The Effect of TBP-Related Factor 2 on Chromocenter Formation and Chromosome Segregation in Drosophila Melanogaster

Chromosome nondisjunction in meiosis causes the gene disbalance and a number of anomalies in development and fertility. Otherwise, genetically programmed sex-ratio meiotic drive occurs in a number of species. One of the forms of eukaryotic genome organization is a chromocenter evolutionally involved in the regulation of chromosome behavior in dividing cells among insects, plants, mammals, mollusks, and even yeast. In Drosophila , TBP related factor 2 (Trf2) belongs to a conservative Tbp (TATA box-binding protein) gene family and encodes a basic transcription factor. Recent data demonstrates that a decrease in TRF2 expression can result in the abnormalities of chromatin conden- sation; however, no details of this process have been studied. We demonstrated that a decrease in the TRF2 expression damaged proper chromocenter structure and abolished chromatin condensation and it was a reason for the chromosome nondisjunction. We found that compact chromocenter and correct homologue pairing were abolished in flies with a lower Trf2 expression in germline and in somatic cells. We conclude that TRF2 can not only be involved in transcription activation, but also may perform structural function in pericentromeric heterochromatin organization. The possibility of TRF2 to regulate the evolutionary genetically programmed sex-ratio meiotic drive is discussed.


Introduction
Chromosome nondisjunction during meiosis causes the gene disbalance and, consequently, a number of anomalies in development and fertility. On the other hand, genetically programmed sex-ratio meiotic drive occurs in a number of animal species when mainly males or females are born, which is normal within the given species [1]. The genetic regulation of these processes is actively being studied. There are many factors that can result in the incorrect chromosome segregation. The correct segregation of sister chromatids between daughter cells depends on the coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibrils, topoisomerases, proteolytic processes, and motor proteins [2]. On the other hand, chromosomes must be "prepared" (or structurally organized) when they enter meiosis (or mitosis). Structural disorganization of chromosome or same their regions that control the correct pairing of homologs during meiosis frequently results in the incorrect chromosome segregation. The one way of eukaryotic genome organization is chromocenter, which is evolutionally involved in the regulation of chromosome behavior in dividing cells not only among insects but also among plants, mammals, mollusks, and even yeast [3][4][5][6][7]. This nuclear structure arises in differentiated somatic and germ cells during interphase and meiotic prophase. The chromocenter is generated by the association of pericentromeric regions of all or separate groups of chromosomes and plays an important role in spatial organization of chromosomes [8]. Studies on Drosophila have clearly demonstrated that its disorganization leads to genomic disbalance [9,10]. The screening for genes that control the formation and reorganization of chromocenter is performed [11,12]. The high frequency of chromosome nondisjunction in the progeny of mutant parents is a main characteristic of mutations in these genes.
In Drosophila, TBP-related factor 2 (Trf2) encodes an alternative basic transcription factor that is homologous to vertebrate Trf2 protein and belongs to a conservative TATA box-binding protein (Tbp) gene family [13]. It was shown that previously discovered lawc p1 (leg-arista-wing complex) mutation [14] appeared to be the only viable mutation that decreases Trf2 gene expression [15]. The high conservatism of the Trf2 protein allows us to study its functions on Drosophila.
In the previous studies, we demonstrated that the lawc p1 mutation suppresses the phenotype of mutations in genes that encode polycomb group (PcG) proteins, which are negative epigenetic regulators of transcription via chromatin modification [16]. At the same time, we have found that lawc p1 increased the effect of transvection (or allelic complementation [17]) caused by disruptions of the homologous chromosome pairing at a number of loci.
Recent data demonstrated that a decrease in Trf2 gene expression could result in the disruption of chromatin condensation [18]; however, almost no details of this process have been studied. At the same time, we have noted frequent cases of chromosome nondisjunction during genetic experiments with hypomorphic Trf2 mutations in Drosophila melanogaster. The question is whether a decrease in the Trf2 gene expression really increases the frequency of chromosome nondisjunction in the female meiosis and if it is so, is this anomaly associated with an abnormal chromatin packaging (and particularly with the disruption in the chromocenter structure)?
Data of genetic experiments for the analysis of the frequency of X-chromosome nondisjunction in mutant lines and of cytogenetic experiments studding the structure of chromosomes in germ and somatic cells are presented below.

Analysis of frequency of X-chromosome nondisjunction in lines with lethal Trf2 mutations
We calculated frequencies of X-chromosome nondisjunction in two groups of lines that contain lethal Trf2 mutations. The lines of first group were obtained from Bloomington Drosophila Stock Centre: l(1)G0039/FM7a; l(1)G0356/FM7a; l(1)G0424/FM7a; l(1)G0376/FM7a; l(1)G0425/FM7a; l(1)G0332/FM7a; l(1)G0152/FM7a; l(1)G0166/FM7a; and l(1)G0178/FM7a [19]. Subsequently, we will call these lines "museum" lines. In museum lines, the lethality is caused by the integration of the p{lacW} transposon in the regulatory noncoding Trf2 region ( Figure 1A). Previously, we demonstrated that these lethal mutations did not complement lawc p1 mutation suggesting that they are in the same gene region [15]. The system is composed of two independent parent transgenic lines, the Gal4 driver line in which the yeast transcription activator Gal4 gene is expressed in a tissue-specific manner and the Upstream Activating Sequence (UAS) responder line in which the gene of interest is under UAS control. Mating of the UAS-containing responder flies with the Gal4 drivercontaining flies results in progeny bearing the two components, in which the UAS-transgene expresses dsRNA hairpins in a transcriptional pattern that reflects that of the Gal4 driver. In our experiment the Gal4 driver is Sgs3 which express in larvae salivary glands. The lines of second group were obtained in our laboratory: l(1)lawc 4 /FM4, l(1)lawc 16 [20]. We will further call these lines "laboratory" lines. They carry lethal Trf2 mutations obtained after the destabilization of the mobile P element in the initial lawc p1 allele [20].
In lines with lethal mutation, the X chromosome is maintained on the In(1)FM balancer chromosome. This chromosome carries a dominant Bar (B) marker mutation (narrow eyes) and recessive allele of the yellow (y) gene (yellow body). We crossed y + l(1)/In(1)FM, yB females with males that carried the X chromosome marked by the y 1 mutation (y 1 /Y) in order to identify exceptional classes of descendants and estimate the frequency of X-chromosome nondisjunction in these lines. Males and females of normal classes (the phenotype of which is easily identified) appeared in descendants of this crossing, including In(1)FM, yB/Y males with narrow eyes and yellow body and two classes of females including (1) In(1)FM, yB/yB + (yellow body and kidney-shaped eyes) and (2) y + l(1)/yB + (grey body and normal oval eyes).
When X-chromosome nondisjunction occurred, males and females of exceptional classes (that always differ phenotypically) were detected in descendants. These were X/0 males with normal oval eyes and yellow bodies and XX/Y females with grey bodies and kidneyshaped eyes. Males of the normal class hemizygous for the X chromosome with a lethal allele-l(1)/Y-die. Exceptional classes of Y/0 males and XX/X super-females also die. Therefore, the frequency of X-chromosome nondisjunction (Q) was calculated according to the formula, Q = 100% ⋅ 2 (X0 + XXY) ________________ XX + 2XY + 2X0 + 2XXY , where X0 and XXY are the number of flies of exceptional classes; XX and XY are the number of flies of normal classes. The sum of exceptional classes in the numerator was multiplied by two in order to take into account lethal classes with the XX/X and Y/0 genotype. The number of XY males in denominator was multiplied by two in order to take into account the class of lethal l(1)/Y males [21].
To estimate the influence of the In(1)FM balancer chromosome on the frequency of X-chromosome nondisjunction and compare it with the frequency of Q nondisjunction calculated for our lines, a control experiment was performed. For this, we crossed In (1) To determine the influence of p{lacW} transposon on X-chromosome nondisjunction in museum lines and to take into account the genetic background of laboratory lines, additional control experiments were performed. As a control for museum lines, we calculated the frequency of X-chromosome nondisjunction in l(1)G0071 line with lethal mutation caused by the insertion of p{lacW} transposon not to Trf2 gene region. As a control for laboratory line, we used line with lawc p1+ reversion and unknown lethal mutation (complemented to Trf2), which we obtained after the destabilization of a mobile P element in the initial lawc p1 allele.
All experiments were repeated three times, and the average frequency of X-chromosome nondisjunction ΔQ was calculated for each line. As a result of the experiment, it was found that the frequency of X-chromosome nondisjunction was increased in lawc mutants with decreased expression of the Trf2 protein. The maximal frequency of X-chromosome nondisjunction was in the line l (1)

Control lines
In (1) First column indicates Trf2 alleles. Next two columns indicate the amount of viewed males and females of normal classes. X/X-total amount of females with y + l(1)B + /yB + and yB/yB + genotypes; X/Yx2-doubled amount of males with yB/Y genotype. Next two columns indicate the amount of detected males and females of exceptional classes: X/0, males with yB + /0; XX/Y, females with y + l(1)B + /yB/yB + genotype. Q-frequency of X-chromosome nondisjunction.

Study of the origin of chromosome nondisjunction in lawc mutants
To identify the source of chromosome nondisjunction, we decided to study the meiosis of mutant females. We performed the cytological analysis of the oocyte nucleus in mutant lawc p1 /l(1)EF520 females (the frequency of X-chromosome nondisjunction is 5.2%) with low Trf2 expression. Squash preparations of ovaries were prepared by modified Puro and Nokkala method [10,22].
In germarium, the oocyte passes through the premeiotic DNA replication, meiosis prophase I, prometaphase I, and metaphase I. In mature oocyte of stage 14, division arrest usually occurs at the stage of metaphase I; chromosomes are collected in karyosome; and only achiasmatic chromosomes (IV and rarely X chromosome) are already oriented to opposite poles (Figure 2A).
We found that in anaphase I the chromocenter in mutant oocyte was often split and the compact karyosome structure was often broken (Figure 2B). The split karyosome assumes the disruption of the chromocenter; therefore, we performed an analysis of the early oocyte at the stage of meiosis prophase I when oocyte chromosomes were held together by pericentromeric heterochromatin, and the compact chromocenter was easy to distinguish. As a result, we found that chromosome compaction and homolog pairing were disturbed in mutant females, and the splitting of the chromocenter was proved to exist (Figure 2C and D).
Thus, a decreasing of Trf2 gene expression leads to failure of chromocenter formation and chromatin condensation required for proper homolog paring at premeiotic stages, and it is evidently a reason for the chromosome nondisjunction that we observe in genetic experiments.

Trf2 participates in pericentromeric heterochromatin formation
Chromocenter splitting assumes the disruption of interchromosomal ectopic contacts in the pericentromeric heterochromatin region. We decided to examine Trf2 influence the pericentromeric heterochromatin formation. We used the line with paracentric inversion on X chromosome In(1)w m4 [23]. This inversion transfers the white locus next to the pericentromeric region, and as a result, w m4 mutants get a red-white mosaic colored eyes due to the position-effect variegation. To determine the ability of the lawc p1 mutation to modify the position-effect variegation, we performed a genetic experiment using w m4 mutation as a sensitive test system. The combination of w m4 with the hypomorphic lawc p1 mutation resulted the restoring of eye coloration in compound w m4 lawc p1 flies ( Figure 3A). This suggests that decrease in the concentration of Trf2 protein causes the decompaction of normally tightly packed pericentromeric heterochromatin that results in white gene derepression. Thus, the Trf2 is normally required for the formation of pericentromeric heterochromatin, which in turn participates in the chromocenter organization.
As the Trf2 protein is a transcription factor and can indirectly influence the chromatin structure (through the activation of genes responsible for chromatin compaction), the question arises: whether Trf2 protein can directly participate in chromocenter formation? To answer this question, we used the y 1 w 1 ; P{w + , [GFP~Trf2-1]} flies express the hybrid GFP:Trf2 protein  (short Trf2 isoform fused with green fluorescent protein [GFP]) under the control of the constitutive Hairy wing (Hw) gene promoter. We performed the immunofluorescence staining of y 1 w 1 ; P{w + , [GFP~Trf2-1]} larvae salivary gland polytene chromosomes by antibodies to GFP and analyzed the distribution of fusion GFP:Trf2 in chromocenter. As a result, we found sites of Trf2 localization in pericentromeric heterochromatin regions ( Figure 3B). These data allow us to confirm the direct participation of the Trf2 in the chromocenter formation.

The effect of Trf2 knockdown on salivary glands polytene chromosome morphology
We demonstrated in the above described experiments that the decrease in Trf2 concentration influences proper chromatin compaction and chromocenter structure in germ cells.
However, Trf2 localization found in pericentromeric salivary glands heterochromatin region assumes the involvement of this protein in the compaction of chromosomes also in somatic cells.
As considered, polytene chromosomes are very favor objects for the analysis of numerous features of interphase chromosome organization and the genome as a whole [24]. To confirm our hypothesis, we decided to use UAS-GAL4 two-component system [25] for specific RNA interference (RNAi)-mediated Trf2 depletion in salivary gland. We obtained two Drosophila UAS-containing transgenic lines using P-element-mediated transformation. These lines contain constructs that express double-stranded RNA hairpins that are complementary to either 5′UTR Trf2 regulatory (P{w + ; UAS-Ri}13) or encoding (P{w + ; UAS-TRIS}) Trf2 gene region ( Figure 1A). Both UAS-Ri13 and UAS-TRIS constructs are able to express RNA hairpins under the control of inducible UAS yeast promoter element ( Figure 1B).
For specific Trf2 depletion in somatic cells, we used the Sgs3-GAL4 driver (the line w 1118 ; P{Sgs3-GAL4.PD}TP1) that expresses yeast GAL4 activator in larvae salivary glands. After crossing UAS-containing flies with Sgs3-GAL4 driver flies, the morphology of polytene chromosomes in descendant larvae Sgs3>Ri13 and Sgs3>TRIS was analyzed. Larvae from UAS/+ lines and larvae from Sgs3-GAL4/+ line were used as the controls.
Normally, polytene chromosomes are present in salivary glands in singular due to the somatic synapsis occurs when two homologous chromosomes remain consistently conjugated. Polytene nonhomologous chromosomes in the nucleus are joined by their centromeres to form the most compact common region-chromocenter (Figure 4A, C, and E). Studies of Trf2-depleted salivary gland polytene chromosomes show a number of structural aberrations in the polytene chromosomes morphology. Its banding patterns are changed, the pairing is significantly disturbed, and asynapsis frequently involves very extensive regions (almost the entire chromosome; Figure 4B, D, and F). These defects were found approximately in 95% of analyzed nuclei (N = 100) in the experimental sample and approximately in 5% (N = 50) of analyzed nuclei in the control sample.  It is known that partial asynapsis is not a consequence of squashing of nuclei and variations in methods used to make preparations do not affect the frequencies of asynapsis [26]. So, we concluded that high frequency of chromosome asynapsis was induced by Trf2 depletion. However, the main trait of nuclei in lines with depleted Trf2 was the failure of chromocenter formation. Thus, the suppression of the Trf2 expression in salivary glands reveals the involvement of Trf2 gene in a chromocenter organization and in the correct pairing of homologous chromosomes not only in meiosis but also in somatic cells.
It is known that the chromocenter is responsible for the chromosome co-orientation during cell division and facilitates the paring of homologs [9,27]. The disturbance of paring affects the transvection (or allelic complementation)-the phenomenon in which gene regulatory elements located in one of the homologs control a promoter of the same gene but located in another homolog [28,29]. It is interesting to note that hypomorphic lawc mutations suppress transvection effect induced by zeste mutations [16]. This fact confirms the existence of abnormal homolog paring in lines with lower Trf2 expression.
The study a set of mutations that cause chromosome nondisjunction allowed to conclude that the chromocenter is a genetically programmed structure, that is, there are genes that control its formation and reorganization [11]. For example, it was demonstrated that the recessive mutation of crossover suppressor on 3 of Gowen (c(3)G) gene influences the structure of the lateral element and the length of meiotic chromosomes [30][31][32].
The Syntaxin 13 (Syx13) gene mutation (ff16) causes sterility and chromosome nondisjunction in males and females meiosis. Oocytes of mutant ff16 females demonstrate a split karyosome and the disruption in the chromocenter formation. The product of this gene is homologous to the receptor of synaptosomal-associated protein of 25 kDa (SNAP-25) and is involved in cytokinesis [32,33].
Another gene-no distributive disjunction (nod)-is involved in the organization and orientation of the spindle during mitosis and meiosis and is required for its binding to chromosomes in the Drosophila oocyte nucleus. This gene encodes the protein that contains DNA-binding domain and the conservative motor domain homologous to the Kinesin. nod mutations disrupt chromocenter formation in germ and somatic cells and cause achiasmatic chromosome nondisjunction in Drosophila females meiosis [30,34]. All these proteins have distinct functions; however, a decrease in their activity leads to a similar result, that is, the disruption of chromocenter formation and chromosome nondisjunction.
It was shown that Trf2 may be the part of the macromolecular chromatin-remodeling complex NUcleosome Remodeling Factor (NURF) which is correlated with transcriptional activation [35]. Nevertheless, the data we obtained have demonstrated that Trf2 could not only be involved in transcription activation but also could perform structural functions in chromatin organization. This idea is supported by the observation that there is no proper chromatin condensation in early spermatids of mice with null Trf2 mutation and, in particular, the chromocenter formation is disturbed [36]. Thus, we may conclude that the role of Trf2 in the organization of chromocenter structure and chromatin condensation is evolutionarily conservative.
In yeast, it was demonstrated that kinetochores-large protein complexes assembled on the centromeric region of the chromosomes, to which spindle microtubule is attached during cell divisionare formed by the epigenetic mechanism. This mechanism involves the generation of specialized nucleosomes in which a canonical histone H3 is replaced by its centromere-specific homologscentromere protein A (CENP-A). This protein served as a landmark for kinetochore assembly to define the identity of centromeres [37,38]. The high frequency of chromosome nondisjunction induced by decondensation of pericentric heterochromatin in lawc mutants allows us to assume that Trf2 may be involved in the epigenetic regulation of kinetochores formation in Drosophila.
As it was mentioned above, the correct distribution of chromatids between daughter cells depends on the coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibrils, topoisomerase, proteolytic processes, and motor proteins. The error of accurate spatiotemporal interactions between any of these factors results in a genomic disbalance. We cannot completely exclude the probability that Trf2, being transcription factor, can indirectly influence the process of cellular division through the regulation of genes that control mitosis and meiosis. In previous experiments, while looking for interactions between the Trf2 and other genes, we performed genetic screening to detect cytological regions that are sensitive to a decreased level of Trf2 expression [39]. Table 2 shows genes of meiosis and mitosis localized in these regions. The genes involved in chromatin compaction are the largest group.  This does not mean that Trf2 interacts with each of them; nevertheless, we cannot exclude the probability that decompaction of pericentric heterochromatin and defects in chromosome segregation in mitosis and meiosis in lawc mutant are induced by low expression of some of these genes. However, the localization of Trf2 in the chromocenter supports the idea that this factor can be independently involved in the organization of chromatin structure.

Conclusion
We demonstrated that a decrease in the Trf2 expression damages proper chromocenter structure and abolishes chromatin condensation required for correct homologs pairing at premeiotic stages and is evidently a reason for the chromosome nondisjunction that we observed in genetic experiments. Moreover, we found that compact chromocenter and correct homolog pairing were abolished in flies with a lower Trf2 expression not only in germline but also in somatic cells. As Trf2 is localized in pericentromeric regions, we conclude that Trf2 can not only be involved in transcription activation but also may perform structural function in pericentromeric heterochromatin organization that is responsible for a chromocenter formation.
In conclusion, we would like to note that in the recent screening for genes that control the sex-ratio meiotic drive in Drosophila simulans (closely related species to D. melanogaster), the Trf2 was suggested as the candidate for the factor responsible for this natural phenomenon typical for some animal species [40,41]. It is interesting that in studied D. simulans population Trf2 locus underwent the tandem duplication [41]. Thus, the function to control the specific X-chromosome nondisjunction may be adapted during the evolution by one of Trf2 copies.