Genetic and physical interaction of Drosophila Ino80 with Polycomb Responsive Element

The chromatin remodeling protein, dIno80 (Drosophila Ino80) regulates homeotic genes. We show that Ino80, along with Trx and ETP (Enhancer of Trithorax and Polycomb) proteins, interacts with two Polycomb/Trithorax Responsive Elements (PRE/TRE), iab-7 and bxd PRE in flies and the larval imaginal discs. In S2 cells, dIno80 localizes to the endogenous iab-7 and bxd-PREs. The localization of Ino80 and Pleiohomeotic (Pho) at the PRE is sensitive to the cellular abundance of each other; when levels of Ino80 are limiting, there is increased Pho enrichment, and Pho knock-down leads to increased enrichment of Ino80. We demonstrate that over-expression of dIno80 rescues the pupal lethality in pleiohomeotic (pho) deficient flies, which suggests that dIno80 has a role in cellular memory. The apparent competition between Pho and Ino80 for binding at the PRE indicates that Ino80 may act as a potential recruiter of the regulatory complex in addition to being a chromatin remodeler. Author Summary The null mutants of Pho and dIno80 show lethality at different stages of development in the fly, implying that they may function independent of each other. The observation that Pho-lethality can be rescued by overexpression of dIno80 with significant penetrance and that Ino80 has its own DNA binding domain, led us to predict that Ino80 may have Pho-independent functions, perhaps through non-canonical complexes. In the current study, we show that dIno80 interacts with bxd and iab-7 PRE in cooperation with Polycomb and Trithorax proteins and regulate the homeotic genes. The effect of knock-down or mutation of dIno80 results in altered phenotype in adult flies and rescue of Lac-Z expression in imaginal discs, in parallel with similar effect of Pho mutation or knock-down. We provide evidence of direct interaction of dIno80 with iab7- and bxd-PRE using chromatin immunoprecipitation. The dIno80 localization in and around the PRE sequence was enhanced in the absence of Pho, indicating competition between Pho and dIno80 for binding at the PRE.


Introduction
The Homeotic (Hox) genes control body patterning in animals and are highly conserved across phyla [1,2]. The level and domain of expression of Hox genes, control segmental identity in Drosophila [3]. The transcription factors such as Krüppel [4] and the segment polarity genes in Drosophila regulate Hox gene expression during development. The maintenance of the expression is through the interaction between specific cis-elements, the PREs/TREs (Polycomb/Trithorax Responsive Elements) and the Polycomb (PcG) and the trithorax (Trx) complexes that constitute the Cellular Memory Module [5,6]. The proteins of the PcG complex (Polycomb Repressive Complexes PRC1 and PRC2) are highly conserved among the organisms [7]. The PcG and TrxG complexes are epigenetic modifiers that bring about heritable changes in gene expression from one generation of cells/organisms, to the next [5,[8][9][10][11].
The PREs were first identified in the Bithorax complex of D. Melanogaster [12][13][14][15]. PREs and TREs are often overlapping and contain multiple motifs for sequence-specific DNA binding proteins of the PcG/ TrxG complex [16]. In Drosophila, PREs are known to exert their effect on target genes that are located at long distances, ranging from a few bases to several kilo-bases [17].
The PRE/TREs contain the binding sites for Pleiohomeotic (Pho) and other recruiters of Polycomb/Trithorax complexes such as DspI, Grh, SPI/KLF, Zeste, GAGA factor, Pipsqueak and Grainyhead [11,18]. It is known that there are other factors, such as modified histones, long non-coding RNAs and short RNAs that mediate the recruitment of Polycomb/Trithorax complex [19,20]. The PRC2 member, EZH2 catalyzes the methylation of histone H3 at K27 and this modification, in turn, is recognized by PRC1 which brings about ubiquitylation of H2A at K119 leading to repression [21][22][23]. There are other mechanisms known for the recruitment of PcG/TrxG complex at the PREs [24][25][26]. As such, there are no reports of chromatin remodeling complexes interacting with PREs/TREs; however, there are a few examples where the chromatin remodeling proteins such as the Brahma (Brm) genetically interact with TrxG members. The chromatin remodeler complex further mediates transcriptional activation through their interactions with trithorax group protein Zeste [27].
dIno80 is a member of the ETP group [28,29]. The ETP members are also known to interact with PREs, for interact Corto interacts with iab-7 PRE [30]. dIno80/hINO80 interact with Pho/YY1 in Drosophila and human, respectively [31,32]. However, the interaction of this complex with PRE is not demonstrated. In Drosophila, the Pho-independent function of dIno80 during development and the localization of dIno80 on the polytene chromosomes in the absence of Pho is known [29]. In the present study, we provide evidence of genetic interaction and also direct interaction of dIno80 with iab-7 and bxd PRE. We demonstrate that the enrichment profile of Pho and dIno80 are related to the relative concentration of each other in the cells.

dIno80 regulates Hox genes
In order to dissect the role of dIno80 in development, we examined the interaction of heterozygous mutants of dino80 with different genes. In a previous study from our lab, we demonstrated that the null mutants of dIno80 led to alteration of Hox gene expression at the late embryonic stages [28]. The study was taken forward to examine the consequence of the genetic interaction of dIno80 with Hox genes (Abd-B, abd-A and Ubx of the bithorax complex) in adult flies. The dIno80 heterozygous knock-out (dIno80 Δ4 /Sb 1 ) flies were crossed with Hox mutants (Fig 1 and Table1). The abnormalities occurring at high penetrance, are the fusion defects in the abdominal segments (segments 3-4 and 4-5) and altered wing margins (Table 1). Genetic interaction between dIno80 mutant and mutants of the bithorax complex members produced progeny with abdominal fusion of segment A3 and A4 in females ( Fig   1A'), an extension of the dark pigmentation in males from A5 to A4 segment (Fig1B') and shrunken wings and brown patches on the wings (Fig 1D, E & F). Thus, suggesting that dIno80 functions as a regulator during development.
Since the regulation of Hox genes is through the PcG and TrxG complexes that interact with PRE/TRE sequences, we examined the interaction of dIno80 with PRE sequence.

dIno80 interacts with Polycomb Responsive elements (PRE)
We investigated the genetic interaction of dIno80 with two known PREs of D. melanogaster; iab7-PRE that regulates the expression of abd-A and Abd-B genes and bxd-PRE, which governs the Ubx expression [33]. We tested different iab-7 PRE transgenic lines, including wt15 and wt44, and observed significant changes in the expression of the mini-white gene in the background of dIno80 mutation (Fig 2 and S1 Fig). Increase in the eye color in the mutant context indicates that dIno80 is required for the repressive function of the PRE.

dIno80 cooperates with PcG/TrxG complex for interaction with PRE
We studied the effect of PcG, TrxG, and ETPG mutations on the interaction of dIno80 with the PREs. We generated double heterozygotes of iab7-PRE transgenic flies in the background of dIno80 mutation along with mutants of different members of the maintenance group proteins including PcG, TrxG and the ETP group (Table S1).
We estimated the pigment content in each line following extraction from an average of 10 flies of each sex in three independent biological replicates (Fig 3 and S2 Fig). A significant difference in pigment level was observed between iab-7-PRE line in the background of dIno80 mutant (iab-7/+ vs iab-7/dIno80) and also between double heterozygotes of iab-7/dIno80 TrxG, PcG and ETPG genes [(iab-7/Trl R85 vs iab-7/Trl R85 ; +/dIno80), (iab-7/Pho b vs iab-7/Pho b ; +/dIno80), (iab-7/Asx XF53 vs iab-7/Asx XF53 ; +/dIno80)]. We carried out similar assays in the background of several mutations (S2 Fig and Table S2). We have compared all the genotypes with iab7-PRE (wt15)/+ for statistical significance (Table S2). These results suggest that dIno80 regulates homeotic gene expression through its interaction with PRE and in combination with the PcG, TrxG, and ETPG proteins. However, we cannot formally rule out the independent function of dIno80 in this regulation.

Role of dIno80 in PRE mediated-regulation during development
PcG/ TrxG complexes also mediate the regulation of homeotic genes during larval stages through their interaction with PRE/TRE [34]. We examined the effect of dIno80 knockdown and deletion mutation in imaginal discs of third instar larvae of D. melanogaster. Since the wt15 line with iab-7 PRE has only mini-white as the reporter gene, we used the bxd-PRE transgenic with Lac-Z reporter gene driven by ubiquitin promoter (Fig 4A). We compared heterozygous deletion mutant lines of dIno80 and Pho (bxd-PRE/dIno80 Δ4 and bxdPRE/+;Pho b /+) and dIno80RNAi lines (bxdPRE/+;UASIno80RNAi/ ActGAL4 and bxdPRE/+;UAS PhoRNAi/ActGAL4) for the expression of β-galactosidase in the imaginal discs.
In transgenic lines with bxd-PRE/+ in the background of dIno80 4 , the repression of the βgalactosidase gene is reversed ( Fig 4B). We observed a similar effect, when dIno80 was knocked-down (bxd-PRE: UASIno80RNAi/ActGal4) or when Pho is mutated (Pho b ) or knocked-down (UASIno80RNAi/ActGal4; Fig 4B). The similarity in the outcome of dIno80 and Pho depletion in conjunction with the genetic interaction of dIno80 with members of PcG, TrxG, and ETPG in iab7-PRE transgenic line strongly suggest the interaction of dIno80 with PREs through PcG complex.
Thus, we demonstrate that dIno80 regulates reporter genes (mini-white and Lac-Z) via its interaction with the iab-7 and bxd-PRE, however, it needs to be elucidated whether dIno80 protein directly interacts with the PRE sequences.

dIno80 protein is enriched at the Polycomb Response Elements (PREs)
The localization of dIno80 on chromatin has been demonstrated earlier by immunostaining of polytene chromosomes [28,29]. Since anti-dIno80 antibodies are not commercially available, we carried out the Chromatin immunoprecipitation experiments in S2 cells transfected with full-length cDNA of dIno80 with FLAG tag. The expression of FLAG-dIno80 was confirmed using anti-FLAG antibody (S3 Fig). dIno80 is known to form a complex with Pho along with other proteins [31,32]. Therefore, we investigated the localization of both Pho and Ino80 at iab7-PRE and bxd PRE.
The enrichment of dIno80 at Bxd-PRE ( Fig 5) and iab-7PRE (Fig 6) was analyzed in the transfected cells using anti-FLAG antibody within the PRE and also the flanking regions. The enrichment varied between the regions. The regions flanking Bxd-PRE showed higher enrichment of dIno80 than the PRE itself ( Fig 5). On knock-down of Pho, the enrichment of dIno80 further increased in the flanking regions while it remained unchanged within the PRE.
While the knock-down of dIno80 decreased enrichment in all cases. This indicates that the binding of dIno80 is independent of Pho. The reason for differential enrichment in these regions remains to be investigated. Differential enrichment of dIno80 was observed in case of iab-7PRE and its flanking regions, which was sensitive to the presence of dIno80 and was enhanced in the absence of Pho (Fig 6). The reduced FLAG-Ino80 interaction on knockdown of Ino80, confirms the specificity of anti-FLAG antibody. The knock-down is around 80% for dIno80 and 50% for Pho (S4 Fig). The enrichment of Pho in both Bxd-PRE and iab-7PRE was assayed using Anti-Pho antibody (Figs 7A and B). On over-expression of dIno80, Pho enrichment decreases, however on knock-down of the over-expressed dIno80, the enrichment of Pho within Bxd-PRE and region downstream is observed but in the upstream flanking region it remains poor. This is consistent with the significantly high localization of dIno80 at this region when Pho is knocked down (Fig 5). In the iab-7 PRE, the overexpression of dIno80 decreases the enrichment of Pho in all the regions, which is partially reversed on dIno80 knock-down.
These results show the Pho independent binding of dIno80, but the relative enrichment of the two proteins at the regions is not uniform across the regions examined. This may have its bearing on the existence of variable affinity of the sites known in other cases [35].

Functional complementation of Pho by dIno80
The lethality due to the lack of Pho and dIno80 proteins manifest at different stages of development in the fly [3], suggesting that they have developmental functions independent of each other. As both dIno80 and Pho are involved in transcriptional regulation and both are temporally differentially expressed, we examined whether dIno80 can rescue the pharate/pupal lethality caused by pho knockdown. The dIno80 was over-expressed in Pho knockdown background using UAS-RNAi approach with nos>Gal4 as the driver. The scheme of the cross set up is given in Supplemental Figures S5 and S6. The over-expression of dIno80 in pho knockdown background led to 10% rescue of pharate lethality in F 2 and 46% rescue in the F 3 generation ( Table 2,

INO80 interacts with hPRE-PIK3C2B
hPRE-PIK3C2B is a well-characterized PRE/TRE element that is known to interact with both the activating (TrxG) and repressive (PcG) complex members [38,39]. hPRE-PIK3C2B is present in the 1 st intron of PIK3C2B gene. We performed genetic interaction studies using the hPRE-PIK3C2B transgenic fly line  in the background of dIno80 Δ4 mutation and observed a decrease in eye pigmentation in comparison to PI-17/+ (Fig. 9A).
Further, we examined the interaction of human INO80 with hPRE-PIK3C2B in HEK cell line ( Fig 9B). The repressive effect of YY1 (the human homolog of Pho) and INO80 on the expression of endogenous PIK3C2B gene was reversed when either hINO80 or YY1 expression was knocked-down using siRNA (Fig 9B). The chromatin immunoprecipitation experiments demonstrated enrichment of INO80 at hPRE-PIK3C2B, which drastically decreased upon knockdown of YY1 (Fig 10C). Unlike the PREs in the fly, the human INO80 shows YY1 dependent recruitment to the hPRE-PIK3C2B. We have earlier demonstrated that hPRE-PIK3C2B has multiple repeats of YY1 binding site [38].

Discussion
INO80 subfamily of chromatin remodelers was identified as the regulator of transcription, and the mechanism involves nucleosome re-arrangement which in turn leads to the increase in the accessibility of the chromatin [40]. The present study further adds to the already known diversity of Ino80 protein. The Ino80 protein is a versatile protein that is involved in multiple functions [29,[41][42][43][44]. The interaction of dIno80 with promoters of pluripotency genes which are dependent on Oct4/WDR5 binding is also known [45]. In the current study, we have shown that dIno80 directly interacts with PREs, strongly indicating that the basis of the previously demonstrated role of Ino80 in development is mediated by its interaction with chromatin [28,29]. The fusion of abdominal segments as a consequence of the interaction of dIno80 with Abd-B and also the altered wing patterning indicates the interaction of dIno80 with homeotic genes. Earlier, we reported the dual regulation of Scr, by dIno80 in different imaginal discs; being an activator of Scr in wing imaginal disc while in the leg and salivary gland disc, it acts as a repressor [29]. Thus , dIno80 interacts with several homeotic genes and based on our results, we predict that this interaction could be through the PRE/TRE sequences. The effect of dIno80 on PRE-mediated expression of the β-galactosidase reporter gene in imaginal discs through, bxd-PRE is very similar to that of Pho. This can be due to the canonical complex of Ino80 where Pho acts as the recruiter; however, the direct interaction of dIno80 with bxd-and iab7 PRE even in the absence of Pho and the fact that dIno80 can rescue Pho null mutants with considerable penetrance (43%) indicates the involvement of a non-canonical complex. The genetic interaction strongly suggests that the effect of dIno80 on the transcription of genes driven by PRE sequences is through PcG, TrxG and ETPG members. Thus, as an ETP protein, dIno80 enhances the effect of Trl, Pho, and Asx on the reporter expression. It remains to be established whether Ino80 interacts with PREs directly or indirectly through other recruiters.
In the light of our results, we predict the direct interaction of dIno80 with DNA.
We have shown that the DBINO domain of hIno80 as well as dIno80 bind to a consensus DNA sequence [46,47]. Therefore, we analyzed the presence of this motif and the motifs for binding of other proteins such as Pho, Dsp 1, GAF, Pipsqueak known to be involved in the recruitment of the PcG/TrxG complexes to the PREs of D. melanogaster (Table 3). It is observed that the putative Ino80-binding motif is present in many PREs and therefore, the direct interaction of dIno80 with PREs could be through this motif in addition to the known mechanism whereby Pho/YY1 recruits the Ino80 complex.
There are other modes of interaction of chromatin remodelers with chromatin, BPTF (bromodomain PHD finger transcription factor), a subunit of NURF complex recognizes H3K4me3, while CHD1 recognizes both H3K4me3 and H3K4me2 [48,49].
The localization of dIno80 in both the PREs and the neighboring regions is seen by the steady enrichment of Ino80 as well as Pho. The dependence of this interaction on dIno80 is indicated by the lack of enrichment on knock-down of dIno80. Interestingly, we observe that there is an increase in Pho localization on knock-down of dIno80. These results not only prove the direct interaction of Ino80 with PRE but also suggest concentration-dependent recruitment of the two proteins to the PRE sequences. In conjunction with the genetic interaction studies, it emerges that there can be the recruitment of either activating or the repressive complex through dIno80, which needs to be analyzed further. This is unlike Pho, which is known to recruit PRC2 complex at the PRE. The variable enrichment of dIno80 at the two PREs and their flanking region is similar to the variability in enrichment of PRC complex and the distribution of H3K27me3 observed in other cases where the strong and weak binding sites for PRC proteins is observed in large genomic regions of greater than 145kb [35]. It has been shown that the deletion of the major PcG regulated invected-engrailed (inv-en) gene complex does not affect regulation, which was attributed to the presence of several low-affinity PRElike sequences in the region [35]. Even though the region we have examined is only 800-430bp, the results indicate that there is interaction beyond the limits of Bxd and iab-7 PRE.
The developmental genes and their regulators are known to be highly conserved through evolution. dIno80 binds to hPRE-PIK3C2B in transgenic flies to bring about activation, as we observe a decrease in eye pigmentation in the background of Ino80 Δ3 mutation. In terms of localization of hINO80 at hPRE-PIK3C2B, as measured by ChIP in HeLa cells, this interaction is completely lost when YY1 was knocked down. Thus, indicating that INO80 is recruited to hPRE-PIK3C2B by YY1. This is in contrast to the results we obtained where Pho and dIno80 are enriched at iab-7 PRE and bxd-PRE in the absence of the other; knockdown of ino80 leads to increased enrichment of Pho. This strongly supports the Pho-independent role of Ino80, shown earlier in flies [29]. Previously, it has been shown that YY1 (Human homolog of Drosophila Pho) knock-down led to an increase in the expression of PIK3C2B [38]. Here, we show that INO80 knockdown also rescues the expression of PIK3C2B in HEK cells.
Our results do not formally rule out the role of ATP dependent chromatin remodeling activity of dIno80 in transcription regulation. However, the partial complementation of dIno80 of Pho function raises the possibility that Ino80 could function as a recruiter of PcG/Trx complexes. The over-expression of dIno80 can rescue Pho mutant with significant penetrance (40%), while Pho null mutation shows 100% lethality. Thus dIno80 appears to complement the function of Pho at certain developmental stages. The number of known DNA-binding recruiters coded in the fly genome is limited in the context of the diversity of developmental regulatory complexes and the sites of their recruitment. There is a report of dINO80-Pho complex, in which Pho recruits dINO80 complex to the genome [52]. Pho is essential for the completion of development in Drosophila and is a well-characterized polycomb group member and its knock out is pupal lethal [53]. In spite of the well-known essentiality of Pho in development, it is interesting to note that over-expression of dIno80 can rescue Pho lethality.
This strongly suggests the functional complementation by dIno80 in terms of the recruitment of regulatory complexes on the genome.
Apart from its role in the development and DNA repair, Ino80 also plays a vital role in maintaining the boundary between euchromatin and heterochromatin. Ino80C blocks Dot1mediated H3K79 methylation [54]. Thus the functional diversity of chromatin remodeling proteins can be mediated by their interaction with different protein complexes. The diversity of protein complexes formed by INO80/dIno80 protein is currently being investigated in our laboratory.
It can be hypothesized that proteins belonging to various functional classes may be brought together to achieve a unique functional outcome in a manner very similar to the concept of a "lego set." Rvb1 and Rvb2 proteins are partners of Ino80 in the chromatin remodeling complex and are also known to interact with Tip60 complex (histone acetyltransferase), telomerase complex, snoRNP complexes and the mitotic spindle assembly [55]. Brahma, which is a part of the SWI/SNF chromatin remodeling complex is associated with MeCP2 and plays a role in transcriptional silencing [56] and DNMT3B interacts with HDAC1 and 2 along with hSNF2H (chromatin remodeling enzyme) and co-localize at heterochromatin regions [57]. Thus, proteins diverse in nature associate with each other to accomplish a novel function.
The regulatory function of Ino80 in development through interaction with PRE demonstrated here may be a tissue-specific function, and it remains to be seen whether this function is the result of a novel interaction of Ino80 either with proteins and/or RNA molecules. However, it is clear from several examples that there is a crosstalk between chromatin remodelers and well-known silencing mechanisms mediated either by Polycomb proteins or the HP1 protein.

Knockdown/over-expression of genes using UAS/Gal4 system
The UAS/Gal4 system was utilized as required either for over-expression or the RNAi for the desired gene [60,61]. For RNAi mediated knock-down of genes, we used UAS-Ino80-RNAi

Immunostaining of embryos
10-14 hours old embryos of the desired genotype were used for immunostaining as per the published protocol [62]. Anti-abd-A (Santa Cruz; sc-27063) primary antibody and Donkey polyclonal to Goat IgG -H&L (HRP) (ab6885) secondary antibody were used at a dilution of 1:100 and 1:200, respectively. In the case of HRP chromogenic reaction was performed to stain the embryo and then CNS of the embryos of the appropriate stage were dissected out, and images were captured by using Zeiss AxioScop 2.

Cloning full-length dIno80 in an expression vector
The full-length dIno80 (SDO2886, from the pOT2 vector-5117 bp) was cloned in pBUF- Dissecting imaginal discs β-Galactosidase assay (X-gal staining of imaginal discs) The protocols followed for dissection and immunostaining of imaginal discs were as described by Spratford and Kumar (2014) [63].
The imaginal discs isolated from each genotype were processed within 2 hrs. The discs were washed with 1X PBS and treated with a fixative. The standard protocol was followed for staining the discs for β-galactosidase expression [53]. The stained discs are mounted in 50% glycerol and viewed under Nikon Eclipse E600 microscope.

Eye Pigmentation Assay
Eye pigmentation was quantified as described by Wald and Allen, (1946) using male flies The expression of dIno80 (full-length dIn80 in pBUF vector) was analyzed by Western blotting using anti-FLAG ® antibody (Sigma Aldrich ® , cat. No. F7425). The band intensity was measured using densitometry.

Knock-down of Pho and dIno80 in S2 cells
For carrying out the knock-down of Pho and dIno80 in S2 cells, we have followed the protocol described by Celotto and Graveley (2004) [65]. In brief, we used two different pools of duplex siRNA oligonucleotide (Eurogentec © , Belgium), each for dIno80 and Pho. We

Chromatin Immunoprecipitation (ChIP) assay
The ChIP assay was performed as described by Kuo and Allis (1999) [66]. Approximately 8.0 X 10 6 Schneider 2 (S2) cells were taken and were cross-linked at a final concentration of 1% formaldehyde G and glycine was added to a final concentration 0.125mM to quench the