1 Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, 134 ShinChon-Dong, SeoDaeMoon-Ku, Seoul, Korea 120-752
2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
3 Department of Biology, Kon-Kuk University, Seoul, Korea 143-701
4 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
5 Genome Sciences Department, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail stop 84-171, Berkeley, CA 94720, USA
*Author for correspondence (e-mail: mdbiggin{at}lbl.gov)
Accepted 17 December 2001
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SUMMARY |
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Key words: Polycomb group, Trithorax group, Homeotic, Zeste, GAGA, Drosophila
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INTRODUCTION |
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The PcG are a coherent set of genes whose sole function is to maintain repression of homeotic and other developmental control genes. Repression is maintained only in those cells in which transcription has not been activated in the pregastrual embryo. PcG repression has been likened to form of a molecular memory because the PcG proteins must continuously mark those genes that are initially repressed in the early embryo (Bienz and Muller, 1995; Cavalli and Paro, 1999
; Pirrotta, 1998
). The mark on the gene must be continuous as there are no spatially restricted regulators in older embryos that can reinitiate the correct pattern of homeotic expression. PcG proteins are physically associated with their target genes, suggesting that they form a stable structure that is propagated through multiple rounds of cell division (Sinclair et al., 1998
; Strutt and Paro, 1997
). The Polycomb system is conserved in most animals, including mammals, and is important for maintaining the determined state of cells (Hashimoto et al., 1998
; Strouboulis et al., 1999
).
The trxG are ubiquitously expressed activators of one or more of the homeotic genes and are not as homogenous as the Polycomb group (Kennison, 1995; Tillib et al., 1999
). By definition, the trxG are distinct from the spatially restricted activators that initiate the early patterns of homeotic transcription. However, unlike the PcG, which are dedicated to a shared set of targets, the trxG act on a wide range of different genes; there is much less overlap in the genes that are regulated by each of the trxG members.
It has often been assumed that the PcG and trxG are mutually exclusive. However, several genetic experiments have hinted at the possibility that some trxG proteins are also involved in Polycomb repression (Gildea et al., 2000; Hagstrom et al., 1997
; LaJeunesse and Shearn, 1996
; Wu et al., 1989
). For example, larvae mutant for the PcG gene Enhancer of Zeste show reduced expression of homeotic genes in some modified backgrounds (LaJeunesse and Shearn, 1996
). But because the trxG members have broad pleiotropic effects, it could not be ruled out that the regulation seen in these experiments was indirect and that the PcG and trxG are therefore distinct.
We show that the trxG proteins Zeste and GAGA play a direct role in maintaining repression of the homeotic gene Ultrabithorax (Ubx). Based on these data and on previous results demonstrating that Zeste binds to a promoter regardless of its activation state, we propose a new model for the establishment of Polycomb repression in the early embryo. We suggest that the selective recruitment of the PcG to their targets requires proteins that function in both transcriptional activation and repression in the following manner. On inactive promoters in the early embryo, these duel activity proteins are bound to the DNA but are not sequestered in a regulatory complex with other activators. In cells where the promoters are transcribed before gastrulation, however, we propose that these factors make protein/protein interactions with an activation complex. Thus, surfaces on the dual activity factors would be differently exposed, depending on the transcription state, providing a unique tag that the PcG factors could read.
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MATERIALS AND METHODS |
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Drosophila strains and P-element-mediated transformation
Germline transformation, analysis of expression patterns, and crosses into zv77h and zae(bx) mutant embryos were performed as described previously (Laney and Biggin, 1992; Spradling, 1986
; Patel, 1994
), except that the host microinjection stock was w1118, and chromosomal linkage of inserts was determined by crosses with the balancer fly, w/Y; CyO;MKRS/apXa. Four to 13 homozygous independent transgenic fly lines for each Ubx-lacZ fusion construct were obtained: six for 22UZ GAGA, 11 for 22UZ Deletion, 13 for 22UZ ZESTE, four for 22UZ NTF-1, four for 22UZ ZESTE/NTF1 and six for 22UZ GAGA/NTF-1. All lines for a given construct give essentially the same pattern of expression.
To analyze 22UZ GAGA transgene expression in Pc3 embryos, flies homozygous for a 22UZ GAGA transgene located in the second chromosome were crossed to w; st in ri Pc3 pp/TM3, Sb Ser to produce w /w; 22UZ GAGA; Pc3 flies. These were then self crossed to generate w/w; 22UZ GAGA; Pc3/Pc3 embryos. To analyze expression of 22UZ Zeste transgenes, a 22UZ Zeste transgene inserted on the third chromosome was recombined with Pc3. The resulting w/w; 22UZ Zeste Pc3/TM3 flies were crossed to produce w/w; 22UZ Zeste Pc3/22UZ Zeste Pc3 embryos.
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RESULTS AND DISCUSSION |
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As ectopic expression of Ubx in anterior and posterior regions is generally caused by a failure of the initiating repressors or the Polycomb maintenance system (Chan et al., 1994; Simon et al., 1993
; Zhang and Bienz, 1992
). One interpretation of the above result is that Zeste and GAGA are required for at least one form of repression, while NTF-1 is not. It is also possible, however, that Zeste and GAGA are not repressors. Instead, it may be that they are unable to activate expression in anterior or posterior regions, even though they are expressed at similar levels throughout the embryo (Bhat et al., 1996
; Pirrotta et al., 1988
). To distinguish between these two possibilities, we first examined constructs that contained either Zeste and NTF-1 sites or GAGA and NTF-1 sites. These constructs are expressed in the central region of the embryo; but, importantly, they are not significantly expressed in anterior or posterior regions (Figs 1, 2) (M.-W. Hur, unpublished). As NTF-1 can activate Ubx transcription in these terminal regions (Fig. 1E), the absence of terminal expression is consistent with GAGA and Zeste directly repressing transcription in addition to their activation function.
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Consistent with this idea, transgenes containing only Zeste sites at the proximal promoter fail to express in zeste mutant embryos, whereas constructs containing only GAGA or NTF-1 binding sites are expressed in this same genetic background (Fig. 2A,B,E,F). Thus, this genetic experiment confirms that Zeste bound at the proximal promoter is required to activate transcription of the 22UZ constructs in the normal domain of Ubx expression. To test the role of Zeste in repression, constructs containing binding sites for both Zeste and NTF-1 at the proximal promoter were compared in wild type and zeste mutant embryos. In the normal domain of Ubx expression, these constructs are expressed at similar levels in mutant and wild-type embryos. Importantly, these constructs are derepressed in anterior and posterior regions of embryos lacking zeste (Fig. 2G-L). Thus, Zeste actively represses transcription in terminal regions of the embryo via binding sites at the proximal promoter.
The embryos shown in Fig. 1 and Fig. 2A-I are at late stages of development, well after the Polycomb maintenance system has become active. To distinguish if Zeste is required for the initiation or the maintenance of repression, we examined expression of the 22UZ ZESTE/NTF-1 construct at an earlier stage. As Fig. 2K shows, in embryos that lack zeste, the 22UZ ZESTE/NTF-1 transgene is almost fully repressed in anterior and posterior regions at this earlier stage. Only weak derepression is observed in a few isolated cells. Thus, the transiently expressed factors that initiate repression in the early embryo must be active, and the extensive derepression observed later must be due to a failure in the maintenance system.
The PcG genes are an essential part of system that maintains repression of the endogenous Ubx gene. To confirm that these genes also act on our transgenes, the 22UZ Zeste and 22UZ GAGA constructs were crossed into PcG mutant embryos. Fig. 3 shows that both transgenes are derepressed in late stage embryos lacking the Polycomb gene. Similar results were obtained in embryos lacking another PcG gene, extra sex combs (M.-W. Hur, unpublished). Thus, Zeste and probably also GAGA act together with the Polycomb system to maintain repression of Ubx.
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The data presented in this paper are consistent with the earlier genetic data that suggested that some trxG and PcG proteins may have dual activities (Gildea et al., 2000; Hagstrom et al., 1997
; LaJeunesse and Shearn, 1996
; Wu et al., 1989
). Further support for this idea comes from recent biochemical experiments that have shown that GAGA is complexed with two PcG proteins in Drosophila nuclear extracts (Horad et al., 2000
) and Zeste is part of a multisubunit complex that contains Polycomb (Saurin et al., 2001
). In addition, PcG proteins are frequently associated in vivo with promoter regions that include Zeste or GAGA DNA recognition sites, including the Ubx proximal promoter examined in this paper (Orlando et al., 1998
). Most PcG proteins do not recognize specific DNA sequences; thus, the interaction with Zeste and GAGA may serve to recruit PcG proteins to promoters.
But is it essential that some proteins, such as Zeste and GAGA, participate in both repression and activation, or is it mere coincidence? We suggest that it may be essential. At the transition between the initiating repressors and the Polycomb system, one possibility is it that Polycomb proteins are recruited to or activated on only those genes that are bound by initiating repressors; the initiating repressors may physically bind to PcG proteins to recruit them. However, Poux et al. have shown that Polycomb repression can be established on Ubx promoter constructs that lack initiating repressors elements, provided that initiating enhancer elements are also absent (Poux et al., 1996). In other words, at the transition between the establishment and maintenance of the Ubx expression pattern, the Polycomb systems reads the absence of activation, rather than the presence of repression or repressors.
Endogenous Zeste protein binds to Ubx promoter constructs in vivo whether they are transcribed or not (Laney and Biggin, 1997). We suggest that in the early embryo in the cells in which Ubx is activated, Zeste is complexed, directly or indirectly, with initiating activators on the Ubx promoter. These complexes mask surfaces on Zeste that would otherwise be bound by components of the Polycomb system. By contrast, in those cells where Ubx is not activated, Zeste is still bound to the promoter but is not be part of an activating complex. Surfaces on Zeste protein would then be exposed and could serve as the signal that the Polycomb system reads to initiate the maintenance phase of repression. The dual activities of Zeste and GAGA could be a key to understanding this fascinating regulatory mechanism.
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ACKNOWLEDGMENTS |
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REFERENCES |
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