BIOLOGICAL OVERVIEW

zeste is a gene only a geneticist could love. There seem to be few if any aspects of developmental biology affected by this gene, in contrast to the useful examples of genetic effects it provides. Zeste is a DNA binding protein, activating genes by binding to regulatory regions and forming multiprotein complexes. Typically, the regulatory regions sit on the same chromosome as the gene activator, often alongside the gene being regulated (cis-configuration). Zeste, in its peculiarly lovable manner, can act on a copy of a gene located entirely on another chromosome (trans-configuration). This property is termed transactivation.

Zeste's ability to activate transcription at a distance, as it were, was tested using a phenomenon called position effect variegation (PEV). PEV will inactivate, in a capricious manner, a gene when the gene is placed near heterochromatin by chromosomal rearrangement. Heterochromatin is the genetically silent area of a chromosome, a quiet haven in what is otherwise a noisy workzone. Variegation produces a quixotic expression of a gene, with the gene now turned on, now turned off. Variation in expression is found from fly to fly, and even between different cells in the same fly. This on-off gene status is passed on from a cell to that cell's offspring. Zeste is a recessive enhancer of the PEV phenomena. Polycomb Group mutants are suppressors of PEV.

Two genes subject to PEV are white and Notch. Wild type Pc-G proteins silence genes, so in Pc-G mutants genes that would otherwise be silenced when placed next to heterochromatin, are in fact transcribed. This is due to Pc-G mutants failing to inactivate transcription of genes usually subject to their gene silencing effects. Because Zeste normally can activate transcription, mutations in zeste enhance random silencing because they fail to activate transcription properly, or put another way, zeste mutation enhances position effect variegation.

Transactivation is the induction of gene transcription by regulatory regions positioned in trans, on another chromosome, in contrast to the usual cis activation by regulatory regions held on the same chromosome as the structural gene. Transactivation is known to take place with genes such as white, Ubx and dpp. zeste function in transactivation is not required by the cell, because mutating zeste or otherwise prohibiting its expression has no deleterious effect on the fly. Despite the binding sites for Zeste in the promoter regions of these genes, despite the fact that Zeste can activate Ubx transcription in vitro, zeste function is not considered vital (Biggin, 1988).

Zeste is considered a member of the trithorax group, because, as though by analogy to Trithorax, Zeste can activate transcription. zeste functions are important to understand because they foster an understanding of the role of chromatin in gene activation and silencing (Judd, 1995 and Kennison, 1995).

The trithorax group activators and Polycomb group repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. The requirements for transcriptional activation by Zeste have been dissected in this study. Reconstituted transcription reactions have established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. The BRM complex appears to act after promoter binding by Zeste. Purification of the BRM complex has revealed a number of novel subunits. Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and Osa. The leucine zipper of Zeste mediates binding to Mor. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting (Kal, 2000).

The BRM complex is not required for promoter binding by Zeste, suggesting that it functions at a later step during the transcription cycle. Restructuring of the local chromatin environment by the recruited BRM complex may allow for the subsequent recruitment of other coactivators and the transcription machinery. In yeast cells, such an ordered recruitment has been observed at the HO promoter. The notion that Zeste recruits the BRM complex is further supported by the catalytic amounts of BRM complex needed to mediate Zeste-directed transcription. A BRM-to-nucleosome molar ratio of less than 1:50 is estimated. Recently, direct recruitment of the yeast SWI/SNF complex by an acidic activation domain has been reported. Because Zeste contacts the BRM complex through different protein motives, it will be of interest to determine what subunits of the yeast SWI/SNF complex are contacted by acidic activators. This may establish whether different activators target distinct subunits in SWI/SNF-type remodeling complexes (Kal, 2000 and references therein).

Can the recruitment mechanism described here be generalized? Although the majority of PcG and trxG proteins associate with specific chromosomal sites, they do not appear to bind DNA directly. Response elements for PcG and trxG proteins (PREs) are poorly defined sequences of several hundred base pairs. In addition to Zeste, there are a few candidate sequence-specific tethering factors such as Pleiohomeotic and GAGA. Thus far it has been impossible to reduce PREs to a number of simple sequence motives, therefore it is likely that there will be additional DNA-binding proteins that function as anchors for PcG and trxG proteins (Kal, 2000 and references therein).

Zeste and BRM both belong to the trxG proteins that have been identified as transregulators of homeotic gene function in Drosophila. The majority of Brahma-associated proteins (BAPs) are not encoded by trxG genes and several other trxG proteins have been found to be part of separate protein complexes. Thus, it has been unclear whether distinct trxG proteins may cooperate in a single biochemical pathway. This study now establishes a direct physical interaction between four distinct trxG proteins during transcriptional activation. Previous analysis of osa and mor has revealed a strong genetic interaction of these genes with brm. MOR and OSA are shown to be integral constituents of the BRM complex that are directly contacted by Zeste. Genetic studies have indicated that MOR, OSA, BRM, and Zeste share at least some target genes. These results now provide a biochemical basis for the functional relationship between these trxG proteins (Kal, 2000 and references therein).

Purification of the BRM complex and stringent coimmunoprecipitation experiments have suggested the presence of two novel core BAPs in addition to OSA: BAP170 and BAP26. Moreover, it appears that there are several less tightly associated proteins. The idea is favored that the interaction of the majority of these proteins with the core BRM complex is specific, because they copurify over several columns and remain associated during selective coimmunoprecipitation. Moreover, Zeste specifically interacts with the p400 complex component, supporting the notion that its association with the BRM complex is functional (Kal, 2000).

Do distinct remodeling factors perform different functions or are they redundant? A number of recent studies shed light on the basic mechanisms by which SWI/SNF and ISWI remodelers catalyze nucleosome mobilization. However, their potential roles as regulators of transcription are still poorly understood. Presented here is a clear example of functional differentiation among chromatin remodelers. The BRM complex is an essential coactivator for Zeste, whereas the ISWI family members are not required. Reversibly, at least some ISWI remodeling factors appear to be more efficient at ordering nonperiodic nucleosomal arrays than the BRM complex. Thus, this side-by-side comparison of distinct endogenous Drosophila remodeling factors shows that each performs distinct specialized functions (Kal, 2000).

The SWI/SNF-type remodelers appear to function in a highly selective manner. For example, the human SWI/SNF-related chromatin-remodeling complex, E-RC1 is required for the activation of the beta-globin gene by the activator EKLF but does not work with another transcription factor, TFE3. Moreover, it is pertinent to note that the yeast and Drosophila SWI/SNF family members were first identified by genetic screens for gene-specific regulators. Thus, studies in yeast, mammals, and Drosophila all point to an integral and essential role for SWI/SNF remodelers in gene-specific transcriptional regulation. Although the ISWI remodelers are not required for Zeste function, they have been implicated in transcriptional activation by other regulators such as GAL4-VP16. Several lines of evidence suggest that ISWI remodelers may act by a mechanism that is fundamentally distinct from that of the SWI/SNF family complexes. For example, unlike NURF, SWI/SNF does not seem to require the histone tails for remodeling. Moreover, studies on NURF suggest that it remodels chromatin in a transient nonspecific manner, creating an opportunity for transcriptional activators to bind DNA. Such a mode of action does not involve the direct physical interactions between remodeler and activator described in this study for the BRM complex. In conclusion, all available evidence points to an extensive functional specialization of ATP-dependent chromatin remodeling factors. An attractive possibility is that different genes require the action of distinct subsets of remodeling complexes, histone acetyl transferases, and other coactivators. Such a combinatorial arrangement would vastly expand a cell's potential for precise and coordinated regulation of individual genes (Kal, 2000).

GENE STRUCTURE

Bases in 5' UTR - 504

Exons - three

Bases in 3' UTR - 345

PROTEIN STRUCTURE

Amino Acids - 555

Structural Domains

The amino terminal DNA binding domain is a helix turn helix motif, rich in basic and acidic amino acids. A second region starting at residue 154 is strongly acidic, a third region is almost devoid of basic or acidic residues, and is slightly hydrophobic, contining a large number of Gln and Ala. The carboxyl terminal is densely packed with basic and acidic residues, identified as a leucine zipper (Pirotta, 1987 and Chen, 1993a and 1993b).

Zeste is a Drosophila sequence-specific DNA-binding protein that performs a variety of functions during chromatin-directed gene regulation. Its DNA-binding domain (DBD) has been identified, but no similarities to established DNA-binding structures are known. This study presents sequence comparisons suggesting that the Zeste-DBD is a novel variant of the tri-helical Myb-DBD. Using band shift assays, the Zeste-DBD has been mapped to 76 residues, corresponding to a single Myb repeat of only 50 residues. All residues involved in formation of the hydrophobic core of the Myb domain are conserved in Zeste, suggesting it forms an extended Myb domain. Mutagenesis studies determined (T/C/g)GAGTG(A/G/c) as the consensus Zeste recognition sequence. Reconstituted transcription experiments established that deviations from this optimal consensus compromise transcriptional activation by Zeste. In addition, flanking DNA is critical because Zeste-DBD binding requires a DNA sequence of minimally 16 base pairs, which is much longer than the consensus site. The DNA flanking the consensus is contacted by Zeste through sequence-independent backbone contacts. Interestingly, hydroxyl radical footprinting revealed that the Zeste-DNA backbone contacts all map to one face of the DNA. The DNA-binding properties of Zeste are compared with those of classical tri-helical DBDs harboring a helix-turn-helix motif and suggest a model for Zeste-DNA recognition (Mohrmann, 2002).

date revised: 15 November 98

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