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?

Polycomb and trithorax group proteins mediate the function of a chromatin insulator

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 trx^B11 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:

One class of protein that inhibits apoptosis, represented by baculovirus P35, can block apoptosis by binding to and inactivating members of the interleukin-1beta-converting enzyme (ICE)-like cysteine proteases or caspases, which reside at critical positons in a number of apoptotic pathways (see Death caspase-1). Members of a second family of apoptosis blocking protein, the IAP family, can substitute for P35 in blocking certain apoptosis events. The IAPs have no structural similarity to the P35 protein but instead possess two types of zinc finger motifs, the baculovirus IAP repeat (BIR) and a carboxyl-terminal RING finger, both of which are required for antiapoptotic activity. The RING finger motifs are found in a number of proteins involved in such diverse cellular activities as transcription, DNA repair, and cell proliferation, whereas the BIR motif is unique to IAPs and is tandemly repeated in the amino-terminal and central portions of the protein.

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).

EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin

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 EAST^933-2362. 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 receptor and its downstream targets, Mod(mdg4) and Hsp70, work as a signaling pathway modulating cytotoxic damage in Drosophila

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).

Drosophila SUMM4 complex couples insulator function and DNA replication control

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. This study developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. This findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins (Andreyeva, 2022).

Replication of metazoan genomes occurs according to a highly coordinated spatiotemporal program, where discrete chromosomal regions replicate at distinct times during S phase. The replication program follows the spatial organization of the genome in Megabase-long constant timing regions interspersed by timing transition regions (Marchal, 2019). The spatiotemporal replication program exhibits correlations with genetic activity, epigenetic marks, and features of 3D genome architecture and subnuclear localization. Yet the reasons for these correlations remain obscure. Interestingly, the timing of firing for any individual origin of replication is established during G1 before pre-replicative complexes (pre-RC) are assembled at origins, suggesting a mechanism that involves factors other than the core replication machinery (Andreyeva, 2022).

Most larval tissues of Drosophila melanogaster grow via G-S endoreplication cycles that duplicate DNA without cell division, resulting in polyploidy. Endoreplicated DNA molecules frequently align in register to form giant polytene chromosomes. Importantly, in some cell types, genomic domains corresponding to the latest replicated regions of dividing cells, specifically pericentric (PH) and intercalary (IH) heterochromatin, fail to fully replicate during each endocycle resulting in underreplication (UR). These regions are depleted of sites for binding the Origin of Replication Complex (ORC), and thus, their replication primarily relies on forks progressing from external origins in both dividing and endoreplicating cells, which suggests that both cell types utilize related mechanisms of regulation of late replication. Although cell cycle programs are dissimilar between endoreplicating and mitotically dividing cells, they likely share the components of core biochemical machinery for DNA replication. Thus, underreplication provides a facile readout for late replication initiation and delayed fork progression (Andreyeva, 2022).

The Suppressor of UnderReplication (SuUR) gene is essential for polytene chromosome underreplication in intercalary and pericentric heterochromatin (Belyaeva, 1998). In SuUR mutants, the DNA copy number in underreplicated regions is partially restored to almost reach those for fully polyploidized regions of the genome. SuUR encodes a protein (SUUR) containing a helicase domain with homology to that of the SNF2/SWI2 family. The occupancy of ORC in intercalary and pericentric heterochromatin is not increased in SuUR mutants (Sher, 2012), and, thus, the increased replication of underreplicated regions is likely not due to the firing of additional origins. Rather, SUUR negatively regulates the rate of replication fork progression (Nordman, 2014) by an unknown mechanism. It has been proposed (Posukh, 2015) that retardation of the replisome by SUUR takes place via simultaneous physical association with the components of the fork (e.g., CDC45 and PCNA) and repressive chromatin proteins, such as HP1a (Andreyeva, 2022).

Using a newly developed proteomics approach, this study discovered that SUUR forms a stable stoichiometric complex with a chromatin boundary protein Mod(Mdg4)-67.2. SUUR and Mod(Mdg4)-67.2 together are required for maximal underreplication of intercalary heterochromatin and full activity of the gypsy insulator, thereby implicating insulators in obstructing replisome progression and the control of late DNA replication (Andreyeva, 2022).

This paper presents a facile method, termed MERCI, to rapidly identify subunits of stable native complexes by only partial chromatographic purification. It allows one to circumvent the conventional, rate-limiting approach to purify proteins to apparent homogeneity. Since a multistep FPLC scheme invariably leads to an exponential loss of material, reducing the number of purification steps in the MERCI protocol allows identification of rare complexes, such as SUMM4, which may be present in trace amounts in native sources. On the other hand, MERCI obviates introduction of false-positives frequently associated with tag purification of ectopically expressed targets that render results less reliable. Notably, MERCI is not limited to analyses of known polypeptides since it is readily amenable to fractionation of native factors based on a correlation with their biochemical activities in vitro (Andreyeva, 2022).

Both subunits of SUMM4 contribute to the known functions of gypsy insulator. Although a SuUR mutation decreased the insulator activity, the suppression was universally weaker than that by mod(mdg4)u1. It is possible that SUUR is not absolutely required for the establishment of the insulator. For instance, the loss of SUMM4 may be compensated by the alternative complex of Mod(Mdg4)-67.2. Furthermore, the mod(mdg4)u1 allele is expected to have an antimorphic function since it can mis-localize interacting partner proteins, including SUUR itself. Interestingly, SuUR has been previously characterized as a weak suppressor of variegation of the whitem4h X chromosome inversion allele, which places the white gene near pericentric heterochromatin. In contrast, SuUR mutation enhances variegation in the context of insulated, heterochromatin-positioned white. Therefore, this phenotype is unrelated to the putative Su(var) function of SuUR but, rather, is insulator-dependent (Andreyeva, 2022).

Discovery and analyses of SUMM4 provide a biochemical link between ATP-dependent motor factors and the activity of insulators in the regulation of gene expression and chromatin partitioning. Insulator elements organize the genome into chromatin loops that are involved in the formation of topologically associating domains [TADs]. In mammals, CTCF-dependent loop formation requires ATP-driven motor activity of SMC complex cohesin. In contrast, CTCF and cohesin are thought to be dispensable for chromatin 3D partitioning in Drosophila. Instead, the larger, transcriptionally inactive domains (canonical TADs) are interspersed with smaller active compartmental domains, which themselves represent TAD boundaries (Rowley et al., 2017). It has been proposed that in Drosophila, domain organization does not rely on architectural proteins but is established by transcription-dependent, A-A compartmental (gene-to-gene) interactions. However, Drosophila TAD boundaries are enriched for architectural proteins other than CTCF, and their roles have not been tested in loss-of-function models. Thus, it is possible that in Drosophila, instead of CTCF, the 3D partitioning of the genome is facilitated by another group of insulator proteins, such as Su(Hw) and SUMM4, that together associate with class 3 insulators (Andreyeva, 2022).

Moreover, SUUR may provide the DNA motor function to promote a physical separation of active and inactive loci and help establish chromosome contact domains. It is proposed that within the SUMM4 complex, SUUR utilizes its putative ATP-dependent motor activity to translocate along chromatin strands, thus facilitating the establishment of higher-order structures that isolate promoters from enhancers and stabilize DNA loops/domains to prevent unrestricted heterochromatin encroachment and penetration of replication forks. The translocation model is consistent with observations of an asymmetric, selective occupancy of SUUR away from its initial sites of deposition via Su(Hw)-Mod(Mdg4) binding toward inside of intercalary heterochromatin regions but not outside, which may be facilitated by physical interactions between SUUR and linker histone H1 enriched in intercalary heterochromatin. It has been reported that another Drosophila BTB/POZ domain insulator protein CP190 forms a complex with a DEAD-box helicase Rm62 that contributes to the insulator activity. Thus, ATP-dependent motor proteins may represent an obligatory component of the insulator complex machinery (Andreyeva, 2022).

This work explains previous observations about biological functions of SUUR. For instance, the initial deposition of SUUR and its colocalization with PCNA has been proposed to depend on direct physical interaction with components of the replisome (Kolesnikova, 2013). The model developed in this study indicates that, instead, the apparent colocalization of SUUR with PCNA throughout endo-S phase may be caused by a replication fork retardation at insulator sites. SUUR is deposited in chromosomes as a subunit of SUMM4 complex at thousands of loci by tethering via Mod(Mdg4)-Su(Hw) interactions. As replication forks progress through the genome, they encounter insulator complexes where replication machinery pauses for various periods of time before resolving the obstacle. Thus, the increased co-residence time of PCNA and SUUR manifests cytologically as their partial colocalization. With the progression of endo-S phase, some of the SUMM4 insulator complexes are evicted and, thus, the number of SUUR-positive loci is decreased, until eventually the replication fork encounters nearly completely impenetrable insulators demarcating the underreplicated domain boundaries (Andreyeva, 2022).

This mechanism is especially plausible given that boundaries of intercalary heterochromatin loci very frequently encompass multiple, densely clustered Su(Hw) binding sites. This study examined the data from genome-wide proteomic analyses for Su(Hw) and SUUR performed by DamID in Kc167 cells. Strikingly, Su(Hw) DamID-measured occupancy does not exhibit a discrete pattern expected of a DNA-binding factor. Instead, it appears broadly dispersed, together with SUUR, up to tens of kbp away from mapped Su(Hw) binding sites. Interestingly, when hidden Markov modeling was applied to the DamID data, Su(Hw), Mod(Mdg4)-67.2, and SUUR occupancies were found to strongly correlate genome-wide in a novel chromatin form ('malachite') that frequently demarcates the boundaries of intercalary heterochromatin. These observations strongly corroborate the translocation model for the mechanism of action of SUMM4. According to this model, upon tethering to DNA-bound Su(Hw), SUMM4 traverses the underreplicated region, which helps to separate it in a contact domain. As DNA within the underreplicated region is tracked by SUUR, it is brought into a transient close proximity with both SUMM4 and the associated Su(Hw) protein, which is detected by DamID (or ChIP) as an expanded occupancy pattern (Andreyeva, 2022).

The deceleration of SUUR-bound replication forks was also invoked as an explanation for the apparent role of SUUR in the establishment of epigenetic marking of intercalary heterochromatin (Posukh, 2015). It is proposed that global epigenetic modifications observed in the SuUR mutant likely do not directly arise from derepression of the replisome as suggested but, rather, result from the coordinate insulator-dependent regulatory functions of SUUR in both the establishment of a chromatin barrier and DNA replication control (Andreyeva, 2022).

This work demonstrates for the first time that insulator complexes assembled on chromatin can attenuate the extent of replication in discrete regions of the salivary gland polyploid genome. Despite distinct cell cycle programs in dividing and endoreplicating cells, the core biochemical composition of replisomes in both cell types is likely similar. Although the putative relationship is limited by a paucity of comparative biochemical analyses of replication factors in different cell types, related insulator-driven control mechanisms for DNA replication may be conserved in endoreplicating and mitotically dividing diploid cells. The data thus implicates insulator/chromatin boundary elements as a critical attribute of DNA replication control. Our model suggests that delayed replication of repressed chromatin (e.g., intercalary heterochromatin) during very late S phase can be imposed in a simple, two-component mechanism. First, it requires that an extended genomic domain be completely devoid of functional origins of replication. The assembly and licensing of proximal pre-RC complexes can be repressed epigenetically or at the level of DNA sequence. Second, this domain is separated from flanking chromatin by a barrier element associated with an insulator complex, such as SUMM4. This structural organization is capable of preventing or delaying the entry of external forks fired from distal origins (Andreyeva, 2022).

An important frequent feature of the partially suppressed underreplication in mod(mdg4) animals is its asymmetry, which is consistent with a unidirectional penetration of the underreplicated domain by a replication fork firing from the nearest external origin. The SUMM4-dependent barrier may be created as a direct physical obstacle to MCM2-7 DNA-unwinding helicase or other enzymatic activities of the replisome. Alternatively, SUMM4 may inhibit the replication machinery indirectly by assembling at the insulator a DNA/chromatin structure that is incompatible with replisome translocation. This putative inhibitory structure may involve epigenetic modifications of chromatin as proposed earlier, linker histone H1 as shown previously and may also be dependent on Rif1, a negative DNA replication regulator that acts downstream of SUUR (Andreyeva, 2022).

In conclusion, this study used a newly developed MERCI approach to identify a stable stoichiometric complex termed SUMM4 that comprises SUUR, a previously known negative effector of replication, and Mod(Mdg4), an insulator protein. SUMM4 subunits cooperate to mediate transcriptional repression and chromatin boundary functions of gypsy-like (class 3) insulators and inhibit DNA replication likely by slowing down replication fork progression through the boundary element. Thus, SUMM4 is required for coordinate regulation of gene expression, chromatin partitioning, and DNA replication timing. The insulator-dependent regulation of DNA replication offers a novel mechanism for the establishment of replication timing in addition to the currently accepted paradigm of variable timing of replication origin firing (Andreyeva, 2022).

Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. This study has analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, these findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes (Kabakci, 2022a).

Meiosis is a key innovation that evolved before the eukaryotic radiation into the extant domain. The canonical program of this conserved process relies on meiotic recombination (MR). MR contributes to the initial pairing of homologous chromosomes and generates crossovers that maintain homologs linked as bivalent chromosomes until the onset of anaphase during the first meiotic division (M I). MR proceeds usually in concert with synapsis, which achieves close homolog pairing all along the chromosomes via formation of the synaptonemal complex (SC). In spite of the eminent significance of MR, diverse meiosis variants have evolved that do not rely on MR. A most thoroughly studied example of such an achiasmate meiosis occurs in Drosophila melanogaster. While meiosis is largely canonical in D. melanogaster females and includes MR, it is achiasmate in the heterogametic males. This sex-specific difference in meiosis is characteristic among higher dipterans. Its evolution is poorly understood, but may be linked to the suppression of recombination between sex chromosomes (Kabakci, 2022a).

In D. melanogaster spermatocytes, not only MR but also SC formation does not occur. Nevertheless, soon after the last spermatogonial mitosis, homologous chromosomes are paired all along their length, according to analyses with a lacO/lacI-GFP system and FISH. It remains to be clarified whether the pairing of homologous chromosomes in early spermatocytes during the S1 stage is driven by the same mechanisms that are responsible for the pervasive somatic homolog pairing in D. melanogaster. Importantly, the extensive pairing of homologs in spermatocytes lasts only a few hours. During the S2b/S3 stages, homolog pairing was no longer detectable at any of the analyzed 14 distinct locations with euchromatic lacO array insertions. Moreover, even sister chromatid cohesion appeared to be lost except at centromeres (Kabakci, 2022a).

The drastic loss of homolog pairing and sister cohesion in mid-stage spermatocytes starts concomitantly with the process of territory formation, which separates three major chromosome territories apart within the interphase nucleus. One of the major territories contains the chromosome (chr) 2 bivalent, another the chr3 bivalent and the third the chrXY bivalent. The additional bivalent of chr4, a small dot chromosome, is often associated with the chrXY territory. Territory formation breaks up all non-homologous associations between the large chromosomes. Such non-homologous associations are extensive in S1 spermatocytes. They arise from a coalescence of large blocks of pericentromeric heterochromatin into a chromocenter. Similarly, centromeres are clustered initially. Disrupting these non-homologous associations during territory formation at the S2b stage depends on condensin II activity and additional unidentified forces. Failure of territory formation leads to persistence of non-homologous associations until prometaphase I and consequential chromosome segregation errors (Kabakci, 2022a).

The mechanisms that break up non-homologous chromosome associations during territory formation disrupt also homolog pairing and sister chromatid cohesion, presumably because of inevitable side effects. However, normally, homolog separation does not proceed to completion already during spermatocyte maturation. Complete premature homolog separation is prevented by residual homolog conjunction maintained by a dedicated special system that serves as an alternative to canonical homolog linkage by crossovers. Large-scale mutant screening has led to the identification of three genes (tef, mnm, and snm) that are specifically required for this alternative homolog conjunction (AHC). A proteomic approach has recently uncovered an additional AHC gene (uno). Loss-of-function mutations in these four genes result in chromosome missegregation during M I, but exclusively in males. In mnm, snm and uno mutant males, both sex chromosomes and autosomes are distributed randomly during M I. In contrast, only autosomes are missegregated in tef mutant males during M I (Kabakci, 2022a).

The TEF protein includes three C2H2-type zinc fingers and is therefore predicted to bind to DNA. The SNM protein is a distant relative of the stromalins (SCC3/SA/STAG protein family). Stromalins are subunits of cohesins, complexes of crucial importance for chromosome organization during interphase and M phase in somatic and meiotic cells. However, SNM is not co-localized with core components of cohesin, indicating that it does not function as a cohesin subunit. MNM is encoded by one of many differentially spliced mRNAs transcribed from the highly complex mod(mdg4) locus. MNM has an N-terminal BTB/POZ motif that is shared among almost all of the more than 30 distinct protein products expressed from the mod(mdg4) locus. In addition, MNM has a unique C-terminal zinc finger motif of the FLYWCH type. These N- and C-terminal motifs of MNM are predicted to mediate protein-protein interactions. UNO does not have obvious similarities to functionally characterized proteins (Kabakci, 2022a).

MNM, SNM and UNO accumulate in early spermatocytes, eventually co-localizing during spermatocyte maturation in multiple subnucleolar foci. At the start of M I, these foci coalesce into a single prominent spot on the chrXY bivalent. In D. melanogaster, chrX and chrY are strongly heteromorphic, lacking extended euchromatic homology that could mediate specific pairing. However, both sex chromosomes harbor rDNA gene clusters in the centromere-proximal heterochromatin and these rDNA clusters function as pairing centers during male M I. The prominent dot formed by MNM, SNM and UNO on the chrXY bivalent at the start of M I is localized on the paired rDNA loci of chrX and chrY. Apart from the prominent dot on the chrXY pairing center, autosomal bivalents, which rely on euchromatic homology for pairing display far weaker dot signals of co-localized MNM, SNM and UNO. These autosomal dot signals were shown to be at least partially dependent on tef function. Strikingly, MNM, SNM and UNO disappear rapidly from all the bivalents within minutes during the onset of anaphase I. Separase, an endoprotease known to eliminate chromosomal cohesin at the metaphase to anaphase transition during mitotic and meiotic divisions, is required for the rapid disappearance of MNM, SNM and UNO from M I bivalents. UNO includes a separase cleavage site. Mutations that abolish this cleavage site prevent the rapid disappearance of MNM, SNM and UNO from M I bivalents and preclude homolog separation (Kabakci, 2022a).

The findings summarized above strongly support the notion that SNM, MNM and UNO function as proteinaceous glue that conjoins chromosomes into bivalents. However, it remains to be clarified how these proteins are recruited to chromosomes. SNM, MNM and UNO do not include known bona fide DNA-binding domains. They might therefore be recruited by other chromatin proteins. The zinc finger protein TEF is clearly an attractive candidate factor for chromosomal recruitment of the other AHC proteins. TEF's pattern of expression and its subcellular localization during spermatogenesis have not yet been characterized. This study closes this gap in understanding. Using transgenes encoding tagged functional versions of TEF, it was observed to be only transiently detectable in early spermatocytes. In contrast to the other known AHC proteins (MNM, SNM and UNO), TEF cannot be detected on bivalents at the start of M I, indicating that it is unlikely a stoichiometric component of the homolog-conjoining glue. However, evidence is provided that TEF can recruit MNM to chromosomes by direct protein-protein interaction. Indirectly, TEF can also recruit SNM-UNO, as they bind to MNM. Moreover, presumably by promoting AHC protein interactions, TEF stabilizes these proteins and controls their levels. AHC protein levels need to be controlled, as suggested by the consequences of simultaneous overexpression of all four AHC proteins in spermatocytes, which resulted in ectopic chromosome conjunction, failure of territory formation and segregation errors during M I. Ectopic expression of the four AHC proteins in somatic cells induced aberrant chromosome conjunction during mitosis, suggesting that AHC might not depend on additional spermatocyte-specific proteins beyond those already known (Kabakci, 2022a).

Genes required specifically for alternative homolog conjunction (AHC) during the achiasmate meiosis of Drosophila males were identified initially by extensive screening of mutants, and the teflon (tef) mutant phenotype was the first to be characterized in detail. The molecular identification of the affected gene revealed that tef encodes a zinc finger protein. This report provides more detailed functional characterization of the TEF protein. Expression pattern and intracellular localization during spermatogenesis were clarified with the help of tagged functional variants. An interaction between TEF and MNM was demonstrated by co-immunoprecipitation, and the responsible binding regions were mapped. Moreover, TEF was shown to bind to chromatin of polytene chromosomes in larval salivary glands. Importantly, TEF recruits MNM to chromatin, and via MNM also the other known AHC proteins SNM and UNO. Moreover, TEF potentiates the chromosome-linking activity of the AHC proteins SNM, UNO and MNM, as revealed by overexpression experiments in spermatocytes and other cell types (Kabakci, 2022a).

The TEF expression pattern was characterized with g-tef-sm_myc. This transgene under control of the tef regulatory region results in expression of a TEF version tagged with a spaghetti monster myc epitope tag (sm_myc). According to mutant rescue experiments, TEF-sm_myc is fully functional. Based on the g-tef-sm_myc expression pattern revealed by anti-Myc immunofluorescence, TEF is absent or low in somatic hub cells of testes but present in germline stem cells, spermatogonial cells and spermatocytes. TEF-sm_myc is also abundant in ovaries, where it is not germline-restricted as in testes. TEF's role in ovaries remains unclear, as no aberrant phenotype has been found in tef mutants so far (Kabakci, 2022a).

The subcellular localization of TEF-sm_myc was unexpected. During spermatogonial mitoses, a strong enrichment on centrosomes was observed. It is noted that mitotic centrosomal localization is also characteristic of CP190, an architectural chromatin protein, which like TEF has zinc fingers and interacts with a Mod(mdg4) protein. During interphase, TEF-sm_myc was primarily in many intranuclear foci of variable size, and a majority of these did not appear to be chromatin-associated. Intriguingly, the presence of TEF-sm_myc in spermatocytes was transient. TEF-sm_myc levels declined during spermatocyte maturation. It was no longer detectable in late spermatocytes (stages S5 and S6) and during the meiotic divisions. This subcellular localization and transient presence in spermatocytes were also observed in case of bamP-GAL4-VP16 driven UASt-tef-EGFP expression, which also rescues tef mutants. Importantly, in contrast to TEF, the other known AHC proteins (SNM, MNM and UNO, abbreviated as SUM) are all detectable on autosomal bivalents, when expressed analogously (as EGFP fusions from UASt transgenes with bamP-GAL4-VP16). These results argue strongly against the notion that homologous autosomes are conjoined by complexes of AHC proteins containing stoichiometric amounts of TEF. Rather than being an essential component of the glue that keeps homologous autosomes linked until onset of anaphase I, TEF might function only in early spermatocytes in the regulation of AHC establishment. It is noted that a presence of functional TEF in bivalents of late spermatocytes at levels below detectability is not excluded (Kabakci, 2022a).

Comparison of the mutant phenotypes caused by loss of tef, on the one hand, and loss of snm or mnm, on the other hand, provides further arguments against the notion that TEF is an essential component of the glue that conjoins autosomal homologs. The two tef alleles present in the transheterozygous mutants that have been analyzed are early non-sense mutations, shown to be amorphic with respect to meiotic chromosome transmission. However, the extent of autosome missegregation was significantly less severe in the transheterozygous tef mutants compared to snm and mnm mutants. This conclusion rests on concurrent findings made by time-lapse imaging of progression through M I and by analyses of meiotic chromosome missegregation with dodeca FISH. In snm and mnm mutants, bivalents are prematurely separated into independent univalents that are segregated randomly during M I. In contrast, in tef mutants there is some residual conjunction of autosomal homologs and their segregation is not completely random. Phenotypic comparisons are therefore consistent with the notion that TEF contributes to AHC establishment in early spermatocytes rather than also to the maintenance of AHC until anaphase I like the SUM protein. According to current observations after ectopic expression of AHC proteins, TEF might contribute to AHC establishment by promoting the recruitment of the SUM proteins to chromatin. TEF is the only AHC protein with a predicted bona fide DNA-binding domain. TEF has three zinc fingers, one in the N-terminal and two in the C-terminal region. Jointly, these N- and C-terminal zinc fingers mediate efficient TEF binding of TEF to polytene chromosomes after ectopic expression in larval salivary glands, as deletion of either the N- or the C-terminal region resulted in a substantial reduction of the chromosome-associated signals. Consistent with the absence of known DNA-binding motifs, none of the other AHC proteins displayed substantial binding to polytene chromosomes when expressed individually. Unexpectedly, however, polytene chromosome binding was clearly observed after co-expression of SNM and UNO. Presumably, these two proteins form a complex (SU) that includes a composite DNA-binding site. The two chromosome-binding entities among the AHC proteins, TEF and SU, have distinct preferences for chromosomal locations. However, both are able to recruit MNM onto polytene chromosomes. TEF and MNM interact directly according to co-immunoprecipitation experiments after transient expression in S2R+ cells, consistent with the previously observed co-purification of TEF with MNM-EGFP from testis extracts. The TEF-MNM interaction is mediated by the N-terminal part of TEF that includes the first zinc finger and by the C-terminal part of MNM. This C-terminal part is uniquely present in MNM. All the many additional isoforms that are generated by differential splicing from the complex mod(mdg4) locus have distinct C-terminal parts, and the three isoforms tested (T, C and P) were unable to bind to TEF. Beyond the TEF-MNM interaction, analyses of polytene chromosome binding and of co-immunoprecipitation suggested that all four AHC proteins (SNM, UNO, MNM and TEF) can co-assemble into SUMT complexes (Kabakci, 2022a).

Clearly, in salivary glands, TEF does not just bind to autosomes but also to the X chromosome. Thus, TEF does not appear to have an autosome-specific chromosome-binding ability that would explain why tef is required in spermatocytes for regular M I segregation of autosomes but not of sex chromosomes. It is suggested that the chromosome-specificity of the tef requirement might be linked to an additional effect of TEF on AHC proteins. According to the quantification of expression levels after Sgs3-GAL4-mediated expression of AHC proteins in salivary glands, formation of AHC protein complexes appears to stabilize these proteins. Levels of TEF and MNM were higher after co-expression compared to individual expression. Analogous observations were made with SNM and UNO. Similarly, after bam-GAL4-VP16-driven overexpression of SUM or SUMT in spermatocytes, the levels of the only tagged protein UNO-mCherry were increased by the presence of TEF. Moreover, MNM-EGFP levels were lower in tef mutant spermatocytes. Overall, these observations indicate a positive correlation between TEF and SUM protein levels. In tef mutants, some of the remaining SUM is presumably still recruited to autosomal bivalents due to the chromosome-binding activity of SU. However, as SUM levels during wild-type meiosis are far lower on autosomal bivalents compared to the chrXY bivalent, autosomal bivalents might be more strongly affected when SUM protein levels decrease as a result of a loss of tef function. TEF increases SUM protein levels presumably by promoting the formation of protein associations that are more stable than the individual proteins. Stimulating effects of TEF on SUM gene transcription are not excluded but unlikely as the analyses included experiments where the AHC proteins were expressed with exogenous regulatory sequences (UASGAL4 and hsp70) (Kabakci, 2022a).

The proposed explanation for the autosome-specific effect of tef mutations remains speculative, also because of the technical difficulties to detect SUM proteins on autosomal bivalents. Even the normal amounts of autosomal SUM proteins during wild-type meiosis are difficult to detect unequivocally and consistently in each spermatocyte. In this study, by analyzing fluorescent versions of UNO, a quantitative estimate is provided for the striking difference in the amount of SUM proteins on autosomes and sex chromosomes in normal spermatocytes. Around 25-100-fold lower amounts of UNO is found on autosomal bivalents compared to the chrXY bivalents. Without future technical improvements of detection sensitivity, a conclusive demonstration of the postulated residual autosomal SUM complexes in tef mutants is not feasible (Kabakci, 2022a).

If detectable, the autosomal SUM proteins appear to be confined to 1-2 dots per bivalent at NEBD I in normal spermatocytes. Do these dots mark the location of autosomal homolog conjunction, or might there be additional SUM complexes at other locations below the limit of detection that contribute to conjunction as well? Recent cytological analyses of meiotic quadrivalents in spermatocytes heterozygous for autosomal translocations have indicated that autosomal homolog conjunction is spatially constrained to dot-like chromosomal locations. The spatial control of autosomal homolog conjunction in spermatocytes appears to be analogous to that of canonical crossovers. As a rule, a single restricted region within the euchromatic portion of each autosomal chromosome arm is linked by AHC protein assemblies to its homologous region. Thus, AHC positions might be controlled by processes analogous to crossover interference. The particularly strong crossover interference in C. elegans has recently been proposed to involve spatially restricted biomolecular condensation of recombination nodule proteins in combination with a regulated coarsening process. It is tempting, therefore, to speculate about the significance of MNM's apparent liquid phase separation potential. Both MNM and TEF include substantial portions that are predicted to be intrinsically disordered. Such regions are thought to favor liquid-liquid unmixing when they confer multivalent interactions. At high levels of expression, MNM-EGFP formed droplets in salivary gland nuclei, while this was hardly observed with MNM-mCherry. The known weak dimerization of EGFP might reinforce multivalent associations. With TEF-EGFP droplets were not obtained when expressed alone but when co-expressed with MNM-mCherry, which was co-localized with TEF-EGFP in the droplets. Thus, droplet formation was stimulated by the EGFP tag when present on either MNM or its binding partner TEF. In case of untagged endogenous proteins, droplet formation might remain restricted to chromosomally recruited AHC assemblies. Accordingly, liquid phase separation of AHC proteins might be involved in the control of establishment or maintenance of alternative homolog conjunction in Drosophila spermatocytes (Kabakci, 2022a).

Experiments with spermatocytes revealed that an excess of AHC proteins is detrimental to regular chromosome segregation during male meiosis. Overexpression of SUMT severely inhibited chromosome territory formation most likely because it results in increased and more widespread conjunction between not only homologous but also non-homologous chromosomes. As a consequence, presumably, chromosomes fail to separate normally, often forming prominent bridges during anaphase and telophase of M I. The meiotic defects observed after SUMT overexpression are highly reminiscent of those caused by a loss of condensin II function. Conversely, absence of SUMT, as in mutants, has very similar phenotypic consequences as overexpression of the limiting condensin II subunit Cap-H2. Evidently, AHC proteins and condensin II have opposing activities that need to be in proper balance (Kabakci, 2022a).

The detrimental effects on meiotic chromosome segregation were much stronger after overexpression of SUMT compared to SUM. Overexpression of individual AHC proteins had barely any effect. These results provide further support for the proposal that in normal male meiosis, TEF assists in the chromosomal recruitment of SUM, the actual glue that maintains homolog linkage in bivalents until anaphase I onset. Accordingly, overexpression of TEF alone might not have severe detrimental effects because potentially low levels of endogenous SUM proteins might not allow excess assembly. Similarly, previous overexpression of individual SUM proteins was not observed to cause severe detrimental effects, perhaps also because low levels of other SUM subunits might limit excess assembly. The finding that ectopic SUMT expression in mitotically proliferating wing imaginal disc cells results in mitotic defects that resemble closely to the meiotic defects observed after SUMT overexpression in spermatocytes might indicate that AHC during male meiosis does not depend on additional spermatocyte-specific proteins beyond the known AHC proteins. The three proteins SNM, UNO, and MNM appear to be sufficient to induce conjunction between mitotic chromosomes and thus interfere with their normal segregation during anaphase. In combination with TEF, SUM had even more detrimental effects on mitotic chromosome segregation. However, much remains to be learned about the regulation that controls the appropriate chromosomal positioning of AHC during male meiosis (Kabakci, 2022a).

Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. This study reports that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, it is suggested that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, this study demonstrates that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease (Kabakci, 2022b).

Drosophila male meiosis is achiasmate and therefore dependent on dedicated proteins (SNM (Stromalin 2), UNO (univalents only) and MNM (modifier of mdg4), together referred to as SUM, that maintain conjunction between homologous chromosomes in replacement for the missing crossovers. The main findings (summarized in Fig 7F) provide insight into the biochemical basis of (1) how the SUM proteins achieve this alternative homolog conjunction (AHC), and (2) how AHC is eliminated in time at the transition from metaphase to anaphase of M I to permit separation of the homologs to opposite spindle poles. In addition, our results are informative concerning the evolution of the AHC system (Kabakci, 2022b).

SNM and the C-terminal domain of UNO form a stable heterodimeric complex (SU_C). Based on sequence comparisons, AlphaFold structural predictions and XL-MS with recombinantly expressed and purified proteins, the SU_C complex is homologous to that formed by stromalin and the stromalin-binding region of α-kleisin. Stromalins and α-kleisins are components of cohesin complexes. While SNM was recognized as highly similar to stromalins early on, the very limited similarity of UNO to α-kleisins has escaped detection until now. The important and conserved N- and C-terminal domains of α-kleisins, which mediate its binding to the SMC heterodimer in cohesin, are not present in UNO. From an α-kleisin precursor, UNO has thus retained only the stromalin-binding region and the previously identified separase cleavage site (Kabakci, 2022b).

Stromalin, via positively charged surface patches, has recently been shown to promote DNA-binding of cohesin in vitro. Purified SU_C also binds DNA. At least one of stromalin’s positively charged surface patches [43] is clearly also present in SNM and contributes to the DNA-binding of SU_C, according to in vitro analysis with mutant versions of SU_C. In addition, a conspicuous, positively charged α-helix at the very C-terminus of UNO, which is absent from α-kleisins, makes a contribution to the DNA-binding of SU_C that is even more important than the basic SNM patch. Apart from DNA-binding, the interactions with the other AHC proteins were still normal in case of UNOchm-EGFP, a mutant with acidic or neutral residues in place of the six basic residues in the C-terminal α-helix. In vivo, UNOchm-EGFP displayed strongly reduced chromosome-binding and failed to provide normal AHC during male meiosis. These results strongly argue for the physiological importance of the DNA-binding activity of SU_C. It is speculated that the C-terminal α-helix of UNO might clamp down on a DNA double helix bound to the basic surface patches of SNM and thereby strongly increase the strength of DNA-binding. The binding of SU_C to DNA does not appear to be sequence specific. Clearly, in competition with the scrambled DNA sequence, this study has not detected increased binding to the 240 bp repeat sequence from the rDNA intergenic spacers, which appears to mediate sex chromosome conjunction (Kabakci, 2022b).

Beyond DNA, SU_C binds to MNM. Neither SNM nor UNO interact with MNM individually, indicating that prior association of SNM and UNO is required for MNM binding. These conclusions are based on co-immunoprecipitation experiments after transient expression in S2R+ cells. Of note, this study has not accomplished SUM complex formation with purified proteins in vitro so far. Attempts at expressing and purifying full length MNM were not successful. Moreover, the successfully purified C-terminal region of MNM (MNM_C), which mediates the binding to SU_C according to co-immunoprecipitation experiments, did not bind to SU_C in vitro. It is conceivable, therefore, that binding of MNM to SU depends on prior post-translational processing steps. At present, the inability to generate SUM complexes in vitro precludes a straightforward clarification of the issue whether SU can bind simultaneously to both MNM and DNA. However, the extended contacts between the C-terminal domain of UNO and SNM over long stretches provide ample space with interface potential, thereby increasing the likelihood of simultaneous binding of MNM and DNA to SU (Kabakci, 2022b).

MNM_C mediates binding not only to SU_C but also to TEF, as revealed by the co-immunoprecipitation experiments. The MNM-TEF interaction also remains to be re-constituted with purified proteins in vitro. However, in case of MNM_C, simultaneous binding of both TEF and SU_C is not feasible according to co-immunoprecipitation experiments (Kabakci, 2022b).

Beyond the interaction domains discussed above, analyses demonstrated the presence of multimerization domains in both UNO and MNM. It ia suggested that these domains are likely of crucial importance for the molecular mechanism whereby the SUM proteins generate AHC. In case of UNO, the N-terminal domain (UNO_N), which is highly conserved in UNO homologs, self-associates, forming dimers and tetramers when expressed and purified from bacteria. This UNO_N region has a predicted structure that is very distinct from that of the conserved N-terminal region of α-kleisins, indicating that the evolution of uno involved substitution of N-terminal in addition to deletion of C-terminal coding sequences in an ancestral α-kleisin gene. Multimerization in case of MNM is also mediated by the N-terminal region MNM_N. The primary sequence of MNM_N is identical to that of the N-terminal region present in the considerable number of alternative isoforms expressed from the complex mod(mdg4) locus. This common part of the Mod(mdg4) protein isoforms (thus also designated as Mod(mdg4)_CP) contains a BTB/POZ domain. This protein interaction domain present in many eukaryotic proteins with diverse functions has been shown to mediate homomeric dimerization. In addition, in case of the particular type of BTB domain that is also present in the Mod(mdg4) protein products, heteromeric and higher order multimerization has been reported based on SEC, native gel electrophoresis and crosslinking studies. These results confirm and extend these findings. Purified MNM_N/Mod(mdg4)_CP was found to form stable hexamers according to SEC-MALS. The hexamers were readily modeled by AlphaFold2 as a ring-like complex with three dimers, and negative-stain electron microscopy revealed ring-like complexes of an appropriate dimension. A recent preprint describing similar structural analyses of the same type of BTB domains (i.e. the TTK-type) derived from other Drosophila proteins (Lola and CG6765) and also from Mod(mdg4) provides further confirmation of the ring-shaped hexameric structure (Kabakci, 2022b).

Because of the multimerization domains (UNO_N and MNM_N/Mod(mdg4)_CP), the SUM proteins have presumably the potential to form extended protein assemblies that include many copies of the SU_C DNA-binding site. Thereby, they might be empowered to effectively and stably conjoin distinct double-stranded DNA molecules and function as a chromosome glue. Accordingly, AHC would not rely on a topological ring-like embrace as proposed to be provided by cohesin in case of sister chromatid cohesion. Since the tetramers and hexamers formed in vitro by purified UNO_N and MNM_N/Mod(mdg4)_CP are stable, SUM protein assemblies formed on bivalents in spermatocytes are expected to adopt a more solid rather than a liquid state. Indeed, FRAP analyses confirmed that the SUM proteins in the dots associated with the sex chromosome pairing regions do not undergo dynamic exchange (Kabakci, 2022b).

The proposed extended SUM protein assemblies with their multitude of DNA-binding sites are unlikely to conjoin exclusively homologous DNA strands. Presumably, sister DNA strands are connected as well (and perhaps even neighboring regions on the same strand). Previous characterizations of meiotic mutant phenotypes are consistent with this view. Absence of AHC function results in premature separation of bivalents into univalents in late spermatocytes and early in M I. The SOLO (Sisters on the loose) and SUNN (Sister unbound) proteins, which appear to function similar to the Rec8 cohesin complexes of other eukaryotes, still assure in these univalents a functional unification of sister centromeres for organization of a single kinetochore unit, as well as well as pericentromeric sister chromatid cohesion. In solo and sunn mutants, sister centromeres and pericentromeric regions lack cohesion, but bivalents are still present until the onset of anaphase I. As sister chromatid cohesion within the regions of chromosome arms is normally lost after territory formation already during spermatocyte maturation, the presence of bivalents in solo and sunn mutants during early M I suggests that the SUM proteins conjoin not just homologous chromatids but also sister chromatids. In support of this interpretation, snm solo double mutants display univalents during early M I. It is emphasizes that a chromosomal glue that conjoins DNA strands indiscriminately, as proposed for the SUM protein assemblies (i.e., sister strands and homologous strands in trans and perhaps also neighboring regions in cis) should be perfectly adequate if it is applied at the right time during spermatocyte maturation, i.e., after disruption of non-homologous chromosomal associations by territory formation but before complete disruption of homolog associations (Kabakci, 2022b).

Clearly, the proposal that AHC relies on extended assemblies of SUM proteins providing a high number of DNA-binding sites remains speculative and requires further investigation. For example, understanding how the formation of SUM protein assemblies is controlled and restricted to limited chromosomal regions will be crucial. The mechanism whereby SUM protein assemblies are targeted to the sex chromosome pairing rDNA loci on chromosome X and Y remains unexplained. In case of autosomal bivalents, TEF is likely involved in the initial establishment of SUM protein assemblies. However, after ectopic expression in larval salivary glands, TEF as well as SUM bind to a large number of polytene chromosome bands In contrast, in mature S6 spermatocytes, the autosomal SUM protein assemblies are spatially restricted to one or two dots per chromosome arm. Targeting of SUM protein assemblies to the sex chromosome pairing site and into autosomal dots might involve interactions with additional chromosomal proteins. Mod(mdg4)_T (also designated as 67.2 or 2.2), the most extensively characterized isoform expressed from the complex mod(mdg4) locus, interacts and co-operates with several chromatin architectural proteins (including CP190, HIPP1 and SuHw) at the gypsy insulator. Moreover, Mod(mdg4)_T in combinations with chromatin architectural proteins in various combinations is generally enriched at boundaries between topologically associated chromatin domains and also at button loci that promote the somatic pairing of homologous chromosomes. Recently, Mod(mdg4) function has been implicated in a striking example of chromosome pairing-dependent regulation of physiological gene expression. Thus, multimerization by Mod(mdg4)_CP is likely crucial for chromosomal associations other than AHC during male meiosis. Accordingly, by recruitment of the Mod(mdg4)_H isoform MNM for AHC, evolution might have co-opted a pre-adaption that achieves chromosomal associations by Mod(Mdg4)_CP multimerization (Kabakci, 2022b).

While Mod(mdg4) isoforms other than MNM were found to be unable of binding to SU, these other isoforms clearly have the potential to form heteromeric associations with MNM according to co-immunoprecipitation experiments. Moreover, based on yeast two-hybrid (Y2H) analyses, various other proteins with TTK-type BTB domains might also form heteromeric associations with MNM. Whether such heteromeric interactions are relevant of AHC remains to be clarified. However, phenotypic analyses with various mod(mdg4) alleles have argued against contributions to AHC by Mod(mdg4) isoforms other than MNM. Moreover, while heteromeric associations of Mod(mdg4)_T with other Mod(mdg4) isoforms can readily be detected by Y2H and co-immunoprecipitation after overexpression in S2 cells, their occurrence on chromosomes without overexpression is questionable according to chromatin-immunoprecipitation (Kabakci, 2022b).

Importantly, AHC must provide conjunction between homologs in bivalents that is very robust and yet also amenable to rapid and complete elimination after biorientation of all the bivalents in the M I spindle, so that homologs can be separated to opposite poles during anaphase I. Efficient destructibility of AHC was achieved by the evolutionary co-option of the α-kleisin-derived protein UNO. Like α-kleisin, UNO includes a separase cleavage site that is highly conserved among UNO orthologs. This cleavage site was shown to be required for AHC elimination and homolog separation during anaphase I. By exchanging the separase cleavage site in UNO with that cleaved by the bio-orthogonal TEV protease, this study provides evidence that UNO cleavage is indeed sufficient to eliminate AHC. In these experiments, TEV was expressed under control of cis-regulatory sequences from exu or betaTub85D in mid spermatocytes. The presence of normal chromosome territories and of a normal subcellular localization of UNOTEV-EGFP at the onset of TEV expression indicated that this TEV expression occurred after successful AHC establishment, which occurs early during spermatocyte maturation. However, as a consequence of TEV expression, bivalents were prematurely converted into univalents, as clearly indicated by cytological analyses and by time lapse imaging of progression into and through M I. UNO cleavage separates the multimerization domain UNO_N from UNO_C, which mediates DNA-binding in conjunction with SNM. Therefore, it is proposed that UNO cleavage dissociates the chromosomal SUM protein assemblies to an extent where the number of associated DNA-binding sites is no longer sufficient for tight linkage of distinct double-stranded DNA molecules. Clearly, alternative mechanisms of AHC elimination by UNO cleavage remain conceivable, and further work will be required to clarify the mechanistic details of AHC and its elimination (Kabakci, 2022b).

GENE STRUCTURE

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).

date revised: 22 June 2023

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