocal microscopy showed the presence of RNF2 in the cytoplasm of the Pgp-negative, drug-sensitive MCF-7 breast cancer cells. However, it was undetectable in the Pgp-positive and drug-resistant MCF-7 cells. We suggest that RNF2 regulates the cellular abundance of Pgp, and plays a key role in the development of cancer drug resistance through its own down-regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Role of Bmi1 in H2A ubiquitylation and Hox gene silencing.

Posttranslational histone modifications play a crucial role in the regulation of chromatin structure and gene activity. In previous studies, we identified the histone H2A ubiquitin ligase as Ring2, together in a complex with Ring1, Bmi1, and HPH2 (human polyhomeotic 2). We report here that the oncogene Bmi1 stimulates H2A ubiquitylation both in vitro and in vivo and that Bmi1-regulated H2A ubiquitylation is required for Hox gene silencing and normal cell growth. Our studies indicate that Bmi1 maintains the integrity of the complex through simultaneous interactions with the other subunits. We reconstituted the functional human H2A ubiquitin ligase complex and a panel of subcomplexes of different subunits. Comparisons of the H2A ubiquitin ligase activities of these different complexes revealed that Bmi1 stimulates the H2A ubiquitin ligase activity of Ring2 (and Ring1). Additionally, we demonstrated that the HoxC5 gene is regulated by ubiquitylated H2A in HeLa cells and that ubiquitylated H2A is localized on 5' regulatory regions of the HoxC5 gene. The role of Bmi1 in H2A ubiquitylation and HoxC5 gene expression in vivo was analyzed by RNA interference experiments. Knockdown of Bmi1 causes a global and loci-specific loss of H2A ubiquitylation, up-regulation of the HoxC5 gene, and slower cell growth. Intriguingly, Ring2 binds to its target regions in Bmi1 knockdown cells. Therefore, our studies reveal that Bmi1 is required for H2A ubiquitylation and suggest that H2A ubiquitylation regulates Bmi1-mediated gene expression.

Animals↗