InteractiveFly: GeneBrief

putzig: Biological Overview | References

The gene putzig (pzg) is a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that exists within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. This study provides in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. These findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation (Kugler, 2007; full text of article).

In a developing organism, cell proliferation and apoptosis must be strictly coordinated with patterning processes to correctly shape the organs. Thus, it is not surprising that all major morphogenetic and developmental signaling pathways have been involved in the regulation of cell proliferation and apoptosis and that they have been linked to numerous cases of cancer formation in mammals. In Drosophila, a large body of work shows that several of these pathways act in concert in the coordination of cell survival and death. For example, overexpression of Notch causes vast overgrowth, whereas inhibition of Notch activity by overexpression of its antagonist Hairless results in tissue loss and apoptosis. Indeed, the combined activity of Hedgehog (Hh), Decapentaplegic (Dpp), and Notch is required to promote reentry into the cell cycle after a developmentally regulated G1 arrest in the eye anlagen of Drosophila larvae. Moreover, it was shown that Hh signaling promotes cellular growth by transcriptional activation of G1 cyclins Cyclin D and Cyclin E. However, to this end, the understanding of the molecular principles that connect these pathways to either control of cell cycle or apoptosis remains largely fragmentary (Kugler, 2007).

Cell cycle entry requires the activity of G1-S cyclins that eventually activate dE2F1, a transcription factor that induces transcription of downstream genes required, e.g., for DNA replication. In Drosophila, transcriptional activation of replication-related genes encoding, for example, proliferating cell nuclear antigen (PCNA) or DNA-polymerase alpha subunit involves also DNA replication-related element factor (DREF) that recognizes DNA replication-related element (DRE) response elements. DREF can be part of a macromolecular complex including TRF2, a TATA-binding protein-related factor that binds to a subset of selected promoters, including one promoter in the PCNA gene. TRF2 has been isolated from several different organisms, where it is required for transcription of replication-related genes and key developmental genes as well (Hochheimer, 2003). The TRF2/DREF complex consists of more than a dozen proteins, including several known chromatin-remodeling components. Three of them confer chromatin activation, whereas two others, including p160, resemble regulators of insulator function (Hochheimer, 2002; Hochheimer, 2003). Interestingly, p160 was recently found to enhance position effect variegation and hence chromatin silencing and to be associated with interband regions of polytene chromosomes (Eggert, 2004). To this end, the biochemical activity and functional specificity of most of the proteins within the TRF2-complex, i.e., their role in transcriptional activation or in chromatin remodeling, however, remain elusive (Kugler, 2007).

This study isolated the Zn-finger protein p160 as a genetic suppressor of Hairless activity, prompting an interest in its role during Drosophila development and especially during Notch signaling. In vivo RNA interference resulted in tiny larvae and developmental delay, which is why the corresponding gene was named putzig (pzg). This study presents in vivo evidence that pzg is essential for fly survival by regulating cell cycle entry and progression. In addition, pzg encodes a key regulator of the Notch signaling pathway and that it is involved in histone modification and chromatin activation. Interestingly, this activity is independent of DREF, suggesting context dependence of Pzg activity (Kugler, 2007).

EP756 was identified in a genetic screen as suppressor of tissue loss caused by an overexpression of the Notch antagonist Hairless (H) during eye development (Müller, 2005). This positive effect was not restricted to the eye, because it was likewise observed during wing development. Moreover, cell growth and proliferation induced by an enforced Notch signal was significantly enhanced (~20%) by a combined overexpression with EP756. Tissue specific overexpression of EP756 caused a very mild enlargement of the respective tissues on its own. These data suggest a more general role of EP756 in the control of cell proliferation as well as an intimate interaction with Notch signaling (Kugler, 2007).

Pzg is one component of a multiprotein complex that contains TRF2 and DREF (Hochheimer, 2002). TRF2 allows transcription initiation from selected promoters independently of TFIID (Hochheimer, 2002; Hochheimer, 2003). DREF is a positive transcriptional regulator of cell cycle and replication-related genes, and it may guide TRF2 to the PCNA and DNA-polymerase alpha promoters (Hochheimer, 2002). Assuming promoter recognition or binding requires Pzg contained within the TRF2/DREF complex, depletion of Pzg might destroy the complex or reduce its activity, easily explaining the dramatic proliferation defects. However, it is noted that only a subset of promoters containing DREF binding sites involves activation through TRF2, suggesting that DREF can act independently of TRF2 (Hochheimer, 2002). Moreover, Pzg activity is found independently of DREF, indicating that TRF2/DREF complex components can act either alone or in conjunction with other factors (Kugler, 2007).

The TRF2/DREF complex contains several proteins involved in chromatin remodeling (Hochheimer, 2002). Notably, Pzg and one other TRF2/DREF component p190 are reminiscent of factors implicated in insulator function. In accordance, Pzg activity has been associated with position effect variegation and chromatin silencing (Eggert, 2004). In contrast, assays reveal an essential function of Pzg in retaining robust K4-trimethylation of histone H3, which is directly associated with open chromatin structures. In accordance with these findings, EP756 was recently identified as a suppressor of the cut allele ct^K. This cut mutation is caused by the insulator activity of a gypsy retrotransposon, which can be relieved by EP756 overexpression (Krupp, 2005). EP756 is shown in this stody to drive Pzg expression, in support of the notion that Pzg's epigenetic activity overcomes gypsy insulator function (Kugler, 2007).

Three of the proteins found in the TRF2/DREF complex have been identified previously in the nucleosome-remodeling factor NURF (see NURF301), which consists in total of four subunits. NURF is associated with chromatin activation by facilitating transcription of chromatin in vivo. In fact, mutations in Drosophila ISWI, the catalytic subunit of NURF, and other nucleosome remodeling complexes caused phenotypes that are very reminiscent of pzg-RNAi-induced defects. Because DREF down-regulation has no effect on trimethylation of H3K4, it seems unlikely that the TRF2/DREF complex as a whole is involved in chromatin activation. Instead, Pzg may be part of a NURF-like chromatin-remodeling complex, depending on the developmental context (Kugler, 2007).

Apart from a role in proliferation, this study has uncovered an important role for Pzg as positive regulator of Notch signaling. Interestingly, it was found that Pzg binds to chromatin in the regulatory region of the Notch target genes E(spl)m8 and vg. This regulation is independent of DREF: albeit DREF binding sites are common to Drosophila promoters, neither Notch nor Notch target genes that were investigated are transcriptional targets of DREF. Thus, reduced transcriptional activity of Notch target genes in pzg-RNAi mutant cells is due to a DREF-independent role of Pzg. Alternatively, Pzg could facilitate formation of the transcriptional activator complex that is assembled on Notch target promoters involving intracellular Notch itself. By using the yeast two-hybrid system, several Notch pathway members were tested; however, no binding to Pzg was detected. It is proposed that Pzg has a dual function that is effected differently. On one hand, it activates proliferation-related genes in conjunction with TRF2/DREF, and on the other hand, it activates Notch signaling by chromatin activation independently of DREF (Kugler, 2007).

Several lines of evidence support the idea that Notch signaling is particularly susceptible to chromatin remodeling. For example, Notch transcriptional activity requires the histone-modifying enzyme dBre1 that is indirectly required for K4-methylation of histone H3. Moreover, chromatin-modifiers were also shown to potentiate Notch activity during Drosophila wing development. Finally, general transcriptional regulators and chromatin remodeling factors were found in several independent genetic screens to influence Notch signaling, indicating to a role of pzg in linking Notch to chromatin remodeling. The bimodal activity of Pzg onto both cell cycle genes and Notch signaling provides further insight into the complex interplay between cell proliferation and differentiation in the fly (Kugler, 2007).

Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure

The subdivision of polytene chromosomes into bands and interbands suggests a structural chromatin organization that is related to the formation of functional domains of gene expression. Use was made of the antibody Z4 to gain insight into this level of chromosomal structure, since the Z4 antibody mirrors this patterning by binding to an antigen that is present in most interbands. The Z4 gene encodes a protein with seven zinc fingers, it is essential for fly development and acts in a dose-dependent manner on the development of several tissues. Z4 mutants have a dose-sensitive effect on w^m4 position effect variegation with a haplo-suppressor and triplo-enhancer phenotype, suggesting Z4 is involved in chromatin compaction. This assumption is further supported by the phenotype of Z4 mutant chromosomes, which show a loss of the band/interband pattern and are subject to an overall decompaction of chromosomal material. By co-immunoprecipitations, a novel chromo domain protein, which was named Chriz (Chromo domain protein interacting with Z4), identified in Flybase as Chromator, was identified as an interaction partner of Z4. Chriz localizes to interbands in a pattern that is identical to the Z4 pattern. These findings together with the result that Z4 binds directly to DNA in vitro strongly suggest that Z4 in conjunction with Chriz is intimately involved in the higher-order structuring of chromosomes (Eggert, 2004).

The localization of Z4 to all interbands and the concomittant absence from transcriptional active loci as represented by the puffed regions strengthens the view that Z4 predominantly participates in the formation of particular chromatin structures. Several different chromatin components with an impact on chromatin structure have been identified by their dose-dependent effect on the expression of the variegating w^m4-allele. In particular, the genes Su(var)2-5, Su(var)3-7 and Su(var)3-9 with a haplo-suppressor and triploenhancer phenotype were shown to encode proteins associated with heterochromatin. The localization to heterochromatin is in accordance with the presumed function of these proteins to influence the expression of w^m4 at the euchromatin/heterochromatin border by variably establishing highly compacted repressive chromatin structures. In contrast to these proteins, Z4 does not bind to heterochromatin but is distributed exclusively within the euchromatic part of chromosomes in the interbands. Although the detailed structure of chromatin constituting bands and interbands is unknown, it is generally accepted that DNA contained within an interband is less compacted than DNA contained within a band. Therefore, reducing the dosage of Z4 was expected to favor chromatin compaction, resulting in an enhancement of w^m4 PEV. Conversely, the overexpression of Z4 was expected to favor 'open' chromatin structures and lead to a suppression of w^m4 PEV. Surprisingly, Z4 in contrast to these expectations turned out to have a haplo-suppressor and a triplo-enhancer effect. This result indicates that Z4 structures chromosomes by supporting the condensation of chromatin. This conclusion is further substantiated by the analysis of chromosomes from 3rd instar larvae mutant for a hypomorphic allele of Z4. In these animals chromosomes are evident which have lost the organization into bands and interbands and altogether appear as a less compact mass of chromatin. The loss of chromosomal structure could be the result of an unpairing of the chromosomal fibres that are oriented in parallel bundles in polytene chromosomes. However, it is found to be rather unlikely that Z4 might have a primary function in the pairing of chromatids. A null-allele of Z4 is embryonic lethal, which exhibits an essential function of Z4 in diploid cells unrelated to chromatid pairing. A possible role of Z4 could involve the establishment of chromosomal borders that separate chromatin domains of different compaction levels and determine the extent of interband formation. The exact length of DNA included within interbands is still unclear, but has been estimated to range from a few hundred to a few thousand base pairs of DNA. Furthermore it is unknown whether Z4 proteins cover the whole length of interbands or are present only at the borders of bands and interbands to exert a classical boundary function. The latter is supported by the finding that within the hsp70 heat-shock puffs Z4 localizes exactly at one of the borders of each structural domain. This is very reminiscent to the localization of two proteins involved in insulator function, Zw5 and BEAF, to the proximal and distal edges of the 87A puff, and suggests common functions in the definition of structural chromosomal domains (Eggert, 2004).

In addition to Z4, several different proteins have been shown to localize to the interbands of polytene chromosomes. JIL-1, a protein with two conserved serine/threonine kinase domains is present in hundreds of interbands, with a twofold enrichment on the male X-chromosome compared with autosomes, suggesting an involvement of JIL-1 in the hyperactivation of X-chromosomal genes in the male for dose compensation. Hypomorphic mutants of JIL-1 have decreased levels of histone H3Ser10 phosphorylation and chromosomes are highly condensed due to the loss of the euchromatic interbands. These results provided evidence for a role of JIL-1 in the establishment or maintenance of an open chromatin structure correlated with the interbands to facilitate gene transcription. Quite evident from the chromosomal phenotypes of the corresponding mutants, Z4 and JIL-1 have opposite effects on chromosomal structure, despite the fact that both proteins localize to interbands. This indicates that different activities contribute to the formation of the banding pattern. Although the function of JIL-1 seems to be tightly linked to the modulation of chromatin in interbands to achieve a more decondensed state, the function of Z4 could be primarily associated with the establishment of chromosomal borders influencing the chromatin structure of the chromosomal bands as well (Eggert, 2004).

A correlation of transcription taking place in the interbands is supported by the finding that the elongating form of RNA PolII is found in hundreds of interbands. In addition, transcription factors like Spt5 and Spt6, CHD1 and the chromatin remodeling complex including Brahma localize to the less compacted interband regions. An involvement of Z4 in the promoter-selective transcription and/or chromatin remodeling is suggested by the recent finding that Z4 is a component of a macromolecular complex containing the TBP-related factor TRF2, DREF, ISWI and NURF-55 (Hochheimer, 2002). However, the chromosomal localizations of the factors involved in general or promoter-selective transcription differs from the localization of Z4 in that the latter is present in nearly all the interbands, whereas the former are found at only a subset of interbands at a few hundred sites. Owing to this difference Z4 is assumed to perform a unique function which is fundamental to the repetitive organization of chromatin into bands and interbands, which in a subset of interbands is possibly used by the transcriptional machinery. Whether this function of Z4 is related to the formation of boundaries is currently unknown. Proteins that bind to boundary or insulator sequences and are distributed in a subset of the interbands in Drosophila have been identified with the BEAF-32, Su(Hw) and Mod(mdg4) proteins. Su(Hw) and Mod(mdg4) are involved in the nuclear organization of about 500 insulator sequences into 20 to 30 insulator bodies, organizing the chromatin fibre into looped domains. A similar organizing capacity is not evident for Z4, as Z4 shows a more uniform distribution in Kc cells, lacking a pronounced concentration in a small number of discrete foci. However, owing to the greater number of sites bound by Z4, the number of nuclear foci organized by Z4 could exceed those formed by Su(Hw) and Mod(mdg4) and remain undetected in a low resolution analysis of nuclei stained for Z4 (Eggert, 2004).

Regardless of the precise chromatin composition that differs between a band and an interband, a primary distinction can be expected to act at the level of the DNA sequence. In this respect the interband DNA should contain one or more sequence motifs that are specifically recognized by one or more proteins, and Z4 with the seven zinc finger motifs is a potential candidate to exert this function. In vitro, Z4 bound to the interband sequence derived from the 5' region of Notch without sequence specificity. Possibly, the accumulated general affinity of the seven zinc fingers for DNA obscured the specific interaction of one or a few of the fingers with its target site in vitro. Still, the question remains regarding how the targeting of Z4 to the interbands is achieved in vivo. This question is especially relevant as a comparison of the few cases of DNA sequences that were unambiguously mapped to the interband regions revealed that these sequences did not contain a single characteristic shared sequence motif. A possible explanation could be given by the capability of Z4 to bind to a variety of consensus sequences, each specifically recognized by single zinc fingers and/or different combinations of the fingers, as has been shown for the vertebrate zinc finger protein CTCF (Eggert, 2004). Alternatively, or in addition to the interaction with DNA, Z4 could bind to a target protein present in interbands. This requires one or a few proteins covering all the chromosomal binding sites of Z4. Until now the novel protein Chriz is the only candidate displaying a chromosomal localization identical to Z4. Significantly, Chriz contains a chromo domain, a motif that has been found in many chromosomal proteins participating in the maintenance of diverse chromatin conformations. Therefore, Z4 and Chriz seem to be central for the modulation of the higher-order chromatin states distinguishing bands from interbands (Eggert, 2004).

Search PubMed for articles about Drosophila putzig

Eggert, H., Gortchakov, A. and Saumweber, H. (2004). Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure. J. Cell Sci 117: 4253-4264. PubMed Citation: 15292401

Hochheimer, A., et al. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420(6914): 439-45. PubMed Citation: 12459787

Hochheimer, A. and Tjian, R. (2003). Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes Dev. 17: 1309-1320. PubMed Citation: 12782648

Krupp, J. J., Yaich, L. E., Wessells, R. J. and Bodmer, R. (2005). Identification of genetic loci that interact with cut during Drosophila wing-margin development. Genetics 170: 1775-1795. PubMed Citation: 15956666

Kugler, S. J. and Nagel, A. C. (2007). putzig is required for cell proliferation and regulates notch activity in Drosophila. Mol. Biol. Cell 18(10): 3733-40. PubMed Citation: 17634285

M¸ller, D., Kugler, S. J., Preiss, A., Maier, D. and Nagel, A. C. (2005). Genetic modifier screens on Hairless gain-of-function phenotypes reveal genes involved in cell differentiation, cell growth and apoptosis in Drosophila melanogaster. Genetics 171: 1137-1152. PubMed Citation: 16118195

date revised: 5 November 2008

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