org Interactive Fly, Drosophila modifier of mdg4: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - modifier of mdg4

Synonyms - E(var)3-93D, doom

Cytological map position - 93D7--93D7

Function - transcription factor

Keywords - trithorax group

Symbol - mod(mdg4)

FlyBase ID:FBgn0002781

Genetic map position - 3-70.7

Classification - BTB domain protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene | HomoloGene
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
Summary:
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
Summary:
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.
BIOLOGICAL OVERVIEW

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:

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).


GENE STRUCTURE

cDNA length - 1733 (reported for doom: Harvey, 1997)

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)


PROTEIN STRUCTURE

Amino Acids - 610 (Dorn, 1993) and 514 (reported for Doom)

Structural Domains

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).


modifier of mdg4: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 April 98

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