Gene name - modifier of mdg4
Synonyms - E(var)3-93D, doom
Cytological map position - 93D7--93D7
Function - transcription factor
Keywords - trithorax group, involved in chromosome condensation and segregation, regulation of transcription and enhancer blocking
Symbol - mod(mdg4)
Genetic map position - 3-70.7
Classification - BTB domain protein
Cellular location - nuclear
|Recent literature||Golovnin, A., Melnikova, L., Shapovalov, I., Kostyuchenko, M. and Georgiev, P. (2015). EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin. PLoS One 10: e0140991. PubMed ID: 26489095
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. This study has shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.
|Pauli, T., et al. (2016). Transcriptomic data from panarthropods shed new light on the evolution of insulator binding proteins in insects. BMC Genomics 17: 861. PubMed ID: 27809783
Body plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. This study screened ecently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods. The study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore GAF orthologs were found in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection. These results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, this study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.
|Melnikova, L., Kostyuchenko, M., Molodina, V., Parshikov, A., Georgiev, P. and Golovnin, A. (2017). Multiple interactions are involved in a highly specific association of the Mod(mdg4)-67.2 isoform with the Su(Hw) sites in Drosophila. Open Biol 7(10). PubMed ID: 29021216
The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190 was performed. The N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, this study has shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.
|Tikhonov, M., Utkina, M., Maksimenko, O. and Georgiev, P. (2018). Conserved sequences in the Drosophila mod(mdg4) intron promote poly(A)-independent transcription termination and trans-splicing. Nucleic Acids Res. PubMed ID: 30102331
Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3' variable exons generated from alternative promoters. To map the regions required for trans-splicing, this study has developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. It was have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5' splice site in the last common exon with the 3' splice site in one of the alternative exons.
|Sun, M. S., Weber, J., Blattner, A. C., Chaurasia, S. and Lehner, C. F. (2019). MNM and SNM maintain but do not establish achiasmate homolog conjunction during Drosophila male meiosis. PLoS Genet 15(5): e1008162. PubMed ID: 31136586
The first meiotic division reduces genome ploidy. This requires pairing of homologous chromosomes into bivalents that can be bi-oriented within the spindle during prometaphase I. Thereafter, pairing is abolished during late metaphase I, and univalents are segregated apart onto opposite spindle poles during anaphase I. In contrast to canonical meiosis, homologous chromosome pairing does not include the formation of a synaptonemal complex and of cross-overs in spermatocytes of Drosophila melanogaster. The alternative pairing mode in these cells depends on mnm and snm. These genes are required exclusively in spermatocytes specifically for successful conjunction of chromosomes into bivalents. Available evidence suggests that MNM and SNM might be part of a physical linkage that directly conjoins chromosomes. This study analyzed this notion was analyzed further. Temporal variation in delivery of mnm and snm function was realized by combining various transgenes with null mutant backgrounds. The observed phenotypic consequences provide strong evidence that MNM and SNM contribute directly to chromosome linkage. Premature elimination of these proteins results in precocious bivalent splitting. Delayed provision results in partial conjunction defects that are more pronounced in autosomal bivalents compared to the sex chromosome bivalent. Overall, these findings suggest that MNM and SNM cannot re-establish pairing of chromosomes into bivalents if provided after a chromosome-specific time point of no return. When delivered before this time point, they fortify preformed linkages in order to preclude premature bivalent splitting by the disruptive forces that drive chromosome territory formation during spermatocyte maturation and chromosome condensation during entry into meiosis I.
|Galouzis, C. C. and Prud'homme, B. (2021). Transvection regulates the sex-biased expression of a fly X-linked gene. Science 371(6527): 396-400. PubMed ID: 33479152
Sexual dimorphism in animals results from sex-biased gene expression patterns. These patterns are controlled by genetic sex determination hierarchies that establish the sex of an individual. This study shows that the male-biased wing expression pattern of the Drosophila biarmipes gene yellow, located on the X chromosome, is independent of the fly sex determination hierarchy. Instead, it was found that a regulatory interaction between yellow alleles on homologous chromosomes (a process known as transvection) silences the activity of a yellow enhancer functioning in the wing. Therefore, this enhancer can be active in males (XY) but not in females (XX). This transvection-dependent enhancer silencing requires the yellow intron and the chromatin architecture protein Mod(mdg4). These results suggest that transvection can contribute more generally to the sex-biased expression of X-linked genes.
Between 1993 and 1998, the mod(mdg4) gene was cloned three separate times, each time by a different laboratory. The investigators were in pursuit of a jack of all trades gene, one that presented a different functional phenotype for each clone: (1) as E(var)3-93D, mod(mdg4) was cloned based on its function as an enhancer of position-effect variegation, a protein involved in establishing and/or maintaining an open chromatin conformation (Dorn, 1993). (2) As mod(mdg4) the gene was cloned based on its ability to effect the ability of suppressor of Hairy wing (su [Hw]) to act as a chromatin insulator (Gerasimova, 1995). See suppressor of Hairy wing for more information about the boundary function of Mod(mdg4). (3) As doom, mod(mdg4) was cloned based on its ability to code for a protein that induces apoptosis (Harvey, 1997). Even more recently, mod(mdg4) has been shown to be a fully functional member of the trithorax family of genes, able to modify the expression of homeotic genes (Gerasimova, 1998). What is the true character of this gene with multiple personalities and what is the meaning of the multifaceted character of the Mod(mdg4) protein?
Recent work with Mod(mdg4) has raised to possibility that three of the four functions ascribed to this multifaceted protein are related: its role as enhancer of postion-effect variegation, its role as a chromatin insulator and its role as a trithorax family member. Members of the polycomb and trithorax families of proteins, coded for by PcG and trxG genes respectively, are thought to repress or maintain activity of homeotic genes through their action at polycomb response elements (PREs). PREs are a part of the promoter region in genes such as Ultrabithorax. Polycomb-group members act at these promoter sites to establish a repressive protein complex that keeps both the bound enhancer and other distal enhancers repressed in cells where the enhancer sites were initially active and subsequently repressed, maintaining this repressed state for many cell divisions. Boundary elements, typified by the boundary element found in the gypsy retrovirus, are a second class of chromosomal elements which function as insulators conferring position-independent transcription to genes and preventing activation of promoters by enhancers separated from proximal promoters by insulator elements. While polycomb and trithorax family members are known to act at PREs, it is now clear that they also can act at boundary elements (Gerasimova, 1998).
The observation of a shared pathway in the function of a chromatin insulator and trithorax group (trxG) and Polycomb group (PcG) gene activation and silencing is suggestive of a common mechanism at work. If this is the case, mutations in trxG and PcG genes, known to be involved in activation and silencing, might also affect the ability of the insulators to interfere with enhancer-promoter interactions. To test this possibility, the effect of trxG and PcG mutations on the abdominal coloration of flies carrying the yellow2 mutation (affecting coloration) was measured using insertion of an insulator-containing gypsy retrotransposon. Males hemizygous for the y2 allele show brown abdominal pigmentation in the fifth and sixth abdominal segments, instead of the black pigmentation observed in wild-type males, due to the effect of the insulator on the upstream body cuticle enhancer. This insulator effect on the body enhancer is altered by hypomorphic mutations in mod(mdg4), which gives rise to a variegated phenotype resulting from different expression levels of the yellow gene in adjacent groups of cells. In some cuticle cells, the effect of the insulator is reversed, resulting in normal expression of the yellow gene; in other cells, the effect of the insulator on enhancer-promoter communication appears to be enhanced, further repressing yellow gene expression. To examine the effect of trxG mutations on insulator function, the partially nonfunctional insulator, renderend such by hypomorphic alleles of mod(mdg4), was tested. An examination was carried out of the consequence of mutations in trxG genes, such as trithorax, on the frequency and severity of a mod(mdg4) phenotype engendered by Mod(mdg4) action at the gypsy insulator (Gerasimova, 1998).
Both the penetrance and severity of a variegated phenotype due to insulator function are enhanced by mutations in trxG genes. trx mutation results in a decrease in the number of dark spots with respect to that observed in hypomorphilc mod(mdg4) males, with only a few spots visible in a light brown-colored background. A stronger effect can be seen when trx is combined with brahma or ash1. Mutations in polycomb cause the opposite result, reversing the effect of the insulator on enhancer-promoter interactions and resulting in a wild-type expression of the yellow gene in the body cuticle. These results indicate that mutations in trxG genes cause an enhancement of the variegated phenotype induced by mod(mdg4) mutations in the yellow gene, suggesting that decreased levels of these proteins enhance the inhibitory effect of the insulator on enhancer-promoter interactions. In contrast, mutations in Pc impair the ability of the insulator to inhibit enhancer-promoter interactions, restoring normal expression of the gene. The effects of trxG and PcG mutations on insulator function at the yellow gene are not a result of homeotic transformations in abdominal segments that cause changes in the pigmentation of the cuticle, since these effects are not observed in flies carrying a wild-type copy of the yellow gene. In addition, the same effect can be observed with other gypsy-induced mutations such as scute-1 and cut-6. Flies of the genotype ct6; brm+ trx+ mod(mdg4)T16/brm2 trxB11 mod(mdg4)+ display a much stronger cut phenotype than ct6; mod(mdg4)T16/mod(mdg4)+ individuals, suggesting that the effect of TrxG and PcG proteins on gypsy insulator function is general and does not depend on the nature of the affected gene. A similar result was obtained with the sc1 mutation. The effects of trxG and PcG mutations on insulator function suggest that the proteins encoded by these genes might be structural components of the gypsy insulator or they might regulate its function (Gerasimova, 1998).
The Mod(mdg4) protein is present at approximately 500 sites on polytene chromosomes of third-instar larvae from strains that lack gypsy elements. Many or all of these sites might represent endogenous insulators. Since both Mod(mdg4) and su(Hw) associate with the gypsy insulator, it is possible that they colocalize at many of these sites. su(Hw) is a DNA binding protein and Mod(mdg4), unable to bind DNA, has been shown to be able to physically interact with su(Hw), thus facilitating the association of Mod(mdg4) with the insulator (Gerasimova, 1995). The su(Hw) protein is present at approximately 200 sites on polytene chromosomes, and Mod(mdg4) is found at every one of these sites. Since the gypsy retrotransposon is not present at these sites, it is hypothesized that these chromosomal locations contain sequences similar to those present in the gypsy insulator and are thus functionally equivalent. The Mod(mdg4) protein is present in approximately 300 additional sites without su(Hw), suggesting that Mod(mdg4) can interact with DNA-binding proteins other than su(Hw), either to form a different type of insulator or to play a different role in gene expression. Indeed, Trithorax group and Polycomb group proteins are found to colocalize with Mod(mdg4) at some sites on polytene chromosomes. Mutations in trithorax, absent small or homeotic discs1 (ash1) and brahma reduce the levels of Mod(mdg4) protein in polytene chromosomes. The punctated pattern of Mod(mdg4) in the nuclei of follicle cells is lost in su(Hw) mutants. It is thought that the functional domains represented by these subnuclear regions is nuclear matrix. This opens the possibility that insulator sequences act as matrix attachment regions and that su(Hw) and Mod(mdg4) mediate the interaction of boundary elements with the nuclear matrix. Interestingly, in the background of a null mutation in the su(Hw) gene, the Mod(mdg4) protein is not found at those sites that are common with su(Hw), whereas localization at other sites appears normal. The subnuclear distribution of Mod(mdg4) and su(Hw) is dramatically altered in the background of trithorax Group mutations, with a loss of the punctated pattern. In trxG mutants Mod(mdg4) localizes mostly to the cytoplasm. In polycomb mutants Mod(mdg4) and su(Hw) localize to the central region of the nucleus instead of the nuclear matrix. The alterations in the subnuclear localization of Mod(mdg4) and Su(HW) proteins as a consequence of mutations in trxG and PcG genes correlate with the effects these mutations cause on insulator function (Gerasimova, 1998).
A large number of mod(mdg4) cDNAs, representing 21 different isoforms generated by alternative splicing, have been isolated. The deduced proteins are characterized by a common N terminus of 402 amino acids, including the BTB/POZ-domain. Most of the variable C termini contain a new consensus sequence, including four positioned hydrophobic amino acids and a Cys2His2 motif. Using specific antibodies for two protein isoforms, different distributions of the corresponding proteins on polytene chromosomes have been demonstrated. Mutations in the genomic region encoding exons 1-4 show enhancement of PEV and homeotic transformation and affect viability and fertility. Homeotic and PEV phenotypes are enhanced by mutations in other trx-group genes. A transgene containing the common 5' region of mod(mdg4) that is present in all splice variants known so far partially rescues the recessive lethality of mod(mdg4) mutant alleles. These data provide evidence that the molecular and genetic complexity of mod(mdg4) is caused by a large set of individual protein isoforms with specific functions in regulating the chromatin structure of different sets of genes throughout development (Buchner, 2000).
A differential distribution of at least two Mod(mdg4) proteins, Mod(mdg4)-58.0 and Mod(mdg4)-67.2, along polytene chromosomes, has been demonstrated. Whereas Mod(mdg4)-67.2 is found at the majority of sites, labeled by the antibody anti-Mod(mdg4)-58.0BTB-534 that detects all protein isoforms, the other isoform is restricted to a small subset of sites. The binding of Mod(mdg4)-58.0 and Mod(mdg4)-67.2 at different sites suggests that at least these two Mod(mdg4) isoforms participate in transcriptional regulation of different sets of genes. It is supposed that the specific C-terminal domains play a critical role in directing the isoforms to different binding sites, possibly through specific interactions with other proteins. Two other observations are consistent with this hypothesis. There is an interaction of Mod(mdg4)-67.2 [Mod(mdg4)2.2] with Su(Hw), a zinc finger protein that binds to gypsy sequences. Both proteins are implicated in the function of chromatin insulator sequences present in the gypsy transposon. One of the Mod(mdg4) isoforms, DOOM [Mod(mdg4)-56.3], interacts with the baculovirus inhibitor of apoptosis protein. Together these results suggest that the large number of protein isoforms generated from mod(mdg4) reflects the functional diversity of individual Mod(mdg4) proteins. The GAGA factor, encoded by the Trl gene, has been shown to be involved in nucleosome remodeling in regulatory regions of many genes. Mutations in Trl and mod(mdg4) display very similar genetic properties, e.g., enhancement of PEV, paternal effects, and homeotic transformation. The generation of different GAGA isoforms containing a common N terminus of 377 amino acids with an N-terminal BTB/POZ domain has been demonstrated. However, in contrast to mod(mdg4), a colocalization of two different GAGA isoforms on polytene chromosomes and their ability to form heterodimers has been demonstrated by coimmunoprecipitation (Buchner, 2000 and references therein).
The protein consensus sequence that contains a Cys2His2 motif within the specific protein domains of most Mod(mdg4) isoforms may be of functional importance. In contrast to canonical zinc-finger motifs of the Cys2His2 type, the one found here has distinct features. The two histidine residues are separated by only one amino acid residue, and the consensus sequence extends N-terminal with additional conserved aromatic amino acid positions. The presence of the conserved sequence in the specific protein domain of DOOM implicates its putative involvement in protein-protein interaction with IAP. Disruption of this interaction by mutagenesis of the highly conserved amino acid positions could test this hypothesis. However, five of the different isoforms do not contain the identified consensus sequence, including Mod(mdg4)-58.0 and Mod(mdg4)-67.2. The functional significance of the presence of several Cys and His residues in these isoforms remains unknown (Buchner, 2000).
Genetic analysis of several mod(mdg4) mutant alleles has revealed pleiotropic effects. All mutations are dominant enhancers of PEV and display paternal enhancer effects. Additionally, mod(mdg4) mutations have been demonstrated to display properties typical for trx group genes. Enhanced homeotic transformation has been observed in trans and cis combinations with several mutations in other trx group genes, suggesting a possible interaction of the corresponding proteins. This is supported by the observed interactions in PEV enhancement. Based on the finding of different distribution of Trx and Mod(mdg4) proteins in diploid interphase nuclei and the altered distribution of Mod(mdg4) proteins in the background of trx mutations, a two-tier model for chromatin assembly has been proposed. According to this model, the formation of complexes containing Trx precedes the assembly of Mod(mdg4) proteins (Buchner, 2000).
Most of the molecularly characterized mod(mdg4) mutations involve sequences within the common 5' region. These mutations would be expected to affect all Mod(mdg4) protein isoforms, explaining the observed pleiotropic mutant effects. Although the differential distribution of two protein isoforms has been demonstrated, it is not known if the loss of single isoforms causes distinct mutant phenotypes. This would be expected if Mod(mdg4) proteins have specific functions in chromatin. Mutations within the specific protein domains of different isoforms should allow a further functional dissection of mod(mdg4) (Buchner, 2000).
mod(mdg4) is expressed at high levels during oogenesis: the presence of large amounts of Mod(mdg4) protein in all stages of oogenesis and early embryogenesis indicates a strong maternal component. The significantly reduced amounts of Mod(mdg4) protein detected in egg chambers of homozygous mod(mdg4) mutant females and the failure of eggs to foster further development indicate important functions of mod(mdg4) during oogenesis and early embryonic development. The presence of Mod(mdg4) in both nurse cell and follicle cell nuclei and the supposed role as a general transcriptional regulator suggest that mod(mdg4) is required for control of maternal genes during oogenesis. This is in agreement with the supposed role of Mod(mdg4) protein in mediating the function of chromatin insulator sequences as a prerequisite for correct promoter-enhancer interactions. In the embryo, Mod(mdg4) protein does not become localized to the nuclei until cleavage cycle 9, further arguing against a function in chromatin organization during early cleavage cycles (Buchner, 2000).
A transgene containing the common part of Mod(mdg4) can partially rescue the recessive lethality of mod(mdg4) mutant alleles. This result can be explained by the expression of a truncated protein containing the 402-amino-acid common N-terminal region and the ability to partially replace the function of full-length Mod(mdg4) protein. However, a protein of the expected molecular size could not be detected in the transgenic animals, which may be due to the limited sensitivity of Western blot analysis. Expression of a tagged protein under control of the hsp70 promoter from a transgene will be required to prove the proposed function of the common N-terminal peptide (Buchner, 2000).
What of the involvement of Mod(mdg4) in cell death? A screen was carried out for proteins able to interact with baculovirus inhibitor-of-apoptosis (IAP) proteins. Mod(mdg4) is able to bind Baculovirus IAP proteins and by so doing, induces apoptosis.
A brief sidetrack is taken here to describe the nature of IAP proteins:
IAPs are able to block some reaper-dependent death in the developing Drosophila eye. Murine cellular IAP1, along with human c-IAP1 and c-IAP2) has been identified by its tethering to a Tumor necrosis factor receptor (TNFR2) through binding to the TRAF1-TRAF2 (TRAFs are TNF-receptor associated factors) heterocomplex or by its homology to baculovirus IAPs. c-IAP1 is able to partially block reaper- and hid-dependent apoptosis in the developing Drosophila eye (Hay, 1995). A Drosophila IAP homolog (DIAP1), was identified in a screen for mutations that enhance the effect of reaper- and hid-dependent apoptosis in the developing eye (Harvey, 1997 and references).
Returning to the main discussion of Mod(mdg4), an alternatively spliced Mod(mdg4) protein, Doom, was obtained in a two hybrid screen using Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) as bait. Doom possesses a C-terminal Doom specific domain (DSD) not found in other Mod(mdg4) splice varients. Two doom cDNAs obtained in the two-hybrid screen lack most or all of the BTB coding sequences of Mod(mdg4). However, Doom possessing the BTB domain is able to induce apoptosis in cultured cells as efficiently as Doom lacking the BTB domain. It has been shown that the DSD of Doom and the BIR of the IAPs interact to form a Doom-IAP complex; the IAP localizes to the nucleus of cultured cells in the presence of Doom. Coexpression of the BIR region of IAP with Doom does not block apoptosis. Thus, the RING finger of the IAP is crucial for antiapoptotic function even though it is not necessary for Doom interaction. The function of the baculovirus RING finger in blocking apoptosis is not known, but it may mediate IAP binding to another factor. If IAPs normally localize with Doom in the nucleus, then one can envision IAPs normally associating with Doom to inhibit Doom from inducing apoptosis, but when Doom is overexpressed, the balance between IAPs and Doom is disrupted, resulting in apoptosis. Alternatively, IAPs may localize to the cytoplasm and be translocated to the nucleus following Doom activation and/or expression. It is possible that the proapoptotic activity of doom is due to Doom being a regulator of chromatin structure and that overexpression of doom mimics or triggers a response similar to DNA damage or genomic dysfunction. Currently, it is unknown if this is a normal means by which cells trigger apoptosis (Harvey, 1997).
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. This study has shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization (Golovnin, 2015).
Insulators belong to the class of regulatory elements that organize the architecture of chromatin compartments. Insulators, or chromatin boundaries, are characterized by two properties: they interfere with enhancer-promoter interactions when located between them and buffer transgenes from chromosomal positions effects. To date, chromatin insulators have been characterized in a variety of species, indicative of their involvement in the global regulation of gene expression (Golovnin, 2015).
The well-studied Drosophila insulator proteins, dCTCF (homolog of vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. The Su(Hw) protein, encoded by the suppressor of Hairy wing [su(Hw)] gene, was one of the first insulator proteins identified in Drosophila. The best-studied Drosophila insulator found within the 5'-untranslated region of the gypsy retrovirus consists of 12 directly repeated copies of Su(Hw) binding sites. Genetic and molecular approaches have led to the identification and characterization of three proteins recruited by Su(Hw) to chromatin-Mod(mdg4)-67.2, CP190, and E(y)2/Sus1-that are required for the activity of the Su(Hw)-dependent insulators. The mod(mdg4) gene, also known as E(var)3-93D, encodes a large set of BTB/POZ protein isoforms. One of these isoforms, Mod(mdg4)-67.2, by its specific C-terminal domain interacts with the enhancer-blocking domain of the Su(Hw) protein. The BTB domain is located at the N-terminus of Mod(mdg4)-67.2 and mediates homo-multimerization (Golovnin, 2015).
Su(Hw), dCTCF, and most of other identified insulator proteins interact with Centrosomal Protein 190 kD (CP190). This protein (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc finger motifs, and a C-terminal E-rich domain. The BTB domain of CP190 forms stable homodimers that may be involved in protein-protein interactions. In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis. It has been shown that CP190 is recruited to chromatin via its interaction with the DNA insulator proteins in interphase nucleus (Golovnin, 2015).
The Su(Hw), dCTCF, Mod(mdg4)-67.2, and CP190 proteins colocalize in discrete foci, named insulator bodies, in the Drosophila interphase cell nucleus. Contradictory reports have been published in which the insulator bodies are described either as protein-based bodies in the interchromatin compartment or as chromatin domains. As shown recently, insulator proteins rapidly coalesce from diffusely distributed speckles into large punctate insulator bodies in response to osmotic stress (Golovnin, 2015).
Cell exposure to hypertonic treatment, which enhances molecular crowding, makes it possible to discriminate between nucleoplasmic bodies formed mainly of RNA and proteins (such as PML bodies) and chromatin compartments such as Polycomb bodies formed due to the interaction of distantly located chromatin regions bound by Polycomb proteins. Nucleoplasmic bodies disappear under less crowded conditions and reassemble under normally crowded conditions, which can be interpreted as a consequence of increased intermolecular interactions between components of nucleoplasmic bodies. Similar to PML bodies, insulator bodies are preserved under hypertonic treatment, in contrast to chromatin-based structures that disappear as proteins dissociate from chromatin. The CP190 protein is suggested to be critical for the activity of insulators and to regulate the entry of other insulator proteins into the speckles. At the same time, CP190 associates with centrosomes throughout the nuclear division cycle in syncytial Drosophila embryos. Nuclear localization of CP190 is also sensitive to various kinds of stress, suggesting that this process is highly regulated. However, the mechanisms and proteins responsible for localization of CP190 in different nucleus compartments are unknown. This study has shown that the nuclear distribution of CP190 depends on the level of EAST, which is located mainly in the interchromatin compartment of the nucleus. EAST is a nuclear protein of 2362 amino acids which, except for 9 potential nuclear localization sequences and 12 potential PEST sites, contains no previously characterized motifs or functional domains. Together with Skeletor, Chromator, and Megator proteins, EAST forms the spindle matrix during mitosis. In the interphase nuclei, EAST localizes to the extrachromosomal compartment of the nucleus and is essential for the spatial organization of chromosomes (Golovnin, 2015).
Despite that the bulk of interphase EAST resides in the interchromosomal domain, the current model assumes that EAST can transiently interact with chromosomes. EAST physically interacts with Megator, a 260-kDa protein with a large N-terminal coiled-coil domain capable of self-assembly. It has been speculated that Megator can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The results show that EAST interacts with CP190 and Mod(mdg4)-67.2 proteins and modulates their aggregation into the nuclear speckles. In case of EAST overexpression, CP190 binding to chromatin is reduced; consequently, the binding of Mod(mdg4)-67.2 and Su(Hw) is reduced as well, since CP190 is essential for it. On the basis of these results, it is hypothesized that EAST regulates localization of CP190 and insulator protein complexes in the interchromatin compartment, with these complexes subsequently determining organization of chromatin insulators (Golovnin, 2015).
The results suggest that insulator bodies are sensitive to the concentration of EAST in interphase cells. The properties of insulator bodies described previously and in this study suggest that they are formed by multiple interactions between proteins and resemble nuclear bodies composed of aggregated proteins and RNAs. As shown previously, the CP190 and Mod(mdg4) proteins interact with Su(Hw) and dCTCF and help the latter to enter the insulator bodies (Golovnin, 2015).
Taking into account the high level of dCTCF and Mod(mdg4) co-binding to chromosomes, it appears that dCTCF interacts with an as yet unidentified Mod(mdg4) isoform. Mod(mdg4)-67.2 and CP190 conjugate to the small ubiquitin-like modifier protein (SUMO). Specific interactions mediated by SUMO, the ability of Mod(mdg4) BTB to form oligomers, and the interaction between the BTB domain of Mod(mdg4)-67.2 and CP190 contribute to specific aggregation of the Su(Hw)/Mod(mdg4)-67.2/CP190 and dCTCF/CP190 complexes into the insulator bodies (Golovnin, 2015).
According to current views, the Megator protein can form polymers that, together with EAST, may serve as a structural basis for the nuclear extrachromosomal compartment. The overexpression of EAST leads to an extension of the EAST-Megator compartment, with consequent reduction in the effective volume available for the insulator proteins in the cell. As a result, the concentration of the insulator proteins increases, contributing to stabilization of the compact protein conformations visualized as insulator bodies. By interacting with Mod(mdg4)-67.2 and CP190, EAST may also be directly involved in nucleation of insulator bodies. It is possible that the truncated version of EAST (from 933 to 2362 aa) can more easily interact with the insulator proteins, which leads to noticeable enlargement of insulator bodies in S2 cell expressing EAST933-2362. The overexpression of EAST leads to segregation of the CP190 protein in independent speckles. The results suggest that EAST interacts with the CP190 region that includes BTB, D, and M domains. These domains are also required for CP190 interactions with other insulator proteins (Golovnin et al., in preparation). Thus, an increase in the EAST concentration may lead to displacement of the insulator proteins from the complex with CP190 (Golovnin, 2015).
The results do not exclude the possibility that EAST overexpression directly leads to dissociation of CP190 from chromatin. During mitosis, CP190 colocalizes with EAST in the spindle matrix, and the increase in the amount of EAST may well be responsible for dissociation of CP190 prior to chromosome condensation (Golovnin, 2015).
According to the current model, the insulator bodies help to form protein complexes that subsequently bind to regulatory elements such as insulators and promoters. In view of this hypothesis, it is likely that disturbances in the insulator bodies caused by EAST overexpression are responsible for the decrease in CP190 binding to the regulatory regions such as dCTCF- and Su(Hw)-dependent insulators and promoters. As shown recently, CP190 is required for recruiting Su(Hw) and Mod(mdg4)-67.2, but not dCTCF, to chromatin. Accordingly, it was observed that EAST overexpression affects the chromosomal binding of Su(Hw), but not of dCTCF. CP190 specifically interacts with the Mod(mdg4)-67.2 isoform, and Mod(mdg4)-67.2 at all Su(Hw) binding sites is colocalized with CP190. Thus, CP190 may be essential for recruiting the specific Mod(mdg4)-67.2 isoform to the Su(Hw) binding sites, with subsequent decrease in the amount of CP190 at the Su(Hw) binding sites, which leads to the substitution of Mod(mdg4)-67.2 by other Mod(mdg4) isoforms, as has been observed in this study (Golovnin, 2015).
Strong inactivation of EAST in S2 cells reduces the entry of the Mod(mdg4)-67.2/ Su(Hw) complex, but not of CP190, into the nucleus. It appears that EAST is involved in the regulation of nuclear localization of Mod(mdg4)-67.2, whose BTB domain can form multimeric complexes. Further study is required to elucidate this issue (Golovnin, 2015).
Adenosine (Ado) is an important signaling molecule involved in stress responses. Studies in mammalian models have shown that Ado regulates signaling mechanisms involved in "danger-sensing" and tissue-protection. Yet, little is known about the role of Ado signaling in Drosophila. This study observed lower extracellular Ado concentration and suppressed expression of Ado transporters in flies expressing mutant huntingtin protein (mHTT). Ado signaling was altered using genetic tools; the overexpression of Ado metabolic enzymes, as well as the suppression of Ado receptor (AdoR) and transporters (ENTs) were found to minimize mHTT-induced mortality. The downstream targets of the AdoR pathway were identified, the modifier of mdg4 (Mod(mdg4)) and heat-shock protein 70 (Hsp70), which modulated the formation of mHTT aggregates. Finally, a decrease in Ado signaling affects other Drosophila stress reactions, including paraquat and heat-shock treatments. This study provides important insights into how Ado regulates stress responses in Drosophila (Lin, 2021).
Tissue injury, ischemia, and inflammation activate organismal responses involved in the maintenance of tissue homeostasis. Such responses require precise coordination among the involved signaling pathways. Adenosine (Ado) represents one of the key signals contributing to the orchestration of cytoprotection, immune reactions, and regeneration, as well as balancing energy metabolism. Under normal conditions, the Ado concentration in blood is in the nanomolar range; however, under pathological circumstances the extracellular Ado (e-Ado) level may dramatically change. Ado has previously been considered a retaliatory metabolite, having general tissue protective effects. Prolonged adenosine signaling, however, can exacerbate tissue dysfunction in chronic diseases. As suggested for the nervous system in mammals, Ado seems to act as a high pass filter for injuries by sustaining viability with low insults and bolsters the loss of viability with more intense insults (Lin, 2021 and references therein).
Adenosine signaling is well-conserved among phyla. The concentration of Ado in the Drosophila melanogaster hemolymph is maintained in the nanomolar range, as in mammals, and increases dramatically in adenosine deaminase mutants or during infections. Unlike mammals, D. melanogaster contains only a single Ado receptor (AdoR) isoform (stimulating cAMP) and several proteins that have Ado metabolic and transport activities involved in the fine regulation of adenosine levels. D. melanogaster adenosine deaminase-related growth factors (ADGFs), which are related to human ADA2, together with adenosine kinase (AdenoK) are the major metabolic enzymes converting extra- and intra-cellular adenosine to inosine and AMP, respectively. The transport of Ado across the plasma membrane is mediated by three equilibrative and two concentrative nucleoside transporters (ENTs and CNTs, respectively) similar to their mammalian counterparts. Ado signaling in Drosophila has been reported to affect various physiological processes, including the regulation of synaptic plasticity in the brain, proliferation of gut stem cells, hemocyte differentiation, and metabolic adjustments during the immune response (Lin, 2021 and references therein).
The present study examined the role of Drosophila Ado signaling on cytotoxic stress and aimed to clarify the underlying mechanism. Earlier reports have shown that expression of the expanded polyglutamine domain from human mutant huntingtin protein (mHTT) induces cell death in both Drosophila neurons and hemocytes. In this study, the low-viability phenotype of mHTT-expressing larvae and it was observed that such larvae display a lower level of e-Ado in the hemolymph. Furthermore, this study used genetic tools and altered the expression of genes involved in Ado metabolism and transport to find out whether changes in Ado signaling can modify the phenotype of mHTT-expressing flies. Finally, a downstream mechanism was uncovered of the Drosophila Ado pathway, namely mod(mdg4) and heat-shock protein 70 (Hsp70), which modify both the formation of mHTT aggregates and the stress response to heat-shock and paraquat treatments (Lin, 2021).
Adenosine signaling represents an evolutionarily conserved pathway affecting a diverse array of stress responses. As a ubiquitous metabolite, Ado has evolved to become a conservative signal among eukaryotes. In previous studies, Drosophila adoR mutants and mice with a knockout of all four adoRs both displayed minor physiological alteration under normal conditions. This is consistent with the idea that Ado signaling more likely regulates the response to environmental changes (stresses) rather than being involved in maintaining fundamental homeostasis in both insect and mammalian models. This study examined the impact of altering the expression of genes involved in Ado signaling and metabolism on the cytotoxicity and neurodegeneration phenotype or Q93 mHTT-expressing flies. A novel downstream target of this pathway, mod(mdg4), was discovered and showed its effects on the downregulation of Hsp70 proteins, a well-known chaperone responsible for protecting cells against various stress conditions, including mHTT cytotoxicity, as well as thermal or oxidative stress (Lin, 2021).
The low level of Ado observed in da-Gal4>mHTT flies suggests that it might have a pathophysiological role; lowering of the Ado level might represent a natural response to cytotoxic stress. Consistently, experimentally decreased Ado signal rescued the mHTT phenotype, while an increased Ado signal had deleterious effects. Interestingly, a high level of Ado in the hemolymph has previously been observed in Drosophila infected by a parasitoid wasp. A raised e-Ado titer has not only been shown to stimulate hemocyte proliferation in the lymph glands, but also to trigger metabolic reprogramming and to switch the energy supply toward hemocytes. In contrast, the experiments show that a lowered e-Ado titer results in increased Hsp70 production. Increased Hsp70 has previously been shown to protect the cells from protein aggregates and cytotoxicity caused by mHTT expression, as well as some other challenges including oxidative stress (paraquat treatment) or heat-shock. The fine regulation of extracellular Ado in Drosophila might mediate the differential Ado responses via a single receptor isoform. Earlier experiments on Drosophila cells also suggested that different cell types have different responses to Ado signaling (Lin, 2021 and references therein).
The data also showed that altered adenosine signaling through the receptor is closely connected to Ado transport, especially to ent2 transporter function. It was observed that adoR and ent2 knockdowns provide the most prominent rescue of mHTT phenotypes. In addition, the overexpression of adoR and ent2 genes results in effects that are opposite to their knockdowns, thus supporting the importance of these genes as key regulators of mHTT phenotypes. Arevious report showed that responses to adoR and ent2 mutations cause identical defects in associative learning and synaptic transmission. The present study shows that the phenotypic response of mHTT flies to adoR and ent2 knockdowns are also identical. The results suggest that the source of e-Ado for inducing AdoR signaling is mainly released by ent2. Consistently, the knockdown of ent2 has previously been shown to block Ado release from Drosophila hemocytes upon an immune challenge, as well as from wounded cells stimulated by scrib-RNAi (Poernbacher, 2018) or bleomycin feeding. These data support the idea that both adoR and ent2 work in the same signaling pathway (Lin, 2021).
The results revealed that lower AdoR signaling has a beneficial effect on mHTT-expressing flies, including increasing their tolerance to oxidative and heat-shock stresses. The effect of lower Ado signaling in mammals has been studied by pharmacologically blocking AdoRs, especially by the non-selective adenosine receptor antagonist caffeine. Interestingly, caffeine has beneficial effects on both neurodegenerative diseases and oxidative stress in humans. In contrast, higher long-term Ado concentrations have cytotoxic effects by itself in both insect and mammalian cells. Chronic exposure to elevated Ado levels has a deleterious effect, causing tissue dysfunction, as has been observed in a mammalian system. Extensive disruption of nucleotide homeostasis has also been observed in mHTT-expressing R6/2 and Hdh150 mice (Lin, 2021).
This study identified a downstream target of the AdoR pathway, mod(mdg4), which modulates mHTT cytotoxicity and aggregations. This gene has previously been implicated in the regulation of position effect variegation, chromatin structure, and neurodevelopment. The altered expression of mod(mdg4) has been observed in flies expressing untranslated RNA containing CAG and CUG repeats. In addition, mod(mdg4) has complex splicing, including trans-splicing, producing at least 31 isoforms. All isoforms contain a common N-terminal BTB/POZ domain which mediates the formation of homomeric, heteromeric, and oligomeric protein complexes. Among these isoforms, only two [including mod(mdg4)-56.3 (isoform H) and mod(mdg4)-67.2 (isoform T)] have been functionally characterized. mod(mdg4)-56.3 is required during meiosis for maintaining chromosome pairing and segregation in males. mod(mdg4)-67.2 interacts with suppressor of hairy wing [Su(Hw)] and Centrosomal protein 190 kD (CP190) forming a chromatin insulator complex which inhibits the action of adjacent enhancers on the promoter, and is important for early embryo development and oogenesis. This study showed that mod(mdg4) is controlled by AdoR which consecutively works as a suppressor of Hsp70 chaperone. The downregulation of adoR or mod(mdg4) leads to the induction of Hsp70, which in turn suppresses mHTT aggregate formation and other stress phenotypes. Although the results showed that silencing all mod(mdg4) isoforms decreases cytotoxicity and mHTT inclusion formation, it was not possible clarify which of the specific isoforms is involved in such effects, since AdoR seems to regulate the transcriptions of multiple isoforms. Further study will be needed to identify the specific mod(mdg4) isoform(s) connected to Hsp70 production (Lin, 2021).
In summary, the data suggest that the cascade (ent2)-AdoR-mod(mdg4)-Hsp70 might represent an important general Ado signaling pathway involved in the response to various stress conditions, including reaction to mHTT cytotoxicity, oxidative damage, or thermal stress in Drosophila cells. The present study provides important insights into the molecular mechanisms of how Ado regulates mHTT aggregate formation and stress responses in Drosophila; this might be broadly applicable for understanding how the action of Ado affects disease pathogenesis (Lin, 2021).
Bases in 5' UTR - 137 and 123 (reported for doom)
Exons - 4 (plus a doom exon: Harvey, 1997)
Bases in 3' UTR - 206 and 64 (reported for doom)
In Drosophila, modifying mutations of position-effect variegation have been successfully used to genetically dissect chromatin components. The enhancer of position-effect variegation E(var)3-93D [formerly E-var(3)3] encodes proteins containing a BTB domain common to Tramtrack and the products of the Broad complex, all of which are the transcriptional regulators. The two cDNAs isolated code for proteins that are identical in the N-terminal region but differ at their C-termini. The C-terminus is rich in charged amino acids (Dorn, 1993).
Amino acids 403 to 514 of Doom encode a novel domain unique to Doom, which is designated DSD. DSD is an alternatively spliced form of Mod(mdg4) (Harvey, 1997).
All the evidence so far points to a gene's protein-coding information being contained in only one of its two DNA strands, with this strand serving as a template for transcription of the precursor RNA that is eventually translated into protein. Structural evidence is presented showing that the protein-coding information of the modifier of mdg4 [mod(mdg4)] gene of the fruitfly Drosophila is provided by both of its complementary DNA strands, and not by just one. This novel organization means that RNA precursors generated from two DNA templates need to be joined subsequently into a single messenger RNA, a surprising feature that raises new questions regarding genome complexity and evolution (Labrador, 2001).
date revised: 15 June 2021
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