putzig: Biological Overview | References
Gene name - putzig
Synonyms - Z4
Cytological map position - 78C5-78C6
Function - transcription factor
Symbol - pzg
FlyBase ID: FBgn0259785
Genetic map position - 3L: 21,279,163..21,283,410 [+]
Classification - zinc finger
Cellular location - nuclear
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 ctK. 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).
Drosophila putzig was identified as a member of the TRF2-DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. This study shows that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig-NURF chromatin complex required for epigenetic activation of Notch targets (Kugler, 2010).
Putzig is a component of a large multiprotein complex that includes the TATA-box-binding-protein-related factor 2 (TRF2) and the DNA-replication related element (DRE) binding factor DREF. The TRF2-DREF complex has been associated with the transcriptional regulation of replication-related genes that contain DREF binding sites. Accordingly, Pzg acts as a positive regulator of cell cycle and replication-related genes. In addition to this, Pzg is also required for Notch target gene activation in a DREF-independent manner. Presumably, Pzg functions at the level of chromatin activation, because the open chromatin structure typical of active Notch target genes is no longer detectable in a pzg mutant background (Kugler, 2010).
The TRF2-DREF complex consists of more than a dozen of proteins and the biochemical function of most of them remains still elusive. Interestingly, it also contains three members of the nucleosome remodeling factor (NURF), imitation switch (ISWI), Nurf 55 and Nurf 38. NURF is a multisubunit complex that has been associated with chromatin activation and repression. NURF triggers nucleosome sliding thereby provoking changes in the dynamic properties of the chromatin. The subunit ISWI is a member of the SWI2/SNF ATPase family and is thought to provide energy for nucleosome remodeling. Nurf 38 encodes an inorganic pyrophosphatase, which catalyzes the incorporation of nucleotides into a growing nucleic acid chain during transcription, replication, and DNA repair mechanisms. Nurf 55 harbors WD-40 repeats, which allow interaction with other proteins and protein complexes. The fourth and largest subunit Nurf 301 is specific to the NURF complex, whereas all other members are shared with other chromatin modifying complexes. Accordingly, Nurf 301 is not a component of the TRF2-DREF complex. Nurf 301 exhibits a number of protein motifs that typify transcription factors and other chromatin modifying proteins. In addition, the N-terminal region of Nurf 301 shows homology to the DNA-binding protein HMGA (high mobility group A) implying that Nurf 301 mediates the contact with the DNA or provides a platform to recruit other transcription factors. In this context it has already been shown that Nurf 301 is required for the transcriptional activation for example of homeotic genes and notably of Ecdyson-receptor (EcR) and Wingless target genes (Kugler, 2010).
The DREF independence of Pzg during the activation of Notch target genes raised the possibility that it may instead involve the NURF complex for chromatin activation. This study provides evidence for a functional interplay between Pzg and the NURF complex with regard to Notch target gene activation. Coimmunoprecipitations revealed that Pzg is present in protein complexes containing the known NURF subunits. Moreover, Pzg binding on Notch target genes is neither detectable in mutants of the NURF-specific subunit Nurf301, nor in mutants affecting other subunits of NURF. In addition, Nurf301 is required for Notch target gene expression, which is impaired in Nurf301 mutant cell clones. Consistent with this, Nurf301 mutants enhance the Notch mutant wing phenotype, strongly arguing for an involvement of the NURF complex in Pzg-mediated epigenetic Notch target gene activation (Kugler, 2010).
This work shows that Pzg is associated with at least two different types of protein complexes that are involved in transcriptional activation: the TRF2-DREF complex and the NURF complex. Interestingly, these two complexes share several members apart from Pzg despite their different roles in core promoter selection versus nucleosome sliding and chromatin activation. However, the specific role for Pzg in the promotion of Notch target gene transcription involves NURF and not the TRF2-DREF complex. Notably, NURF also promotes efficient expression of a subset of Wingless target genes. In this case, a direct interaction between ISWI and Armadillo, the major transcriptional coactivator of Wingless targets, was shown. There is no indication however, that pzg is involved in the regulation of wg, suggesting that the NURF complex recruits Pzg only onto specific promotors. Furthermore, the NURF subunit Nurf 301 contacts the Ecdysone receptor (EcR), thereby modulating the activity of ecdysone signaling during the larval and pupal stages of Drosophila development. How is NURF recruited to Notch target sites? Notch target gene activation involves a ternary complex containing the DNA-binding protein Suppressor of Hairless [Su(H)], intracellular Notch, and Mastermind, plus other more general coactivators. There is no indication of a direct contact of Pzg to either Notch or Su(H), tested by coimmunoprecipitations as well as yeast two-hybrid assays. However, contacts between the other components, notably Mastermind or ISWI cannot be excluded. Mastermind has been shown to interact with several chromatin modifying proteins, for example, with the histone acetyltransferase p300 or with cyclin-dependent kinase 8 (Kugler, 2010).
Several studies in Drosophila and vertebrates have shown that many Notch-responsive target genes are regulated by combinatorial signal inputs, which need the Notch ternary complex and additional cooperators bound to sites nearby. In contrast to cofactors within the transactivation complex, these other factors do not physically interact with the Notch ternary complex but instead synergize during transcriptional activation at Notch target gene promoters. It is conceivable, that a Pzg-NURF complex is likewise needed in conjunction with the Notch transactivator complex for full Notch target gene expression (Kugler, 2010).
It is well established, that chromatin modification complexes share several components. For example, ISWI is not only contained in NURF and TRF2-DREF complexes but also in chromatin-remodeling and assembly factor (CHRAC) and ATP-utilizing chromatin-remodeling and assembly factor (ACF) in Drosophila, where it serves to increase the accessibility of nucleosomal DNA. Nurf 55, also known as CAF-1, forms a stable complex with Drosophila Myb and E2F2/RBf and regulates the transcription of several developmentally important genes. Like ISWI and Nurf 55, also Nurf 38 is present in the TRF2-DREF complex. Pzg is contained within the TRF2-DREF and within the NURF complex serving the activation of proliferation related genes and N target genes, respectively. Not all NURF complexes, however, require pzg, for example, as during the activation of Wg target genes. Sharing components raises the question, how specificity of the different complexes is achieved. Obviously, specificity is mediated either by unique subunits or by certain combinations of shared subunits. These subunits may specifically modulate the activity of the ATPase subunit or, more likely, may help to target the remodeling complexes to particular promoters. Two members of the NURF complex, ISWI and Nurf 301, have been shown to directly target transcription factors. It is tempting to speculate, that Pzg might be a specific cofactor needed to realize some of the operation spectrum of NURF, notably during the epigenetic regulation of Notch target genes (Kugler, 2010).
Drosophila telomere maintenance depends on the transposition of the specialized retrotransposons HeT-A, TART, and TAHRE. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. This study describes two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of Drosophila telomeres, the HeT-A retrotransposon, thus guaranteeing genome stability. Mutant alleles of JIL-1 were found to have decreased HeT-A transcription, putting forward this kinase as the first positive regulator of telomere transcription in Drosophila described to date. The decrease in HeT-A transcription in JIL-1 alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the HeT-A promoter. Moreover, Z4 mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at Drosophila telomeres. Interestingly, a biochemical interaction was detected between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, it is demonstrated that a phenotype of telomere instability similar to that observed for Z4 mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. A model is proposed to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of Drosophila telomeres, such as HP1a (Silva-Sousa, 2012).
Although in Drosophila the role of JIL-1 in activating transcription has remained controversial, at least in the HeT-A, TART, and TAHRE (HTT) array it could act as a positive regulator of transcription for three different reasons: 1) When telomere elongation is needed, a fast activation of HeT-A transcription should be expected. Accordingly, the mammalian JIL-1 orthologous MSK1/2 have been shown to rapidly induce gene expression on the face of stress or steroid response. 2) HeT-A is embedded into the HTT array, a domain that needs to be protected from the influence of the repressive heterochromatin of the neighboring TAS domain. JIL-1 has been suggested to protect the open chromatin state from the spreading of neighboring repressive chromatin at certain genomic positions. 3) The decrease in expression that was observed in the JIL-1 mutants is moderate. Recent data at genomic level revealed that JIL-1 function agrees with a reinforcement of the transcriptional capability of a particular genomic domain rather than net activation (Silva-Sousa, 2012).
Phalke (2009) suggest that JIL-1 has a role in retrotransposon silencing in general and has no effect on telomere transcription. A possible explanation for this discordance with the current results and hypothesis is that the mutant allele of JIL-1 assayed by Phalke, the JIL-1Su(var)3-1 allele, corresponds to a C-terminal deletion of the JIL-1 protein that causes the protein to miss-localize and phosphorylate ectopic sites. The ectopic phosphorylation caused by the JIL-1Su(var)3-1 allele would activate the expression above wild type levels in those genes that normally are not targeted by JIL-1, as it happens to be the case for the Invader4 retrotransposon. The current study has assayed the JIL-1Su(var)3-1 allele obtaining similar result than for the wild type stock, likely for similar reasons. Supporting this, in addition of the JIL-1Su(var)3-1, data from two more JIL-1 alleles, JIL-1z60 and JIL-1z2, is presented that correspond to loss of function alleles and, in both cases, result in a substantial decrease in HeT-A transcription. Moreover, the changes in telomere transcription reported in this study have been assayed directly on the major component of the HTT array, and not through a reporter. The current data demonstrates that JIL-1 is necessary to maintain active transcription of the telomeric retrotransposon HeT-A or, what is the same, transcription from the telomeres in Drosophila (Silva-Sousa, 2012).
Although it was demonstrated that JIL-1 is necessary to maintain transcription from the HTT array, no decrease was detected in telomere length in the JIL-1 mutant alleles. A reasonable explanation for this observation is that the JIL-1 mutant alleles here analyzed (JIL-1z60 and JIL-1z2) have been maintained as heterozygous. It is therefore possible that one copy of JIL-1 is enough to promote enough HeT-A transcription to elongate significantly the telomeres when needed (Silva-Sousa, 2012).
Although in the case of the hypomorph mutation Z47.1 an increase was observed in HeT-A transcription and HeT-A copy number significantly above the control strain (w1118), the null alleles Z42.1 and pzg66 do not show an up-regulation of HeT-A transcription or an increase in its copy number. Although all the stocks were crossed to the w1118 strain to minimize the effects of the genetic background, it could still have a certain influence when comparing the pzg66 allele with the Z47.1. Nevertheless the Z47.1 and Z42.1 alleles come from the same genetic background. A possible explanation could rely on the fact that the Z47.1 mutation is a hypomorph mutation where a small amount of Z4 protein is still present. By ChIP analyses an increase of JIL-1 protein was detected at the HeT-A promoter above control levels, which could explain in part the major transcription of HeT-A in this mutant background, it is possible that although low, the amount of Z4 present in the Z47.1 allele is enough to recruit JIL-1 to the HeT-A promoter. In the pzg66 and the Z42.1 null alleles, JIL-1 cannot be recruited towards the HeT-A promoter and there is no increase in transcription. Nevertheless, with the current data it cannot be concluded that Z4 directly controls the level of HeT-A transcription (Silva-Sousa, 2012).
A phenotype of telomere instability was detected in all three Z4 mutant alleles Z47.1, Z42.1 and pzg66, suggesting a role of this chromosomal protein in guaranteeing telomere stability in Drosophila. Although a number of genes involved in the capping function in Drosophila still remain unidentified, there is no evidence that Z4 directly participates in the protection of the telomeres. Mutant alleles of genes directly involved in the capping function, such as woc or caravaggio (HOAP), show multiple and more numerous TFs in larval neuroblasts than the ones that were observed in the Z4 mutant alleles. Moreover, it has been possible to detect staining for one of the major capping components, the HOAP protein, in the TFs of Z4 mutant neuroblast cells, indicating that the telomere-capping complex is still loaded to a certain degree. Instead of directly participating in the capping, it is hypothesized that the major chromatin changes caused by the lack of Z4 at the HTT array result in a secondary loss of necessary chromatin and capping components like HP1a (Silva-Sousa, 2012).
Results from the ChIP experiments suggest a relationship between JIL-1, Z4 and HP1a in fine-tuning the chromatin structure at the HTT array. HP1a has a dual role at the telomeres explained by its participation in both the capping function and the repression of gene expression that also exerts in other genomic domains. In the HP1a Su(var)2-505 allele, which it is known to have a major transcription of HeT-A and problems of telomere stability, a pronounced decrease was observed in Z4 and JIL-1. In the Z47.1 allele the decrease in Z4 protein is accompanied by a similar decrease in H3K9me3 and HP1a at the HeT-A promoter. Finally in the JIL-1z60 allele the increase in silencing epigenetic marks like H3K9me3 and HP1a is also accompanied by a decrease in Z4. In particular, the pronounced dependence of the presence of HP1a and Z4, points toward the loss of HP1a and H3K9me3 to a possible cause for telomere instability in the Z4 mutant alleles here studied. Interestingly, in the Su(var)2-504/Su(var)2-505 heteroallelic combination (considered a null mutation), 15% of telomeres involved in telomere associations are still able to recruit the HOAP protein. Therefore the data on HOAP localization in the Z4 mutant alleles is still consistent with the TFs being caused by the decreased availability of HP1a in these cells. The above results demonstrate that Z4 in a coordinated manner together with JIL-1 and HP1a is an important component of the telomere chromatin in Drosophila, which upon its reduction causes significant changes in the chromatin of the HTT array, which are the cause of the observed telomere instability in all the Z4 mutant alleles here studied (Silva-Sousa, 2012).
It has been possible to detect a biochemical interaction between JIL-1 and Z4, and the data suggests that these two proteins can be components of the same protein complex. This interaction had been previously suggested because both proteins have been found co-localizing in different genomic locations, but no direct proof existed to date. In each genomic location where the Z4-JIL-1 complex is needed, a special mechanism of recruitment should exist. Importantly, it has been shown how Z4 specifically interacts with HeT-A Gag. HeT-A Gag is the only protein encoded by the HeT-A element and has been shown to specifically localize at the telomeres. HeT-A Gag has been shown to be in charge of the targeting of the transposition intermediates for the HeT-A element and also for its telomeric partner the TART retrotransposon. Interestingly, when the consequences for telomere stability were studied after knocking down the HeT-A gag gene by RNAi, similar TFs were observed than when knocking down the Z4 gene, further relating the action of both genes in telomere stability. Z4 is known to participate in different protein complexes with roles in different genomic locations. Because it has been demonstrated that Z4 is able to associate with a variety of proteins in these complexes, it is thought that the description of a mechanism for its specific targeting to telomeres through one of the telomeric retrotransposon proteins is especially relevant (Silva-Sousa, 2012).
Integrating information from previous literature and the results exposed by this study, a possible model to describe the state of the chromatin at the HTT array in each of these three mutant scenarios; JIL-1, Z4 and Su(var)2-5, as well as in wild type (see Model of the chromatin environment at the HeT-A promoter). The following phenotypes are proposed: (A) Wild type: Z4 defines a boundary at HeT-A promoter that protects from the action of HP1a and other heterochromatin markers. JIL-1 guarantees a certain level of euchromatin inside the HeT-A promoter in order to allow gene expression, (B) JIL-1 mutants: destabilization of the Z4 boundary and the heterochromatin spreads into the HeT-A promoter (enrichment in HP1 and H3K9me3), (C) Z4 mutants: Disappearance of the Z4 boundary, increase in euchromatin marks (H3K4me3) and decrease in heterochromatin marks (HP1a and H3K9me3). Subtle increase in JIL-1 and in euchromatinization of the HeT-A promoter, (D) In Su(var)2-5 mutants: The lack of HP1a allows relaxation of the Z4 boundary causing a JIL-1 and Z4 spread along the HTT array and a relative decrease of these proteins inside the HeT-A promoter. Although the levels of JIL-1 inside the HeT-A promoter are lower than in wild type, the release of silencing caused by loss of HP1a results in increased HeT-A expression (Silva-Sousa, 2012).
It should be taken into account that 1) HP1a has been shown to spread along the HeT-A sequence. 2) The structure and the phenotypes of the different Z4 mutant alleles suggest a possible role of this protein in setting and maintaining the boundaries between heterochromatin and euchromatin in polytene chromosomes. 3) JIL-1 has been extensively shown to be important to counteract heterochromatinization and, when missing, causes a spreading of heterochromatin markers such as H3K9me2, HP1a and Su(var)3-7. 4) JIL-1 has been found to co-localize with Z4 at the band-inter-band transition in polytene chromosomes and also to co-purify with Z4 in different protein complexes. In addition to this, it has been possible to detect a biochemical interaction between JIL-1 and Z4, as well as, a certain dependence on the presence of JIL-1 for the proper localization of Z4, suggesting a possible role of JIL-1 upstream of Z4. Finally, 5) The ChIP analyses in this study suggest a certain dependence of Z4 on HP1a or onto similar chromatin requirements for the loading of both proteins at the HTT array, more specifically at the HeT-A promoter. Summarizing all of the above, it is proposed that the chromatin at the HeT-A promoter could have the following structure: In a wild type situation, the HeT-A promoter contains intermediate levels of HP1a, JIL-1 and Z4. HP1a would be spread along the HTT array, JIL-1 would be concentrated at the promoter region of HeT-A guaranteeing certain level of expression and Z4 would be important to set the boundary between these two opposite modulators (Silva-Sousa, 2012).
In a JIL-1 mutant, the lack of JIL-1 would disturb the Z4 boundary causing a slight decrease in the Z4 presence. This result is in agreement with a Z4-JIL-1 partial interaction. The decrease in JIL-1 presence and the disturbance of the boundary causes a spreading of HP1a into the HeT-A promoter, increasing its presence and repressing transcription from the HTT array (Silva-Sousa, 2012).
In a Z4 mutant, the disappearance of the boundary together with the significant decrease in H3K9me3 causes a decrease in HP1a binding and a substantial modification of the chromatin at the HTT array. The lack of sufficient HP1a at the HTT array causes a destabilization of the chromatin at the cap domain triggering telomere instability as a result. This scenario applies to the three Z4 mutant alleles present in this study, the hypomorph Z47.1, and the nulls pzg66 and Z42.1. On one hand the loss of some Z4 in Z47.1/Z47.1 genotype produces overexpression of HeT-A because in addition to a relaxation of the chromatin, part of JIL-1 is still recruited to the HeT-A promoter and activates transcription in a more effective way than in a wild type situation (Silva-Sousa, 2012).
Finally, in a Su(var)2-5 mutant background, the lack of HP1a along the HeT-A sequence allows a relaxation of the boundary causing a spread of JIL-1 and Z4 from the HeT-A promoter towards the rest of the array and creating as a consequence, permissive chromatin environment releasing HeT-A silencing (Silva-Sousa, 2012).
The model does not completely explain the complex relationships that regulate telomere chromatin, likely because other important components are yet to be described or associated with the ones presented in this study. For example, other chromatin regulatory components that have been associated with Drosophila telomeres included the deacetylase Rpd3, with a regulatory role on chromatin structure, and the histone methyltransferase SetDB1 and the DNA methylase Dnmt2 which by acting in the same epigenetic pathway repress transcription of HeT-A as well as of retroelements in general. Future in depth studies on additional chromatin components will allow completion and detailing even more the description of the chromatin at the HTT array, and allow a better understanding of the mechanism of retrotransposon telomere maintenance and the epigenetic regulation of eukaryote telomeres in general. In the meantime, this study describes a plausible scenario in the view of the transcription and ChIP data (Silva-Sousa, 2012).
The results shown in this study demonstrate the role of JIL-1 as the first described positive regulator of telomere (i.e. HeT-A) expression in Drosophila. Because HeT-A is in charge of telomere maintenance in Drosophila, these results are key to understand how telomere elongation is achieved in retrotransposon telomeres. It was also demonstrated that Z4 is necessary to guarantee telomere stability. The data presented in this study strongly suggest that JIL-1 and Z4 exert these functions by maintaining an appropriate telomere chromatin structure by a coordinated action together with other known telomere components such as HP1a. Moreover, this study shows that JIL-1 and Z4 interact biochemically. Last, and importantly for understanding how the specific role of the Z4-JIL-1 complex at the telomeres is defined and differentiated from its role in other genomic regions, it was shown that Z4 might interact with the HeT-A Gag protein, providing evidence for a targeting mechanism that specifically retrieves this complex to the telomeres (Silva-Sousa, 2012).
The conserved band-interband pattern is thought to reflect the looped-domain organization of insect polytene chromosomes. Previously, it has been shown that the chromodomain protein Chriz (Chromator) and the zinc-finger protein Z4 (Putzig) are essentially required for the maintenance of polytene chromosome structure. This study shows that both proteins form a complex that recruits the JIL-1 kinase to polytene chromosomes, enabling local H3S10 phosphorylation of interband nucleosomal histones. Interband targeting domains were identified at the N-terminal regions of Chriz and Z4, and the data suggest partial cooperation of the complex with the BEAF boundary element protein in polytene and diploid cells. Reducing the core component Chriz by RNAi results in destabilization of the complex and a strong reduction of interband-specific histone H3S10 phosphorylation (Gan, 2011).
The chromatin proteins Chriz and Z4 both are ubiquitous and essential proteins that are required for the maintenance of interphase chromosome structure. Chriz and Z4 directly interact by their central and N-terminal domains, respectively. The Z4 N-terminal domain is required for Z4 interband targeting, mediated by Chriz interaction. When overexpressed, it competes the chromosomal binding of the endogenous Z4 protein. The Chriz N-terminal domain is required for interband targeting of the Chriz protein by an as-yet unknown mechanism. Destabilization of the complex results in strong down-regulation of H3S10 phosphorylation on polytene chromosomes (Gan, 2011).
Surprisingly, neither the Chriz chromodomain nor the Z4 zinc-finger region is needed for chromosomal targeting and no interaction partners for these signature protein domains are yet identified. However, both domains provide important functions. Point mutations in the Chriz chromodomain are unable to complement Chriz mutations. Point mutations within the Z4 zinc-finger region are not yet available, but overexpression of the N-terminal Z4 fragment aa 1-516 including the zinc-finger region results in a 'shrunken' interband phenotype, although neither overexpression of the Z4 full-length protein nor of N-Z4 aa 1-240 affect chromosomal structure. This indicates a dominant negative effect for the zinc-finger region in the aa 1-516 construct (Gan, 2011).
Data from coimmunoprecipitation and colocalization in situ suggest that Chriz and Z4 interact with the boundary element binding protein BEAF and the H3S10-specific histone kinase JIL-1. Chriz and JIL-1 directly interact in vitro, but it remains to be shown if the JIL-1-Z4 interaction is also direct or mediated by Chriz protein. Also, direct interaction between Chriz–Z4 and BEAF still has to be demonstrated. Unlike the Chriz-Z4 interaction, neither the BEAF-Z4 nor the JIL-1-Z4 colocalization is 100%. All four proteins are located in many interbands, but in each case there are sites that are occupied by one or the other protein exclusively. The mechanism explaining this promiscuity in interaction is not yet fully understood. However, it has been demonstrated that interband proteins can be shared between different chromatin protein complexes. For instance, CP190 is present in complexes with BEAF or Su(Hw) but not both. Furthermore, CP190 binds to CTCF with ~50% of CP190-CTCF complexes also bound by BEAF (Gan, 2011).
Mutual interactions could be mediated both by cooperation or competition between different factors for local binding. It is worth mentioning that Chriz also shows a significant overlap with CP190 in polytene chromosome binding in situ and in ChIP on chip experiments on Drosophila cell lines. The overlap with both BEAF and CP190, both well known for their activity as boundary element factors, suggests that Chriz and Z4 may be involved in the control of boundaries, a hypothesis that is currently being tested. The qualitative impression of colocalization gained from polytene chromosome staining is confirmed at higher resolution by ChIP on chip data from diploid S2 cells. Although these data still await a more systematic analysis, it is evident that in some regions the match in BEAF/Chriz binding sites is rather convincing but elsewhere it is more moderate. JIL-1 also colocalizes with Chriz but often shows a broader distribution overlapping several Chriz peaks or extending from a Chriz peak into an adjacent region. Strikingly, for the two cases of well-studied interband regions at 3C6-7 and 61C7-8, binding of BEAF, Chriz and JIL-1 is also observed in S2 diploid cells, suggesting that this stretch of open chromatin is conserved in structure between cell lineages. In the case of 3C6-7, the binding may be correlated with Notch transcription in S2 cells. However, there is no Notch transcription in salivary glands. The transcriptional activity at interband 61C7-8 is not known (Gan, 2011).
The effect of reducing the function of Chriz, Z4 and JIL-1 on polytene chromosome structure was studied by the use of hypomorphic alleles and tissue-specific RNAi knockdown. In general, it results in a phenotype of progressive loss of distinct band interband structure, sometimes concomitant with partial condensation or torsional distortion of polytene chromosomes. It has been reported that targeting of active JIL-1 kinase resulted in ectopic histone H3S10 phosphorylation and local chromosome decondensation that was dependent on JIL-1 kinase activity. It was reasoned that a major role of the Chriz-Z4 complex might be the recruitment of JIL-1 to the chromosome that would result in local phosphorylation of nearby nucleosomes, allowing the ≥30 nm chromatin fibre to unfold and form a less condensed interband chromatin. In support of this hypothesis, gland-specific Chriz-RNAi resulted in decreased levels of chromosomally bound Z4 and JIL-1 and in a significant loss of interband specific H3S10 phosphorylation. However, not all chromosomes exhibited the loss of structure phenotype mentioned above. Conceivably, RNAi induction might have been too mild and there was still enough Chriz protein left to provide for the maintenance of chromosome structure. The experiments were done at 22-24°C, a temperature at which the GAL4 inducer is still not at its maximal activity. Raising the temperature to 29°C early on made the inducer more effective but resulted in a tiny salivary gland phenotype that was not amenable to cytological analysis. Using the Sgs4 enhancer active in late 3rd instar at 29°C was not effective since the Chriz protein with its long half-life masked the RNAi effect. A different explanation for the lack of chromosomal phenotype may be that 10%-20% of the JIL-1 binding sites that do not depend on Chriz/Z4 are not affected by Chriz-RNAi and are sufficient to sustain the chromosome structure (Gan, 2011).
In conclusion, this study provides evidence for a chromatin complex with the chromodomain protein Chriz at its core. The complex, which, besides the Z4 protein, may contain the sequence-specific DNA-binding protein BEAF, is required for H3S10 phosphorylation of interphase chromosomes, presumably by local recruitment of the tandem kinase JIL-1. Local binding and activity of the complex on polytene chromosomes may result in decondensation of interband regions and related sites on nonpolytene diploid chromosomes (Gan, 2011).
The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013).
These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Chromator/Putzig (Chro-Z4/Pzg) protein complex in CNS derived BG3 cells, in which the E(spl)-C has a rare restrained state with a cohesin-H3K27me3 overlap. The E(spl)-C has a highly self-interactive structure that is unexpectedly independent of these protein complexes and the level of gene expression. Depletion of any of these three protein complexes, however, significantly increases E(spl)-C transcription. The effects of these three protein complexes on E(spl)-C expression likely reflect changes in expression of Notch ligands, and in the cases of cohesin and PRC1, potentially direct effects on activator and Pol II activity at the E(spl)-C genes (Schaaf, 2013).
Chromosome conformation capture (3C) analysis revealed that the E(spl)-C has a structure in which all positions within the complex interact with each other at a high frequency, but not with flanking regions. Surprisingly, this study found that this architecture is independent of cohesin, the PcG complexes, the Chro-Pzg/Z4 complex, transcription, and stage of the cell cycle. Thus it is not known which factors establish this striking architecture, which defines the E(spl)-C as a structurally independent domain. It is also not yet known which factors control recruitment of cohesin and PcG complexes to the locus. It is speculated, however, that this architecture coordinates transcriptional control of the entire E(spl)-C, based on the finding that in BG3 cells, cohesin, PRC1, and the ubiquityl-Histone H2 (H2Aub) and H3K27me3 histone modifications made by the PRC1 and PRC2 complexes are co-extensive within this architectural domain. Although no known insulators or boundary elements flank the E(spl)-C, and depletion of the CP190 protein required for activity of all known Drosophila insulators does not alter E(spl)-C expression, it is likely that the unknown factors that form this structure limit the spread of these protein complexes and histone marks. The E(spl)-C architectural domain may be evolutionarily significant, because Notch-regulated Enhancer of split complexes with similar structures are conserved in insects and crustaceans over 420 million years (Schaaf, 2013).
Possible clues to the identities of the factors that control the E(spl)-C architecture and/or the recruitment of cohesin and PcG complexes may arise in genetic screens for factors that alter E(spl)-C sensitive phenotypes, such as the Nspl-1 rough eye and bristle phenotypes. These phenotypes are sensitive to mutations in the E(spl)-C and cohesin genes in a highly dosage-sensitive manner, and modest changes in the E(spl)-C architecture or recruitment of cohesin or PcG proteins may have similar effects (Schaaf, 2013).
There is coordinate regulation of gene complexes by cohesin in mammalian cells. The Protocadherin beta (Pchdb) gene complex is downregulated in the embryonic fibroblasts and brains of mice heterozygous mutant for the Nipbl cohesin loading factor, and brains of mice that are homozygous mutant for the SA1 cohesin subunit, and cohesin is involved in enhancer-promoter looping in the Protocadherin alpha (Pchda) complex, helping determine which genes in the complex are active. While this is a positive role for cohesin, as opposed to the repressive role that occurs in the E(spl)-C, it is possible that the protocadherin gene clusters also have a higher order architecture that dictates how cohesin functions within the gene complex. Recent genome-wide analysis also indicates that there are constitutive higher order looping architectures that may organize cell-type specific interactions on a shorter scale, and that cohesin contributes to both types of structures (Schaaf, 2013).
Prior studies showed that depletion of cohesin or PRC1 increases expression of the Serrate Notch ligand gene. This likely explains part of the increase in E(spl)-C transcription upon cohesin and PRC1 depletion, because the E(spl)-C genes are directly activated by Notch. Consistent with this idea, this study detected increases in NICD association with the HLHmβ and HLHm3 genes upon cohesin or PRC1 depletion. EDTA treatment confirms that increasing Notch activation increases NICD binding to the E(spl)-C genes (Schaaf, 2013).
Because cohesin and PRC1, unlike the Chro-Pzg/Z4 complex, bind directly to the E(spl)-C, it is also possible that they also directly control association of NICD with the Su(H) protein bound upstream of the active genes. For example, they could potentially interact with NICD or the Su(H) complex, and interfere with NICD association, or somehow facilitate ubiquitination and degradation of NICD. The lack of an effect of cohesin or PRC1 depletion on NICD association with E(spl)-C genes after EDTA treatment does not rule out this possibility, because under these conditions, the amount of NICD is no longer limiting (Schaaf, 2013).
It remains to be determined if the multiple effects of cohesin on Notch function seen in Drosophila, including regulation of Notch ligand and target genes, also occur in mammals. If so, this could underlie many of the development deficits seen in Cornelia de Lange syndrome, caused by mutations in NIPBL and cohesin subunit genes. Mutations in Notch receptor and ligand genes cause Alagille and other syndromes that affect many of the same tissues as CdLS (Schaaf, 2013).
The possibility cannot be ruled out that cohesin and PRC1 directly repress E(spl)-C transcription independently of any effects on Notch ligand expression or NICD association with the E(spl)-C genes. This is because both bind throughout the complex, and the PRC1-generated H2Aub repressive histone mark is co-extensive with the E(spl)-C architectural domain. Importantly, all genes in BG3 cells that show rare overlap of cohesin and the PRC2-generated H3K27me3 modification, such as the invected and engrailed gene complex, show substantial increases in transcription upon cohesin or PRC1 depletion, even though they are not Notch activated. It is highly unlikely that cohesin or PRC1 depletion increases the expression of all the diverse activators that control these genes, and more likely that cohesin and PRC1 directly repress their transcription (Schaaf, 2013).
At all genes examined that are strongly repressed by cohesin, cohesin restricts the transition of paused RNA Pol II into elongation, irrespective of whether or not they have the H3K27me3 mark (Fay, 2011). PRC1 restricts entry of paused Pol II into elongation at active genes that bind cohesin and PRC1, but lack PRC2 and the H3K27me3 modification (Schaaf, 2013b). It is thus posited that cohesin and PRC1 together restrict transition of the paused Pol II present at the active E(spl)-C genes into elongation. Because co-depletion of cohesin and PRC1 does not synergistically increase transcription, it is thought likely that they function together at the same step. Cohesin and PRC1 directly interact with each other, and cohesin facilitates binding of PRC1 to active genes that lack the H3K27me3 mark. Cohesin depletion, however, does not significantly alter PRC1 association with the E(spl)-C, likely because PRC1 binding is stabilized by the known interaction of PRC1 with H2K27me3. PRC1 is thus not sufficient to repress E(spl)-C transcription in the absence of cohesin, indicating that cohesin has roles that extend beyond its interaction with PRC1 (Schaaf, 2013).
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 wm4 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 concomitant 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 wm4-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 wm4 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 wm4 PEV. Conversely, the overexpression of Z4 was expected to favor 'open' chromatin structures and lead to a suppression of wm4 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 ID: 15292401
Fay, A., Misulovin, Z., Li, J., Schaaf, C. A., Gause, M., Gilmour, D. S. and Dorsett, D. (2011). Cohesin selectively binds and regulates genes with paused RNA polymerase. Curr Biol 21: 1624-1634. PubMed ID: 21962715
Gan, M., Moebus, S., Eggert, H. and Saumweber, H. (2011). The Chriz-Z4 complex recruits JIL-1 to polytene chromosomes, a requirement for interband-specific phosphorylation of H3S10. J Biosci 36: 425-438. PubMed ID: 21799255
Hochheimer, A., et al. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420(6914): 439-45. PubMed ID: 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 ID: 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 ID: 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 ID: 17634285
Kugler, S. J. and Nagel, A. C. (2010). A novel Pzg-NURF complex regulates Notch target gene activity. Mol. Biol. Cell 21(19): 3443-8. PubMed ID: 20685964
Müller, 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 ID: 16118195
Phalke, S., Nickel, O., Walluscheck, D., Hortig, F., Onorati, M. C. and Reuter, G. (2009). Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2. Nat Genet 41: 696-702. PubMed ID: 19412177
Schaaf, C. A., Misulovin, Z., Gause, M., Koenig, A., Gohara, D. W., Watson, A. and Dorsett, D. (2013). Cohesin and polycomb proteins functionally interact to control transcription at silenced and active genes. PLoS Genet 9: e1003560. PubMed ID: 23818863
Silva-Sousa, R., Lopez-Panades, E., Pineyro, D. and Casacuberta, E. (2012). The chromosomal proteins JIL-1 and Z4/Putzig regulate the telomeric chromatin in Drosophila melanogaster. PLoS Genet 8: e1003153. PubMed ID: 23271984
date revised: 10 October 2013
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