Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S.cerevisiae telomeres
1997; Springer Nature; Volume: 16; Issue: 11 Linguagem: Inglês
10.1093/emboj/16.11.3219
ISSN1460-2075
AutoresGötz Laible, Andrea Wolf, Rainer Dorn, Günter Reuter, Corey Nislow, Angelika Lebersorger, Dan Popkin, Lorraine Pillus, Thomas Jenuwein,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle1 June 1997free access Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S.cerevisiae telomeres Götz Laible Götz Laible Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Andrea Wolf Andrea Wolf Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Rainer Dorn Rainer Dorn Institute of Genetics, Martin Luther University of Halle, Domplatz 1, D-06108 Halle/S, Germany Search for more papers by this author Gunter Reuter Gunter Reuter Institute of Genetics, Martin Luther University of Halle, Domplatz 1, D-06108 Halle/S, Germany Search for more papers by this author Corey Nislow Corey Nislow Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309-0347 USA Search for more papers by this author Angelika Lebersorger Angelika Lebersorger Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Dan Popkin Dan Popkin Search for more papers by this author Lorraine Pillus Lorraine Pillus Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309-0347 USA Search for more papers by this author Thomas Jenuwein Thomas Jenuwein Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Götz Laible Götz Laible Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Andrea Wolf Andrea Wolf Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Rainer Dorn Rainer Dorn Institute of Genetics, Martin Luther University of Halle, Domplatz 1, D-06108 Halle/S, Germany Search for more papers by this author Gunter Reuter Gunter Reuter Institute of Genetics, Martin Luther University of Halle, Domplatz 1, D-06108 Halle/S, Germany Search for more papers by this author Corey Nislow Corey Nislow Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309-0347 USA Search for more papers by this author Angelika Lebersorger Angelika Lebersorger Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Dan Popkin Dan Popkin Search for more papers by this author Lorraine Pillus Lorraine Pillus Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309-0347 USA Search for more papers by this author Thomas Jenuwein Thomas Jenuwein Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria Search for more papers by this author Author Information Götz Laible1, Andrea Wolf1, Rainer Dorn2, Gunter Reuter2, Corey Nislow3,4, Angelika Lebersorger1, Dan Popkin5, Lorraine Pillus3 and Thomas Jenuwein1 1Research Institute of Molecular Pathology (IMP), Dr Bohrgasse 7, A-1030 Vienna, Austria 2Institute of Genetics, Martin Luther University of Halle, Domplatz 1, D-06108 Halle/S, Germany 3Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309-0347 USA 4Department of Biology, Rutgers University, Camden, NJ, 08102 USA 5Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA The EMBO Journal (1997)16:3219-3232https://doi.org/10.1093/emboj/16.11.3219 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes. Introduction Maintenance of different patterns of gene expression is the underlying mechanism by which cell type identity is inherited in eukaryotes and has been correlated with the organization of chromatin domains that modulate gene activity (Zuckerkandl, 1974). Paradigms for chromatin-controlled regulation of key developmental loci include silencing of the mating type loci in Saccharomyces cerevisiae (reviewed in Loo and Rine, 1995) and restriction of expression boundaries within the homeotic gene cluster (HOM-C) in Drosophila (Wedeen et al., 1986; Simon et al., 1993). Moreover, gene expression in eukaryotes is influenced by structural alterations in chromatin, phenomena referred to collectively as position effects. For example, genes placed in the vicinity of yeast telomeres (Gottschling et al., 1990; Nimmo et al., 1994) and centromeres (Allshire et al., 1994), or at cytologically defined heterochromatic positions in the fly (reviewed in Weiler and Wakimoto, 1995), are severely downregulated. In addition, many genes on the inactive X chromosome and several imprinted genes (for reviews, see Efstradiadis, 1994; Migeon, 1994) are repressed in mammals. Basic chromatin components are known to participate in transcriptional regulation because alterations in dosage (Moore et al., 1979; Han and Grunstein, 1988) and modification (Jeppesen and Turner, 1993) of the core histones affect gene expression. However, more complex levels of control are suggested from genetic screens on position effect variegation (PEV) in Drosophila and S.cerevisiae. These screens have identified distinct classes of genes that appear centrally involved in the regional organization of chromatin domains and the regulation of chromatin-dependent gene activity. In contrast to classical transcription factors, many of these chromatin regulators do not seem to bind DNA in a sequence-specific manner, but rather associate with structurally altered chromatin or are recruited by factors already present at target sequences (for reviews, see Loo and Rine, 1995; Orlando and Paro, 1995; Pirrotta, 1995). Models for the function of chromatin regulators have been proposed to explain variegation and the clonal nature of gene expression within a given cell population (Tartof et al., 1984; Henikoff, 1995; Karpen, 1995; Csink and Henikoff, 1996; Dernburg et al., 1996). Consistent with these models, telomeric silencing in yeast and PEV in Drosophila display an epigenetic component, and it has been suggested that the epigenetic fine-tuning of chromatin-controlled gene expression patterns facilitates the generation of cell type diversity in eukaryotes (Zuckerkandl, 1974; Eissenberg et al., 1995; Pillus and Grunstein, 1995). In Drosophila, ∼120 loci have been described genetically that enhance or suppress position-dependent gene expression, but only ∼10% of the corresponding genes have been isolated so far (Reuter and Spierer, 1992). Interestingly, several of the known modifiers of PEV share structural motifs with both activators [trithorax or trx-group (trx-G)] and repressors [Polycomb or Pc-group (Pc-G)] of chromatin-dependent gene activity within HOM-C. The biological significance of chromatin-mediated gene regulation, particularly for controlling mammalian development, has gained further importance by the functional analysis of murine homologues of Drosophila Psc (Pc-G) and trx. Both of these mammalian homologues appear to be involved in specifying expression boundaries within the murine HOX-clusters (Alkema et al., 1995; Yu et al., 1995; Akasaka et al., 1996), and their deregulation has been implicated in inducing leukaemia (Gu et al., 1992; Tkachuk et al., 1992; van der Lugt et al., 1992; Alkema et al., 1995). The structural similarities between gene products that are implicated in controlling gene expression at specific euchromatic locations (HOM-C) and in the vicinity of heterochromatin (PEV) point to a common mechanism(s) for the regulation of chromatin-dependent gene activity. However, only a few modifiers of PEV have been shown to induce homeotic transformations (Dorn et al., 1993; Farkas et al., 1994), and a direct structural involvement of Pc-G genes with heterochromatic regions has not yet been demonstrated. Recently, a 130 amino acid, carboxy-terminal region of high sequence similarity has been identified that is shared between trx and the Pc-G gene Enhancer of zeste [E(z)] (Jones and Gelbart, 1993). This carboxy terminus is also conserved in Su(var)3-9, a dominant suppressor of PEV in Drosophila (Tschiersch et al., 1994). Because of the genetically defined antagonistic relationship between trx and E(z) (Jones and Gelbart, 1993; LaJeunesse and Shearn, 1996), this novel protein domain, termed SET (Tschiersch et al., 1994), may play a role in the assembly of either activating or repressing chromatin complexes, dependent on interactions with accessory proteins from the trx- or Pc-G. Consistent with this notion, E(z) mutants have been shown to reduce immunostaining of some trx- and Pc-G proteins at polytene chromosomes (Rastelli et al., 1993; Platero et al., 1996). Finally, the SET domain is evolutionarily conserved and is present in gene products ranging from yeast to man (Stassen et al., 1995). Based on the above criteria, the SET domain appears to define a new gene family of important chromatin regulators. Here, we describe the isolation of mammalian homologues of the Drosophila Pc-G gene E(z) and demonstrate function of the E(z) gene family in mediating gene repression at well-defined heterochromatic regions in Drosophila and S.cerevisiae chromatin. These data provide a functional link between Pc-G-dependent gene silencing and the organization of inactive chromatin domains. Results Mammalian E(z) homologues are encoded by two loci Based on sequence information from the conserved carboxy-terminal SET domain of Drosophila E(z), we screened a human B-cell-specific cDNA library for mammalian homologues (see Materials and methods). Out of 500 000 plaques, 36 primary phages were selected. Five isolates contained partial cDNA fragments of a gene that was recently mapped to human chromosome 17 and which has been named EZH1 (Abel et al., 1996). By contrast, the majority of the isolates contained novel cDNA sequences from a second human gene, which we designated EZH2 (Enhancer of zeste homologue 2). A full-length human EZH2 cDNA of 2.6 kb encoding a protein of 746 amino acids was obtained by combining DNA fragments from different subclones. Using the human EZH2 cDNA as a probe, we next screened a mouse brain cDNA library for murine homologues of E(z). From the mouse brain library, only cDNAs encoding sequences with highest homology to human EZH1 were found. A 5′ RACE amplification was used to complete the missing 5′ end and, after subcloning, a murine Ezh1 cDNA that encodes a protein of 747 amino acids was obtained. Recently, a murine cDNA homologous to human EZH2 has been isolated by others (Hobert et al., 1996a). To examine directly the number of mammalian E(z) homologues, we hybridized, under reduced stringency, a DNA blot containing Drosophila, mouse and human DNA with a mixed DNA probe that was derived from the SET domains of the human EZH2 and mouse Ezh1 cDNAs (Figure 1A, top). Whereas only a single fragment is detected in Drosophila DNA, 2–4 fragments hybridized with this mixed SET domain probe in both mammalian DNAs. The fragments could either be correlated to predicted sizes from the genomic murine Ezh1 locus (Figure 1A, bottom) or are exclusively detected by the human EZH2 SET domain probe (data not shown). Moreover, homology searches (Altschul et al., 1990) against the currently available human expressed sequence tags (ESTs) (Bassett et al., 1995) indicated an almost perfect match with either EZH2 or EZH1. Thus, we conclude that human and mouse E(z) homologues are represented by not more than two distinct loci which most likely reflect a gene duplication of a single ancestral locus. Figure 1.Mammalian E(z) homologues are encoded by two distinct loci. (A) DNA blot analysis of E(z)-related sequences in 15 μg of digested Drosophila, mouse and human DNA. Digests were performed with the restriction enzymes HindIII (HIII), EcoRI (RI) or with a combination of BglII and EcoRI (BII/RI). The DNA blot was hybridized under reduced stringency with a mixed DNA probe derived from the SET domains of the human EZH2 and murine Ezh1 cDNAs. Mouse DNA fragments that correspond to predicted sizes from the genomic murine Ezh1 locus (see the schematic diagram at the bottom) are indicated by an asterisk, and those exclusively detected by the human EZH2 SET domain probe are highlighted by a solid dot. Position and sizes of DNA marker fragments are indicated at the left. Below, the 3′ portion of the genomic Ezh1 locus, comprising SET domain exons (black boxes), is shown. Numbering of the exons refers to amino acid positions in Ezh1. (B) RNA blot analysis of E(z)-related transcripts in 5 μg of poly(A)+ RNA isolated from human (BJAB) and mouse (PD31 and J558L) B-cell lines and from mouse kidney (KI). The RNA blot was sequentially hybridized under high stringency with DNA probes derived from nearly full-length murine Ezh1 cDNA (left panel), full-length human EZH2 cDNA (right panel) and from murine β-actin sequences (bottom panel). The sizes of the respective transcripts were calculated according to the indicated molecular weight markers. Download figure Download PowerPoint To determine the accurate sizes of the mRNAs encoded by Ezh1 and EZH2, we hybridized an RNA blot containing poly(A)+ RNA from human (BJAB) and mouse (PD31 and J558L) B-cell-specific cell lines and from mouse kidney with DNA probes that were derived from the Ezh1 and EZH2 cDNAs (Figure 1B). The Ezh1 probe detected a specific mRNA of ∼4.4 kb which is present in all RNAs analysed and whose size is in good agreement with the 4.0 kb Ezh1 cDNA that lacks ∼200 bp of 3′ untranslated sequences. Rehybridization of the RNA blot with a DNA probe specific for the full-length EZH2 cDNA identified a mRNA of 2.8 kb which closely matches the size of the EZH2 cDNA in both the human and mouse cell lines, but not in mouse kidney. Structural conservation of mammalian and fly E(z) proteins Human EZH1 and murine Ezh1 (G.Mattei, A.Lebersorger and T.Jenuwein, unpublished), and probably also EZH2/Ezh2, are encoded by orthologous loci in mouse and man, and in both comparisons amino acid identities are 98%. By contrast, inverse comparisons of EZH2/EZH1 or Ezh2/Ezh1 only reach an overall amino acid identity of 67%, suggesting that the presumed gene duplication may have occurred at a relatively early stage after the divergence of vertebrate and non-vertebrate species. Considering the overall cDNA size and sequence comparisons, EZH2/Ezh2 and E(z) appear to be most closely related, whereas EZH1/Ezh1 is the more divergent of the mammalian gene pairs. Over the entire length of the 760 amino acids containing E(z) protein (Jones and Gelbart, 1993), EZH2/Ezh2 is 61% and EZH1/Ezh1 is 55% identical to E(z). Thus, human and mouse homologues of Drosophila E(z) display the highest structural conservation among any of the currently known mammalian and Drosophila chromatin regulators. Alignment of all four mammalian E(z) proteins with Drosophila E(z) reveals four regions of high sequence identity (Figure 2). In addition to the most highly conserved SET domain (86% identity), an 115 amino acid, cysteine-rich region immediately precedes the SET domain (68% identity). Most of the cysteine residues, originally noted for E(z) (Jones and Gelbart, 1993), are conserved in the mammalian proteins. However, these cysteine residues (a total of 18) are unusually spaced and do not show any apparent homology to other well-defined cysteine-rich regions. In the amino-terminal half, two stretches, one of 66 amino acids (domain I; 66% identity) and one of 114 amino acids (domain II; 56% identity), are highly related between mammalian and fly E(z) proteins. Domain II also contains six conserved cysteine residues with unusual spacing and is separated from domain I by a stretch of charged amino acids. Finally, the position and sequence of the nuclear localization signal of the E(z) protein (Jones and Gelbart, 1993) is also conserved in all mammalian homologues. Figure 2.Conserved domains of mammalian and fly E(z) proteins. Amino acid sequences of human (EZH1 and EZH2), mouse (Ezh1 and Ezh2) and Drosophila E(z) proteins were aligned using the PILEUP program of the GCG software package. Amino acids that are identical in all five proteins are shown in green, and conserved cysteine residues are highlighted by a pink colour. Four regions of high sequence similarity are indicated to the right, together with the respective amino acid identities calculated from conserved positions in all five proteins within these individual domains. The domain boundaries (indicated by arrowheads) overlap or are in close proximity to exon/intron positions determined for Ezh1 (data not shown) and E(z) (Jones and Gelbart, 1993). The conserved nuclear localization signal noted for E(z) (Jones and Gelbart, 1993) is underlined. Full-length sequence information for EZH1 was kindly provided by Ken Abel (1996), and the Ezh2 sequence has been reported recently (Hobert et al., 1996a). The full-length EZH2 protein extends the amino terminus of a truncated EZH2 variant (Hobert et al., 1996b) by 133 amino acids. Also indicated by an asterisk above the EZH2 sequence are amino acid positions that are invariant between the E(z) gene family and S.cerevisiae SET1 (1080 amino acids). The position of an additional stretch of 62 amino acids in SET1 that is unrelated to E(z) homologues is shown. Download figure Download PowerPoint Complementary expression profiles of murine Ezh1 and Ezh2 The differences in the abundance of Ezh1 and Ezh2 transcripts in kidney suggested that both loci may be differentially regulated during mouse development. To analyse the temporal and tissue-specific expression profile of the two Ezh genes during mouse development, we performed an RNase protection analysis with antisense RNA probes that are specific for the SET domain of Ezh1 or for the amino-terminal end of Ezh2. Ezh1-specific transcripts were detected ubiquitously during mouse embryogenesis; however, their relative abundance is gradually upregulated during development and reaches 4- to 5-fold higher levels in kidney, brain and skeletal muscle of adult mice (Figure 3, top panel). A 2- to 3-fold increase in the relative abundance (after correction for differences in the amount of RNA as indicated by the S16 RNA control transcripts) of Ezh1 transcripts was also detected after retinoic acid-induced in vitro differentiation of embryonic stem cells. By contrast, Ezh1 transcripts remained at a low level in liver, spleen and thymus. Figure 3.Expression analysis of murine Ezh1 and Ezh2. RNase protection analysis detecting Ezh1 and Ezh2 transcripts during mouse (129/Sv) development. 10 μg of total RNA were hybridized with riboprobes that specifically protect Ezh1 (top panel) or Ezh2 (middle panel). As a quantitation control, an RNA probe that protects murine S16 rRNA sequences was included in the hybridizations. Total RNA was prepared from undifferentiated D3 embryonic stem cells (ES), embryoid bodies derived after retinoic acid-induced in vitro differentiation of D3 cells (EBdiff), whole embryos at day E9.5 and fetal liver (FL), brain (BR) and skeletal muscle (SM) of day E13 or day E17 embryos. In addition, total RNA was also prepared from adult tissues, including kidney (KI), spleen (SP), liver (LI), thymus (TH), brain (BR) and skeletal muscle (SM). Download figure Download PowerPoint The RNase protection analysis of Ezh1 transcript levels during mouse embryogenesis and ES cell differentiation demonstrates a specific upregulation of Ezh1 gene activity at later stages of development. By contrast, Ezh2 transcript levels are downregulated during progressing development and display a more thymus-restricted expression (Figure 3, middle panel; Hobert et al., 1996a), resulting in complementary expression profiles of Ezh1 and Ezh2 that may indicate dosage dependence or distinct functions of the encoded proteins. Further, observed splicing variants for EZH2 (data not shown) or Ezh2 (Hobert et al., 1996a) that remove the amino-terminal half of the conserved cysteine-rich region or part of the SET domain in EZH1 (Abel et al., 1996) could encode functionally distinct protein isoforms. Alternatively, Ezh1 and Ezh2 may provide similar functions, but because of the presence of two loci, transcript levels may be balanced to protect against negative gene dosage effects which, in analogy to other described Drosophila chromatin regulators (Reuter et al., 1990; Eissenberg et al., 1992; Tschiersch et al., 1994), could interfere with normal gene function. EZH2 and E(z) enhance PEV in Drosophila Although Pc-G-dependent gene silencing has been correlated with the establishment of repressive chromatin domains (see Introduction), most of the Pc-G genes do not affect gene repression in the vicinity of heterochromatin (Grigliatti, 1991; G.Reuter, unpublished). However, E(z) mutants have been described recently that reduce immunostaining of several Pc-G proteins at natural euchromatic positions (Rastelli et al., 1993) and prevent the recruitment of Pc-G complexes at ectopic heterochromatic locations (Platero et al., 1996). Together with the extremely high degree of evolutionary conservation between mammalian and fly E(z) homologues, these data suggested a crucial role for E(z) proteins in regulating regional gene activities in chromatin. To demonstrate an involvement of E(z) genes in controlling the organization of repressive chromatin domains, we analysed the potential of EZH2 and E(z) to modify PEV in Drosophila. By P element-mediated germline transformation, we established nine transgenic fly lines that carry the human EZH2 cDNA under the control of the heat shock promoter hsp70. As a control, we also generated transgenic flies carrying the E(z) cDNA (Jones and Gelbart, 1993) (four lines) or a genomic fragment encoding E(z) sequences (Jones and Gelbart, 1993) (one line). For all of the lines, the chromosomal assignment of the transgene was mapped genetically (see Table I) and the transgenic lines were crossed into In(1)wm4h and In(1)wm4h; Su(var)2-1 indicator strains (see Materials and methods). These indicator strains carry an inversion [In(1)wm4h] which places the white gene adjacent to pericentric X heterochromatin, resulting in stochastically repressed, variegated patterns of gene expression that can be easily detected as red or white patches in the Drosophila eye. Mutations in the suppressor of variegation Su(var)2-1, which has been shown to reduce deacetylation of histone H4 (Dorn et al., 1986), derepress heterochromatin-restricted wm4h expression, leading to an almost wild-type red eye phenotype. Table 1. Quantitation of PEV enhancement and paternal imprinting by human EZH2 and Drosophila E(z). Quantitation of PEV enhancement and paternal imprinting by human EZH2 and Drosophila E(z). Offspring of crosses between various fly lines transgenic for human EZH2 or Drosophila E(z) (indicated at left) and wm4h indicator strains that contain an additional suppressor mutation (Su(var)2-1) were genotyped according to phenotypic markers. The chromosomal assignment of the respective transgene has been mapped and is shown. The E(z) gene has been localized to region 67E on chromosome 3 (Kalisch and Rasmuson, 1974; Jones and Gelbart, 1990). Pooled red eye pigments extracted from the eyes of 10 individual siblings representing the respective genotypes were measured for their absorbance at 480 nm. The values given are the mean of three independent measurements with a SD of <10%. The enhancer effect on PEV (indicating repression of wm4h) varied among the lines and resulted in pronounced (5- to 10-fold; +++), intermediate (2- to 3-fold; ++) and weak (∼2-fold; +) reduction in the ratio of the absorbance between wm4h; Su(var)2-1 (0.125) and transgenic red eye pigments. The E(z) transgene of the negative line D was mobilized, resulting in eight sublines (of which only two are shown) with a significant enhancer effect on PEV. By contrast, the E(z)5 null and the antimorphic E(z)1 mutations are weak suppressors (Su) of PEV, whereas the hypomorphic, wild-type like E(z)15 mutation has no effect. In addition, most of the lines exhibit a 2- to 3-fold reduction in the proportion of red eye pigments in outcrossed male offspring, although the transgene is no longer present, indicating paternal imprinting (p) of wm4h repression. Visual inspection of progeny of the respective crosses indicated that six out of the nine EZH2 lines and two out of the five E(z) lines induced an increase in the proportion of unpigmented areas in the eyes, therefore classifying both EZH2 and E(z) as dose-dependent modifiers of PEV (Figure 4). Moreover, after mobilization of the E(z) transgene in a negative line (line D), eight sublines with a significant enhancer effect on PEV could be selected (Table I). PEV enhancement was largely independent of heat shock treatment, and subsequent expression analysis by in situ hybridization demonstrated a low, basal transcription throughout embryonic development of those lines displaying an enhancer effect (data not shown). Figure 4.EZH2 and E(z) enhance PEV in Drosophila. Flies transgenic for human EZH2 (lines D and E) or Drosophila E(z) (line B) were crossed to an indicator strain containing an inversion that places the white gene adjacent to pericentric X heterochromatin (wm4h). In addition, transgenic flies were also mated to a sensitized indicator strain that carries a strong suppressor mutation [Su(var)2-1]. Progeny of the crosses was genotyped according to phenotypic markers, and transgene-dependent repression of wm4h is detected by increased proportions of unpigmented areas in the eyes, thus categorizing EZH2 and E(z) as enhancers of PEV. NT, non-transgenic offspring; TG, transgenic offspring. Download figure Download PowerPoint To quantify the degree of PEV enhancement of the individual transgenic lines, eye pigments were extracted from the progenies of the respective crosses and pigment absorbance at 480 nm was measured. The result of these quantitations, summarized in Table I, shows that the strongest enhancer effect of EZH2 (line E) is similar to E(z) and results in an ∼10-fold reduction in the concentration of red eye pigments as compared with non-transgenic siblings. Thus, the Drosophila Pc-G gene E(z), which has been implicated in restricting gene-specific expression boundaries within the chromatin domains of HOM-C (Jones and Gelbart, 1990; Phillips and Shearn, 1990), is also capable of stabilizing a repressive transcriptional state in the vicinity of constitutive heterochromatin. Moreover, this potential of E(z) to repress heterochromatin-associated gene activity is conserved in its human homologue EZH2. EZH2-mediated enhancement of PEV displays paternal imprinting In the course of the matings to the indicator strains, we observed that non-transgenic In(1)wm4h and In(1)wm4h; Su(var)2-1 male progeny, derived from crosses with most of the transgenic EZH2 or of the transgenic E(z) lines [lines indicated (p) in Table I], also exhibited an ∼2- to 3-fold reduction in the proportion of red eye pigments as compared with unmated In(1)wm4h and In(1)wm4h; Su(var)2-1 control flies (Table I), although the transgene was no longer present. This enhancer effect on wm4h gene repression was strictly dependent upon previous paternal transmission of the transgene. Although the molecular basis for this imprinting-like effect is not known, this result suggests that EZH2 and E(z) can participate not only in the induction, but also in the propagation of an altered chromatin structure which represses wm4h gene activity in trans, and is reminiscent of transvection phenomena described for the zeste1–white interaction (Kalisch and Rasmuson, 1974; Jones and Gelbart, 1990; Phillips and Shearn, 1990). In support of this interpretation, most transgenic EZH2/zeste1 heterozygote females display zeste1 gene variegation after heat shock (data not shown). Thus, together with the enhancement of PEV shown above, these data extend the functional conservation between EZH2 and E(z) in regulating repressive transcriptional states in Drosophila chromatin. EZH2 and Ezh1 restore telomeric silencing in S.cerevisiae As a second assay to examine involvement of
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