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Zygotically transcribed genes
The six Drosophila proteins that belong to the antennapedia-type Homeobox subfamily are Antennapedia (ANTP), Abdominal-A (ABD-A), Deformed (DFD), Proboscipedia (PB), Sex combs reduced (SCR) and Ultrabithorax (UBX). The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the'Homeobox' antennapedia-type protein signature.
Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, it was shown that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system (Schmitt, 2005; full text of article).
Intergenic transcription in the BX-C has a profound phenotypic effect on the expression of the Hox genes. The results with transgenes suggest that the spatially and temporally regulated transcription of noncoding RNAs in the BX-C induces a remodeling of the chromatin, thereby preventing PcG-mediated silencing. The consequence of this is the segment-specific activation of the Hox genes. This is probably especially important in large gene complexes where PREs are located at long distances from the promoters they regulate. It has been shown that the activation of a minimal 219-bp Fab-7 PRE is not accompanied by transcription through the element. However, in this case the minimal PRE was juxtaposed to the promoter, probably benefiting from the open chromatin environment induced by the bound transcription factors. Other results indicate that an 870-bp large Fab-7 PRE, under similar conditions but containing more PcG protein-binding sites, cannot be activated anymore. This suggests that over a certain threshold level of silencing, imposed by the stability of the silencing complexes, chromatin remodeling by transcription is required to remove PcG complexes in order to counteract their silencing activity in a mitotically heritable fashion (Schmitt, 2005).
Hox cluster regulation is only part of the entire PcG/trxG memory system, prompting an analysis of the transcription pattern of characterized en and predicted salm, slou, and tll target genes as well as their respective PRE sequences. RNA in situ hybridizations reveal that these sequences are also transcribed in a pattern that reflects the expression of the cognate target genes. This suggests that the mechanism of epigenetic activation of PREs initially described for the homeotic gene clusters may be required for the regulation of many more PcG target genes than previously thought. Transcription through these PREs can be either uni- (tll PRE) or bidirectional (en, slou, and salm PREs), further suggesting that the induction of epigenetic activation relies on the remodeling of chromatin induced by the transcriptional process, rather than by the noncoding RNAs (Schmitt, 2005).
With the transgenic reporter system, it was shown that the constitutive transcription through the Fab-7 PRE from the actin5c promoter results in the stable epigenetic activation of this element. This raises the question of how this could be achieved mechanistically. As has been proposed for the developmental regulation of globin gene expression and the regulation of VDJH-recombination in mice, the opening of the chromatin structure at a transcribed PRE may be induced by the passing of the transcriptional machinery through the regulatory sequence. It has been shown that the elongating RNA polymerase II complex is associated with the SWI/SNF remodeling complex and a histone acetyl transferase activity. Such enzymatic activities linked with the transcription machinery may catalyze the epigenetic modification of chromatin (Schmitt, 2005).
In this respect, it is interesting to note that most of the trxG mutants were initially uncovered as suppressors of the Pc phenotype, and that the combination of PcG with trxG mutations can restore the typical phenotype of the single mutations to wild type. The molecular mechanism behind this antagonism is not clear. Using clonal analysis, it has been shown that the trxG proteins Ash1 and Trx do not function as coactivators of Hox gene expression, but that they are required as anti-repressors to prevent PcG-induced silencing. In contrast, the Brahma complex containing the trxG proteins Brm, Osa, and Mor to acts as a coactivator of transcription, and a subset of trxG genes encode components of the Mediator complex. The finding that transcription through Fab-7 induces the epigenetic activation of this PRE may explain how trxG complexes involved in more general transcriptional processes antagonize the establishment of PcG silencing (Schmitt, 2005).
Interestingly, in budding yeast, intergenic transcription through a promoter has been shown to prevent the binding of a transcriptional activator to its target sequences. It is possible that, in a similar fashion, the transcription through PREs may lead to the displacement of repressive PcG complexes from the chromatin and/or the prevention of PcG recruitment to the PRE in the first place. Enzymatic activities carried by the RNA polymerase II complex may subsequently or in addition modify histones at PREs with positive epigenetic marks like acetyl or methyl moieties. Interestingly, has been shown that the sequential induction of HoxB gene expression in mouse embryonic stem (ES) cells by retinoic acid correlates with the orchestrated looping out of this locus from chromosome territories. This indicates that, in addition to locus-wide changes in the chromatin structure such as histone modifications, the transcriptional activity of genes may be regulated by an additional order of complexity. It is possible that, in addition to inducing 'small-scale' changes in the chromatin structure, transcription through PREs may lead to a subnuclear relocation of the target gene locus from a repressive into a transcriptionally permissive environment (Schmitt, 2005).
Removal or inhibition of binding of PcG silencing complexes to PREs by tissue-specific transcription appears to be an attractive mechanism to counteract the constant pressure of the repressive system acting by default. However, with such a solution, the problem of epigenetic maintenance is simply moved to another level, since the question arises of what prevents the intergenic transcription from being silenced by the PcG. The simple answer -- promoters of noncoding transcripts are not sensitive to PcG/PRE silencing -- is probably not valid. Noncoding transcripts of the BX-C are activated by the same set of early segmentation genes as the Hox protein encoding mRNAs. As such, their subsequent regulation might be subjected to the same regimen of factors as the protein-encoding transcripts. However, the problem of transcriptional memory might be reduced to the problem of how to inhibit PcG silencing in particular cells/tissues, while the rest will be down-regulated by default (Schmitt, 2005).
With the processive transcription as the central issue, the cell cycle might become an important factor for the maintenance of active transcription. In Drosophila, PcG proteins dissociate from the chromosomes at mitosis. Thus, if after mitosis intergenic transcription starts before PcG proteins rebind to the PREs, the chromatin would be turned into an active mode, and would thus be protected from PcG-mediated silencing until the next round of cell division. At this point, it remains an open question whether continuous transcription of noncoding RNAs is required throughout the cell cycle to prevent the PcG complexes from rebinding, or whether the initial setting of positive epigenetic marks by the early transcription process is sufficient to prevent silencing during interphase. This proposed mechanism further reduces the problem of how transcriptional activity is maintained to the problem of how only a positive epigenetic mark is maintained during DNA replication and mitosis. Here, recent advances in studies of histone variants propose some attractive candidate marks. In particular, the histone variant H3.3 associated with the transcription of active genes could be envisaged as a possible positive signal that is locally maintained and propagated at cell division. As has been suggested before, targeted deposition of the H3.3 variant at sites of active transcription may serve to remove repressive epigenetic marks such as methylation. The establishment of stable PcG silencing complexes not only requires a sequence component, but is also accompanied by the methylation of K9 and K27 of histone H3. In contrast, positive marks, which have been shown to be mainly associated with H3.3 compared to H3, would be specifically enriched at a transcribed PRE and transmitted through mitosis. After cell division, these epigenetic marks may then in turn provide a platform for noncoding transcription through the PRE early in the cell cycle, which itself may re-establish the full active chromatin environment, unsuitable for PcG protein binding and silencing. Additionally, the reported result that an activated PRE is still maintained over a certain period after transcription has ceased (by removal of the promoter by the inducible Cre recombinase) suggests that this positive epigenetic signal is quite stable and is only diluted out by multiple cell divisions (Schmitt, 2005).
In summary, it is proposed that transcriptional maintenance during development by the PcG/trxG system is primarily a process of preventing PcG silencing to occur at those target genes that need to be maintained active in a defined cell lineage. The advantage of this mode of action is that a positive epigenetic mark, surviving DNA replication and mitosis, is sufficient to ensure stable and heritable maintenance of gene expression patterns, since the silenced mode is created by default. As such, transcription of intergenic sequences would serve as an anti-silencing mechanism that would continually counteract the initiation of this PcG-mediated silencing. In the future, it will be important to pursue the involvement of the various trxG components in the establishment and maintenance of regulatory transcription mechanisms and to analyze the link to cell cycle control and the identity and propagation of the positive epigenetic marks required to sustain active transcription (Schmitt, 2005).
Schmitt, S., Prestel, M. and Paro, R. (2005). Intergenic transcription through a polycomb group response element counteracts silencing. Genes Dev. 19(6): 697-708. Medline abstract: 15741315
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