ELYS coordinates NF-κB pathway dynamics during development in Drosophila

ELYS, a nucleoporin spatiotemporally regulates NF-κB pathway dynamics during development in Drosophila and its misregulation in post-embryonic stages leads to apoptosis mediated abnormalities. Abstract Nuclear pores are the exclusive conduit to facilitate the nucleocytoplasmic transport in a precisely regulated manner. ELYS, a constituent protein of nuclear pores, initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores towards the end of mitosis. Using cellular, molecular and genetic tools, here, we report that ELYS orthologue (dElys) plays critical roles during Drosophila development. Through in silico analyses, we find all conserved structural features in dElys except for the presence of non-canonical AT-hook motif strongly binding with DNA. dElys localized to nuclear rim in interphase cells, but during mitosis, it was present on chromatin. RNAi mediated depletion of dElys leads to aberrant development and defects in the nuclear lamina and NPCs assembly at the cellular level. Furthermore, we demonstrate that in dElys depletion NF-κB is activated and accumulates inside the nucleus which results in illimed expression of critical molecules. dElys depletion sustains NF-κB into the nucleus in post-embryonic stages. Prolonged NF-κB inside nucleus induces apoptosis in response to hitherto unknown quality check mechanism and highlights on the under-appreciated apoptotic paradigm of NF-κB pathway.


Introduction:
Nuclear pore complexes (NPCs) are a multi-protein assembly of nucleoporins (Nups), and their size varies from ~60 MDa in yeast to ~125 MDa in vertebrates. Nups are distributed into sub-complexes located on cytoplasmic, nuclear membrane and nucleoplasmic faces of nuclear pores (Hetzer, 2010;Hoelz et al., 2011). In metazoans, however, at the onset of mitosis, sub-complexes of NPCs dissociate from each other and gets redistributed inside the cell. The largest sub-complex of NPC called Y-shaped complex (hereafter Nup107 complex) has nine stoichiometric members (Ori et al., 2013). Nup107 complex plays important roles in NPC assembly, nucleocytoplasmic transport, and gets distributed to the kinetochores during mitosis Zuccolo et al., 2007). ELYS (Embryonic Large molecule derived from Yolk Sac) was characterized in the mouse as a putative transcription factor important for hematopoiesis (Kimura et al., 2002). ELYS, also known as Mel-28, is required for the maintenance of the nuclear morphology and embryonic development in C. elegans (Fernandez and Piano, 2006;Galy et al., 2006). Although in yeast, ELYS is a small protein and carries the minimal ELYS domain, in the higher eukaryotes, its molecular mass ranges from ~190-250 kDa and is also known as AT-hook containing transcription factor 1 (ATCHF1). Mouse ELYS was characterized to contain nuclear localization signal (NLS), nuclear export signal (NES), N-terminal β-propeller region, central helical and most important, C-terminally located AT-hook motifs (Kimura et al., 2002;Okita et al., 2003). The β-propeller like domain present in many nucleoporins mediates interaction with other nucleoporins and facilitates NPC assembly (Bilokapic and Schwartz, 2013). In a breakthrough observation, ELYS was reported to be an integral member of the Nup107 complex (Franz et al., 2007;Rasala et al., 2006;Rasala et al., 2008). In interphase, ELYS is present at nuclear envelope and nucleoplasm, but in mitosis, it associates with chromatin, kinetochores, and spindles (Rasala et al., 2006). Importantly the conserved ELYS domain is required for its NPC and kinetochore localization (Gomez-Saldivar et al., 2016) where ELYS helps in microtubule polymerization (Yokoyama et al., 2014). ELYS tethers Nup107 complex to kinetochores and initiates their assembly into post-mitotic nuclear pores (Rasala et al., 2008). Loss of ELYS in C. elegans causes abnormal nuclear morphology and aberrant chromosome segregation that confers lethality (Fernandez and Piano, 2006;Galy et al., 2006). ELYS is critically required for embryonic development as ELYS null mice die during embryonic stage E3.5 to E5.5, well before the onset of embryonic hematopoiesis (Kimura et al., 2002;Okita et al., 2004). However, conditional inactivation of ELYS locus in adult mice showed reduced effects and mice behaved normally (Gao et al., 2011).
In addition to, playing an important role in NPC assembly, reduction in ELYS activity in HeLa cells leads to phosphorylation-dependent mislocalization of Lamin B Receptor (LBR) from the nuclear periphery (Mimura et al., 2016). ELYS recruits Protein phosphatase-1 (PP1) to the kinetochores and reduction in LBR phosphorylation levels counters the action of mitotic kinases (Hattersley et al., 2016). AT-hook motifs present in ELYS allow DNA binding and the trans-activation domains (acidic region) help in transcription (Kimura et al., 2002). AT-hook motif is rich in positively charged amino acids and harbors -RGRP-residues at its core, is sufficient for binding to DNA (Rasala et al., 2008). ELYS is also known to associate with the promoter region of actively transcribing genes (Pascual-Garcia et al., 2017). Thus, being a DNA binding protein, an effector of proper cell division and a regulator of nucleocytoplasmic trafficking, ELYS may have a significant role to play in early developmental events of an organism.
However, no directed study has been performed on ELYS to obtain the mechanistic knowhow of important roles played by ELYS in cellular homeostasis and early developmental events.
We chose Drosophila melanogaster (fruit flies) to address the importance of ELYS in early developmental processes. Although CG14215 has been reported as ELYS in Drosophila (hereafter as dElys) and shown to be present at the nuclear periphery and in association with promoter region on polytene chromosomes, none of these studies characterized dElys for its role in NPC assembly or other cellular processes in vivo (Ilyin et al., 2017;Pascual-Garcia et al., 2017). Here, through genetic, molecular and cellular characterization we report that the dElys plays important roles in the assembly and dynamics of nuclear pore and nuclear lamina functions. dElys can bind with DNA through its non-canonical AT-hook motif, and RNAi mediated dElys depletion induces severe lack of nucleoporins like Nup98, Nup43 and mAb414 in nuclear pore and perturbs lamin B receptor and lamin B and C incorporation into the nuclear lamina.
Importantly, we observe that in dElys depletion, developmentally important molecule Dorsal is activated and accumulated inside the nucleus and subsequently induces caspases (Dcp-1) to follow the apoptotic pathway. Our observations strongly imply that dElys is a key developmental molecule required in cellular processes integral to normal cellular homeostasis. In addition to maintaining the nuclear architecture, dElys contributes to the development by regulation of the apoptotic arm of dorsal pathway activation.

ELYS is highly conserved in Drosophila:
ELYS is a DNA binding nucleoporin which coordinates NPC assembly, and NPC mediated cellular processes like nucleo-cytoplasmic transport. Loss of ELYS induces developmental defects but how ELYS regulated events affect the complex process of development remains elusive. We have used Drosophila to address these questions.
Although the CG14215 was reported to be Drosophila ELYS orthologue (dElys), however, any analysis to attribute structural elements in the molecule and mechanistic details of ELYS function was absent. Primary sequence alignment of CG14215 exhibits overall ~20-21% identity with mouse and human ELYS (Fig. S1A). ELYS, being an AThook containing transcription factor, we first searched for the presence of AT-hook motifs in CG14215. By utilizing mouse ELYS AT-hook motif (Mm_Elys_AT-hook), we have predicted three AT-hook motifs, but importantly we noticed an evident lack of glycine and proline amino acids in the conserved -RGRP-core (overscored) present in several of AT-hook proteins (Fig.1A) (Aravind and Landsman, 1998). We asked if such a non-canonical AT-hook can bind chromatin. A carboxy-terminal fragment (aa 1858-1962) of dElys bearing all three predicted AT-hook motifs, dElys_AT-1, 2, and 3 helped determine the DNA binding abilities in Electrophoretic gel mobility shift assay (EMSA) (Fig. S1C). dElys AT-hook bound with AT-repeat rich sperm dynein intermediate chain (sdic) promoter oligo in a dose-dependent manner and induced a clear shift in mobility ( Fig.S1B). Our data further established that the presence of glycine and proline is not as important in the -RGRP-core, which is further supported by (Metcalf and Wassarman, 2006). Accordingly, we mutated the arginines in each predicted AT-hook motifs individually (mAT-1, mAT-2, and mAT-3) or combined mutations of arginines to alanines for all three AT-hooks (mAT-1+2+3) (Fig.S1C) and tested the ability of purified proteins (Fig.S1D) to bind with DNA. In these experiments, instead of oligo, we have used larger DNA fragment (~900bp) having an abundance of AT sequence (AT-rich) or a DNA fragment lacking them (Non-AT rich DNA). It is evident from the observations that both mAT-1and mAT-3 show a significant reduction in their ability to bind with AT-rich DNA ( Fig.1B). However, the mAT-3 displayed pronounced perturbation in DNA binding abilities. Our mutational analysis of dElys AT-hook motifs indicated that the AT-3 motif is the prominent DNA binding motif present at the C-terminus of the dElys. Like other two predicted AT-hook motifs, AT-3 lacked glycine and proline in the core, but importantly it had alternate arginine residues as seen in canonical AT-hook of mouse ELYS. This particular feature made AT-3 the most probable AT-hook motif of dElys. Moreover, our DNA binding observation also suggests that the AT-hook motif can serve as a general DNA binding protein also (Fig.S1E). Quantification of the DNA binding data showed that AT-hook motif-1 and 3 are the DNA binding motifs with AT-3 having a stronger affinity for AT-rich DNA sequence ( Fig.1C and Fig. S1F).
Mammalian ELYS was shown to have a strong nuclear localization signal (NLS) and nuclear export signal (NES) sequences. Our in silico predictions indicate for the presence of multiple NLS spread throughout the molecule and two C-terminal NES in dElys. When the N-terminal NLS of dElys (strongest among the predicted NLS) was fused with GFP and expressed in Drosophila S2 cells, it was effectively transported inside the nucleus indicating its functionality (Fig.1D).
Further predictions of structural features present in dElys find a conserved arrangement of N-terminal β-propeller domain (aa 1-488), central helical domain (aa 489-977) and a C-terminal unstructured region (Fig. S1A). Homology-based 3D structure predictions on the N-terminal β-propeller domain of dElys, identified a highly conserved and significantly overlapping seven-bladed beta-propeller structure as seen in the N-terminal domain of mouse ELYS (Fig. S1G) (Bilokapic and Schwartz, 2013;Kelley et al., 2015).
Moreover, we could also predict for the presence of WD repeats at the N-terminal region and an ELYS domain in the central helical region of the dElys (Fig. 1E).
Evolutionary divergence analysis indicates that dElys is present in a clade quite distinct and distant from vertebrate ELYS (Fig. S1H). Our analysis strongly showed that dElys carries all the conserved structural motifs present in canonical ELYS despite low primary sequence homology.
To address the dElys functions in cellular and developmental processes in greater detail, we have raised polyclonal antibodies against the C-terminal fragment (aa 1796-2111). Affinity purified antibodies recognized ~230 kDa band in Drosophila salivary gland lysate (Fig.1E) as well as in lysates obtained from Drosophila S2 cells (Fig.S2A).
We also observed that this antibody could detect a concurrent decrease in dElys protein level when dElys is silenced using RNAi, indicating strongly towards specificity of dElys detection by the antibody. In tissues, dElys antibodies detected specific and strong nuclear rim staining along with feeble cytoplasmic annulate lamellae staining in early Drosophila syncytial embryos ( Fig. 2A and Fig.S2C). This particular observation highlighted about the relatively low abundance of dElys in annulate lamellae as compared to other nucleoporins detected by mAb414 (Fig. S2E). dElys antibodies observed a pattern at nuclear rim overlapping with GFP-Nup107 which is a known interacting partner of ELYS across eukaryotes and also with the nuclear laminaassociated Lamin-B receptor in salivary gland nuclei (Fig. S2B, D).

dElys is absent from kinetochores and localizes around chromosome in mitosis:
ELYS exhibits cell-cycle dependent variations in its localization and is present at the nuclear rim during interphase and to the kinetochores in mitosis (Franz et al., 2007;Rasala et al., 2006;Yokoyama et al., 2014). We probed if dElys also exhibits a cellcycle dependent sub-cellular localization change. The nuclei in Drosophila early embryos divide synchronously and grow in syncytium which provides the desired window to observe cell-cycle dependent localization of proteins. As observed earlier, dElys was present at the nuclear rim during interphase syncytial embryo ( Fig. 2A), but in mitosis, a diffused staining is obvious in the entire presumptive nucleoplasmic region of syncytial embryos (Fig. 2B, Fig. S2E). However, a careful analysis of higher magnification images, indicate that dElys is present around the mitotic chromosomes ( Fig. 2B inset). Interestingly, dElys staining resembled a bracket to chromosomes in Drosophila S2 cells arrested in mitosis, where is showed partial co-localization with αtubulin spindle fibres (Fig. 2C). We further asked if similar to other reported ELYS like molecules, dElys is present at kinetochores in addition to a chromatin bracket in mitosis.
Co-staining of kinetochores with Drosophila CENP-A homolog (CID) (Vermaak et al., 2002) in EYFP-dElys expressing S2 cells exhibited a distinct absence of dElys staining from kinetochores. dElys did not co-localize with CID but were in close vicinity of it (Fig.   2D). The EYFP-dElys, when expressed in salivary glands, produced a localization pattern similar to endogenous dElys (Fig. S2F). It is important to note that ELYS recruits the Nup107 complex to the kinetochores in cell culture, Xenopus egg extracts, and C. elegans embryos. However, the Nup107 complex was reported to be absent from kinetochores in Drosophila syncytial mitotic embryo (Katsani et al., 2008). Our observation that dElys is absent from kinetochores provides a suitable explanation for lack of Nup107 complex staining from Drosophila kinetochores.

dElys is essential for normal development in Drosophila:
After characterizing as a nucleoporin, we asked whether dElys is essential for development in Drosophila. Over-expression of dElys using UAS-dElys transgenic line did not yield any observable phenotypic differences when compared with wild-type (data not shown). dElys was depleted in vivo using RNAi line (v103547) from Vienna Drosophila Resource Centre (VDRC)(dElys KK ) to understand its role in cellular functioning. We also generated TRiP based RNAi line (dElys TRiP ) to validate phenotypes observed by dElys KK RNAi line, which do not have any predicted off-targets. Ubiquitous knockdown of dElys in Drosophila using Act5C-GAL4 had severe consequences on viability with variable lethality at each developmental stages, no adults emerged from one of the RNAi line dElys KK , and only a few viable flies (~4%) emerged out in dElys TRiP (Fig. 3A). We observed that dElys depleted embryos are showing ~4% lethality while ~46% lethality occurring at the larval stage, ~50% of pupae died in the late pupal stage of development (Fig. 3A). The extent of dElys depletion in RNAi lines when assessed by quantitative PCR showed that dElys KK RNAi line caused ~70% knockdown, while dElys TRiP leads to ~55% knockdown in dElys transcript levels (Fig. 3B). Although dElys KK has two predicted off-targets in addition to dElys, we have not observed a significant change in the level of off-targets (Fig.S3). As the dElys KK line is showing maximum knockdown of dElys, we have used dElys KK RNAi line for all subsequent studies. dElys has been contributed in high amount maternally, which makes it difficult to analyze its role in early stages of development. To overcome this problem, we used mat-α-tub-GAL4 which depletes maternally contributed dElys before induction of zygotic transcription. Maternally depleted dElys (dElys KK RNAi line) embryos have an abnormal shape as well as they do not show pole cells development (Fig. 3C, arrowhead).
To avail more insights on the importance of dElys in development, we silenced dElys in a tissue-specific manner with dElys depletion in eyes and wings using eyeless (Ey) and wingless (Wg) drivers respectively. Eyes in dElys depleted flies appeared shrunk with a significant reduction in the area occupied by ommatidia in addition to irregular size and disarrangement of ommatidia leaving behind eye cavity. We also observed that when dElys was depleted significant number of ommatidia show loss of eye bristles, duplication of bristles and their orientation was irregular (Fig. 3E). Similarly, dElys silenced wings look crumpled with signs of vein atrophy and significant cell death in wing blade tissues (Fig. 3F). The severity of these phenotypes varied between the two RNAi lines targeting dElys yet the observations made regarding eye and wing phenotypes were consistent. Our data revealed that the presence of dElys is important for the normal development of Drosophila.

Loss of dElys results in the nuclear pore and nuclear lamina assembly defects:
We proposed that the developmental defect observed in dElys knockdown organisms would be the most probable outcome of compromised NPC assembly. To analyze if dElys is important in NPC assembly, we assessed the localization of various nucleoporins representing various sub-complexes of NPC in the Drosophila salivary gland nuclei. Nup43, a stable member of the Nup107 complex, Nup98, a mobile nucleoporin and mAb414 reacting to nucleoporins with FG repeats and regulate cargo movement through NPCs were absent from the nuclear membrane and presented a diffused cytoplasmic staining in dElys depleted salivary gland tissue ( Fig.4A-D). Lack of nucleoporin sub-complexes and lack of functional nuclear pores in dElys silenced nuclei reinforces the idea that dElys is a critical player in functional NPC assembly.
Concomitantly, we observed a marked increase in cytoplasmic staining for these nucleoporins in dElys depleted salivary gland tissues highlighting possible increase in annulate lamellae (AL) structures in the cytoplasm. Quantitation of these intensities also confirms our observation of loss of these nucleoporins from the nuclear periphery ( Fig.4E-H). We have checked the protein levels of these nucleoporins in larval headcomplex lysate by western blotting, and the observations suggest no significant decrease in the protein levels of tested nucleoporins except for Nup98 (Fig.4Q). Since DAPI intensities didn't change between control and dElys silenced tissues; we have used to normalize the variability observed with the intensity of each molecule tested. We have further confirmed with Histone H3 staining that DNA intensities remain unaltered upon dElys depletion (Fig. S4). Our data with dElys further strengthens the conserved role for ELYS in NPC assembly throughout metazoans. We argued that if dElys depletion perturbs NPC assembly, then it must compromise the nucleo-cytoplasmic transport as well. In situ detection of mRNA showed that dElys depleted salivary gland nuclei had more mRNA compared to control nuclei ( Fig.S6 A  These observations suggested that dElys works independent and upstream of LBR. dElys helps in the incorporation of LBR, Lamin B, and C into the nuclear lamina which is indispensible for the maintenance of the nuclear lamina and ultimately the nuclear architecture. Nucleo-cytoplasmic transport of protein cargo through NPC is coordinated by Ran-GTPase which can be found on the nuclear rim as well. Importantly we have noticed that dElys depleted salivary gland tissues show striking lack of Ran reactivity from nuclear periphery and its redistribution was seen throughout the cell ( Fig. 4L, P). Lack of Ran-GTPase from nuclear periphery may also be one of the reasons for defective nucleo-cytoplasmic transport in dElys silenced condition. The absence of Ran-GTPase in addition to lack of Nups and lamins prompted us to ask if everything associated with nuclear periphery observes re-distribution upon dElys knockdown. However, when we probed for nuclear periphery associated TATA-box binding protein-1 (TBP1, dTBP in Drosophila) (Choudhury et al., 2017) localization in dElys depleted salivary gland tissues, it was unperturbed and marked characteristic nuclear periphery staining pattern ( Fig. S5A, B). To probe changes in the protein level of nuclear lamina molecules upon dElys knockdown, we performed western blotting on larval head complex lysate and with antibodies corresponding to these molecules. Lamin B and Lamin C protein level were unaffected in dElys knockdown while Ran showed little decrease in dElys knockdown (Fig. 4R). These observations approve the important role for dElys in the nuclear pore and nuclear lamina assembly. Consequently, loss of dElys could contribute to a severe reduction in the nuclear transport of various developmentally important molecules leading to abnormal development.

dElys knockdown shows activated NF-B pathway and induced apoptosis:
To deduce the underlying molecular mechanism that drive dElys depletion induced defects, we sought to check for perturbed nucleo-cytoplasmic traffic of various signaling molecules. We observed significant nuclear accumulation of key developmental transcription factor, Dorsal (NF-B) under dElys depleted conditions in salivary gland tissue. Under normal condition, Dorsal stably associates with cactus and is rendered inactive in the cytoplasm but rapidly shuttles inside the nucleus when activated in response to certain stimuli (Rushlow et al., 1989). Shuttling of dorsal inside nucleus is accompanied by ubiquitination-dependent degradation of the inhibitor of NF-B (IB) in the cytoplasm. (Belvin et al., 1995;Whalen and Steward, 1993). We depleted dElys using RNAi, and the assessed Dorsal signal in salivary gland tissues. In control tissues Dorsal signal was diffused and was present prominently in the cytoplasm. However, the dElys depleted cells show intense dorsal signal in close association with chromatin inside the nucleus (Fig. 5A). Quantification of the dorsal signal from dElys RNAi tissues shows a marked increase in nuclear dorsal signal (Fig. 5D). Nuclear localization of Dorsal indicates loss of its cytoplasmic binding partner Cactus, which freed Dorsal. To ensure this, we examined the cytoplasmic level of cactus and observed that cytoplasmic cactus signal decreased significantly in dElys depletion when compared to control tissues (Fig. 5B). This loss of cactus staining was quantified, and the data corroborated the cactus degradation and dorsal activation (Fig. 5E). Our data strongly suggest that dElys silencing trigger the activation of NF-B pathway. Dorsal activation and movement to the nucleus is a proliferative signal and developmentally important event.
However, we observe cell death which is a probable consequence of the unfavorable accumulation of dorsal inside the nucleus. Different studies suggest that activated dorsal can also induce apoptosis in cells (Kaltschmidt et al., 2000;Radhakrishnan and Kamalakaran, 2006;Ryan et al., 2000).
We next probed if the abnormal development of eye and wing tissues as well as lethality observed upon dElys depletion could be a result of increased cell death via apoptosis.
We used acridine orange staining in salivary glands as a primary assay to infer the induction of apoptosis. dElys depleted tissues accumulated significantly more acridine orange than normal tissues which suggested activated apoptosis ( Fig. S6 E, F). To further assert that dElys depletion indces apoptotic response, we probed for Drosophila caspase -1 (Dcp-1) which is critical for normal embryonic development and elevation in Dcp-1 levels is a hallmark for apoptosis (DeVorkin et al., 2014;McCall and Steller, 1998;Song et al., 1997). Dcp-1 antibodies staining showed significantly enhanced reactivity for Dcp-1 and significantly more number of Dcp-1 positive puncta were obvious in dElys silenced salivary gland tissues (Fig. 5C, F). Since the dElys depletion induced significant defects in eye and wing development, we asked if the apoptosis sets in early during the development of these organs in the absence of dElys. We probed for the Dcp-1 levels in Wing and eye imaginal discs obtained from wild-type and dElys depleted third instar larva. Both the eye and wing imaginal discs showed increased punctuate reactivity with Dcp-1 upon dElys depletion. (Fig. 5G, H). We next asked if NF-B causes any alterations in the expression of apoptotic genes. We probed this by quantitative real-time PCR for pro-apoptotic genes Reaper and Hid as well as for anti-apoptotic DIAP-1(Drosophila Inhibitor of Apoptosis-1) transcripts in cDNA prepared from the third instar larval head complex of control and dElys depleted organism. We observed a significant increase in the level of pro-apoptotic Reaper and Hid expression while there was a significant decrease in the level of anti-apoptotic DIAP-1 upon dElys depletion (Fig.6I). The relative levels of DIAP-1, Reaper, and Hid are important determinants of cell survival or cell death. Our qPCR observations and Dcp-1 immunostaining data exhibit undisputable correlation and indicate towards apoptotic induction upon dElys depletion. Dorsal, being a transcription factor and activated in the nucleus in dElys knockdown should bring a change in the levels of its target genes. To investigate this, we checked the levels of primary target genes of Dorsal in cDNA prepared from the third instar larval head complex by quantitative PCR. While relative levels of Snail, Twist, Rho and Sog observed an increase, the levels of Dpp (Decapentaplegic), a gene suppressed by dorsal, decreased in dElys depleted samples as compared to control (Fig.5J). To rule out the possibility of activated NF-B pathway under immune response, we checked for the expression of the anti-microbial peptide, Drosomycin in dElys depleted cDNA. We found that the expression of Drosomycin is unaltered, suggesting that activation of NF-B pathway does not have a microbial infection and immunity component to it (Fig.5J).
Dorsal targets Snail, Twist, Rho, Dpp, and Sog expresses early in development. We used third instar larval head complex for cDNA preparation, which is quite late in the development and still detected the expression of these molecules. Sustained expression of these molecules till late developmental stages might be the cause of apoptosis in dElys depletion which leads to developmental defects. Snail, Twist, Dpp, and Rho are already shown to be activators of the apoptotic response in Drosophila (Campbell et al., 2018;Gullaud et al., 2003;Lim and Tomlinson, 2006;Neisch et al., 2010). It is thus not surprising to find sustained expression of these molecules till late developmental stages resulting into induction of apoptosis asserting developmental defects in dElys depletion conditions.

Dorsal distribution to the nucleus is sustained in post-embryonic tissue and specific to dElys nucleoporin depletion:
Dorsal is one of the maternally contributed molecules which is required for early developmental progress (Belvin et al., 1995;Govind, 1999). Dorsal nuclear localization is affected in all ventral nuclei of developing embryos specifying dorsoventral polarity.
During early development, Dorsal after getting phosphorylated keeps shuttling between cytoplasm and nucleus under the influence of upstream stimuli (DeLotto et al., 2007;Perkins, 2006). The nuclear presence of Dorsal gradually decreases with progress in development beyond the embryonic stage particularly after zygotic induction and Dorsal remains in the cytoplasm through stable association with Cactus. We set out to investigate if nuclear localization of Dorsal a temporal event of later developmental stages or Dorsal entrapped in the nucleus even after the embryonic stage in the dElys knockdown organism. Firstly, we analyzed the dorsal levels in embryonic stages and noticed that there is no observable difference in the nuclear signal of Dorsal in dElys depleted and control embryos (Fig.6A, First vertical panels). We then subsequently looked for dorsal signals in salivary gland nuclei isolated from each successive larval stages. Similarly, no apparent difference was seen in the dorsal signals inside the nuclei isolated from first instar control and dElys depleted larval salivary glands (Fig.6A, second vertical panels). However, we noticed a gradual but steady decrease in nuclear Dorsal signal in embryonic and 1 st instar stage nuclei of control and dElys depletion conditions. While, dElys depleted salivary gland nuclei continued to have more Dorsal in 2 nd and 3 rd larval stages, control nuclei has only residual or no Dorsal (Fig.6A, third-fourth vertical panels). The sustained nuclear localization of Dorsal becomes apparent in the 3 rd instar larval salivary stage. (Fig.6A, third vertical panel). This observation finds a complementary decrease in cytoplasmic signal of Dorsal in the dElys depleted salivary glands. Quantification of nuclear Dorsal signal intensities from control and dElys depleted salivary gland nuclei further establish that Dorsal is retained in the nucleus during post-embryonic developmental stages upon dElys depletion (Fig.6B). Our results strongly suggest that dElys plays important role in shutdown of activated NF-B signaling during post-embryonic stages and its sequestration in the cytoplasm.
We next asked if this sustained nuclear localization of Dorsal is dElys specific or a consequence of lack of nuclear pore complexes upon dElys depletion. To check this, we investigated dorsal localization in salivary gland nuclei of critical nucleoporins Nup160, Nup133, Nup107, and Nup153. Members of the Nup107 complex play a key regulatory role in post-mitotic and interphase NPC assembly while Nup153 is a critical molecule for interphase NPC assembly only Vollmer et al., 2015;Zuccolo et al., 2007). When compared with control depletion Nup153 and Nup133 depletion show no significant difference in Dorsal signals inside nuclei as in control (Fig.6C). Although, Nup160 and Nup107 depletion cause lethality at the 1 st instar larval stage, the Dorsal intensities inside the nucleus were indistinguishable from those observed in the nuclei of 1 st instar larvae of wildtype organism (Fig. S7A). Quantitation of nuclear intensities of the Dorsal signal demonstrate that Dorsal signal inside nucleus increase by ~5 fold when dElys is depleted but the same remains largely unchanged upon depletion of other nucleoporins involved in NPC assembly ( Fig.6D and Fig.S7B). This highlights that sustained residence of Dorsal inside is dElys specific and not the outcome of the absence of NPCs in nuclear envelop.

dElys depletion-induced apoptosis is Dorsal mediated:
We next examined if apoptosis and nuclear localization of dorsal is an independent consequence of dElys depletion. We have created a genetic combination of dElys depletion in the Dorsal null background. However, this imparted lethality at 1 st instar larval stage hence was not useful. We decided to combine the knockdown of dElys and Dorsal and analyzed the apoptotic response from co-depleted salivary glands. dElys depletion induced accumulation of dorsal and enhanced punctate nuclear staining of Dcp-1, but the co-depletion of dElys and Dorsal showed a reduction in Dcp-1 and dorsal staining in salivary gland nuclei (Fig.7A). Quantitation of signal intensities further indicated that Dcp-1 signals return to normal when dElys and Dorsal are co-depleted ( Fig. 7B, C). This also highlighted the fact that induction of apoptosis is dorsal dependent. Moreover, RNAi line used for Dorsal knockdown does not deplete maternally contributed Dorsal and thus do not affect embryonic development.
We next decide to examine if apoptosis can be rescued by overexpressing DIAP-1 in the dElys knockdown. As ubiquitous overexpression of DIAP-1 is lethal, we used localized expression using tissue specific drivers. The salivary gland-specific dElys depletion elicited the increased Dcp-1 signal inside the nucleus, but the DIAP-1 overexpression in dElys depletion background brings the Dcp-1 levels back to normal and subsiding the apoptotic response (Fig.7B). Although the visualized intensity of Dcp-1 is different as compared to Fig.7A, Quantitation of Dcp-1 intensities verified that inhibition of apoptotic response was a consequence of DIAP-1 over-expression in dElys depletion background (Fig.7E). Our data strongly suggest that the induction of apoptosis upon dElys depletion is Dorsal mediated and the DIAP-1 over-expression counters the dElys depletion-induced apoptosis.

Discussion:
ELYS was identified as a putative transcription factor in mouse embryos and was also found in association with chromatin (Gillespie et al., 2007;Kimura et al., 2002).
Subsequently, target genes for ELYS, and cellular function of ELYS remained elusive for quite some time. Studies on ELYS later gained attention when it was found in stable association with a Nup107 complex of nuclear pores (Franz et al., 2007;Galy et al., 2006;Rasala et al., 2006). Nuclear pores present a rim staining around chromatin in interphase cells, and Nup107 complex gets recruited to kinetochores in mitosis.
Similarly, ELYS antibodies recognized specific nuclear rim reactivity in interphase cells and an overlapping kinetochore staining pattern was seen with Nup107 complex (Rasala et al., 2006). Later, it was established that ELYS recruits Nup107 complex to kinetochores and hence helps in regulation of Nup107 complex mediated microtubule formation at kinetochores (Gillespie et al., 2007;Mishra et al., 2010;Yokoyama et al., 2014). Importantly, ELYS binds with promoter regions in chromatin and interacts with Mcm-7 helicase to exert control during DNA replication as well as interacts with Piwi for transcriptional regulation (Gillespie et al., 2007;Ilyin et al., 2017). Loss of ELYS induces severe defects in organism development like skeleton loss in Zebrafish, embryonic lethality in mouse, tissue differentiation defects and aging phenotypes (Cerveny et al., Davuluri et al., 2008;de Jong-Curtain et al., 2009;Gao et al., 2011;Okita et al., 2004). Together, these observations suggested an important regulatory role for ELYS in organism homeostasis and development through a conserved underlying molecular mechanism.
Our study set out to address homeostatic roles for ELYS in Drosophila development.
We report that the Drosophila ELYS orthologue (dElys) encoded by CG14215 has conserved structural and functional elements of a canonical ELYS molecule. We demonstrate that dElys can efficiently bind to AT-rich DNA despite possessing a noncanonical AT-hook motif (Fig. 1). Expectedly, dElys showed nuclear rim staining in interphase which is characteristics of nucleoporins, however, in mitosis, it misses kinetochores and envelopes chromatin. High-resolution imaging for dElys observes discrete chromatin periphery localization absent from the kinetochores in mitotic cells (Fig. 2). This result is in striking contrast with its higher orthologue which showed kinetochore localization during mitosis (Gillespie et al., 2007;Mishra et al., 2010;Yokoyama et al., 2014). This observation concurs with the lack of Nup107 complex from kinetochores reported in Drosophila and provides a suitable explanation for the same (Katsani et al., 2008).
RNAi mediated ubiquitous knockdown of dElys, induced developmental defects and lethality in Drosophila (Fig. 3). The lethality, however became obvious during the later developmental stages (the 3 rd instar larval), and most probable explanation for this delay is the maternal contribution of dElys in embryos. Therefore, dElys depletion in eyes and wing tissues produced more pronounced morphological defects (Fig. 3).
Further, the loss of nuclear pores severely affecting nuclear morphology, and nucleocytoplasmic transport probably is the reason for the observed lethality (Aitchison et al., 1995;Emtage et al., 1997;Fabre and Hurt, 1997;Fernandez and Piano, 2006;Galy et al., 2006). Moreover, we report a conserved interaction between dElys and Lamin B receptor (LBR) which helps in the organization of the nuclear lamina (Clever et al., 2012;Mimura et al., 2016). Nuclear pore assembly after mitosis in Drosophila is a stage-wise process, and mature NPCs are seen largely in interphase. Importantly, different stages of NPC assembly become apparent during different stages of mitosis (Kiseleva et al., 2001). dElys knockdown adversely affects recruitment of various Nups representing different sub-complexes of NPC inducing a gross loss in NPC morphology (Fig. 4). Importantly, we report a novel observation that in addition to LBR, both Lamin B and C are also missing from the nuclear lamina (Fig. 4). LBR interacts with lamin B, and thus the loss of LBR upon dElys depletion may induce loss of lamins too. Moreover, the Ran-GTPase critically required for nucleo-cytoplasmic transport of protein cargoes shows redistribution from the nuclear periphery upon dElys depletion (Fig.4). The Ran-GTPase mislocalization in association with lack of NPCs and compromised nuclear lamina cause the severe nuclear transport defects of important regulatory molecules.
dElys is thus critical for nuclear pores, nuclear lamina organization, and nuclear import/export and together these are integral to the maintenance of cellular equilibrium.
Our search for developmentally important molecular players whose functionality is affected significantly and contributes to dElys phenotypes narrowed down to Dorsal (NF-B). Dorsal disengages from its inhibitor (IB) and accumulates inside nucleus in its activated form (Whalen and Steward, 1993). Induction of Dorsal signaling plays an important role in the growth and development of the organism (Espin-Palazon and Traver, 2016;Govind, 1999). In Drosophila, Dorsal is present inside the nucleus during early developmental stages or when microbial infection elicits an immune response. We observe for the first time that Dorsal is present inside the nucleus even beyond the early development stages, and without any obvious infection which activates death caspase-1 (Dcp-1) (Fig.5). We thus demonstrate that NF-B pathway activation upon dElys depletion independent of microbial infection and even when any evidence of the antimicrobial peptide synthesis is lacking. We further showed that early development targets of Dorsal, namely Snail, Twist, Rho, Dpp, and Sog are expressed even during 3 rd instar larval stage (Fig.5). The persistent and undesired activation of developmental players until the later stages of development might activate the checkpoint alarming the system of a misprogramming upon dElys depletion. The activated checkpoint can subsequently cease the development by inducing apoptosis. Our data corroborated this idea, and an increase in apoptosis was evident in dElys depleted salivary gland tissues, eye and wing imaginal discs (Fig. 5). We attribute critical roles to dElys in early development, and lack of dElys leads to persistent accumulation of activated Dorsal inside nucleus even during post-embryonic developmental stages (Fig.6). ELYS is known to target protein phosphatases to the nucleus, which might dephosphorylate and exports molecules back to the cytoplasm (Hattersley et al., 2016). It is prudent to suggest that a similar phosphatase may be regulated by dElys to control nucleocytoplasmic shuttling of Dorsal in Drosophila. It will be interesting to identify if such a phosphatase is involved in the dorsal shuttling process in Drosophila.

Our observation regarding Dorsal pathway activation is in direct contrast to what has
was shown for another nucleoporin mbo (Nup88) in Drosophila. Depletion of mbo neither affected nuclear import-export nor nuclear lamina assembly. More importantly, nuclear levels of activated Dorsal were reduced in response to infection when Nup88 is deleted (Uv et al., 2000). Nup88 (Genenncher et al., 2016) along with other nucleoporins like Nup62 (Yang et al., 2015), Nup153 and Nup214 (Xu et al., 2002) are shown to be critical in developmental pathways, but detailed analyses emphasizing on their involvement in selective nuclear translocation of activated signals. In contrast, we report sustained activation of NF-B signaling and its accumulation inside the nucleus only upon dElys depletion but not with other nucleoporins involved nuclear pore complex assembly. Our results highlight the key difference between dElys and other nucleoporins and affirm a role for dElys in developmental signaling in addition to NPC and nuclear lamina organization.
Dorsal has lately been reported for inducing apoptotic responses, and emerging ideas suggest that Dorsal, depending on cues, helps in growth versus apoptotic fate choices (Barkett and Gilmore, 1999). Further the activation of the pro-apoptotic function of NF-B is cell-type specific and signal-dependent (Silverman and Maniatis, 2001). The persistent activation and accumulation of NF-B upon dElys depletion could be a result of upstream signaling induced by yet an unidentified inducer. Our observations suggest a nuclear pore independent function of dElys which impinges on pro-apoptotic functions of NF--B. The apoptosis induction and lethality in imaginal discs and wing blade defects (cell death and atrophy) could be dorsal mediated apoptotic outcomes of dElys loss. The prolonged nuclear accumulation of Dorsal shifts balance towards apoptosis causing tissue death and accompanying developmental defects (Fig. 5). Cells may be resorting to the NF-B pathway dependent apoptosis as one of the ways to counter this defect and elimination of cells with abnormal nuclei produced upon dElys depletion.
Moreover, there may be additional signaling cascades that are perturbed by changes in dElys levels, which requires further elucidation.
We suggest that dElys levels help maintain a tight balance between pro-growth and proapoptotic responses to help achieve normal growth and development in Drosophila. dElys does regulate development and could contribute to normal growth in post-mitotic cells by keeping the NF-B trigger under control in a spatiotemporal manner (Fig. 6).
We present dElys levels as few examples which probably can activate NF-B pathway by intracellular cues in contrast to much appreciated NF-B pathway activation by extracellular stimuli for growth or infection (Gillespie and Wasserman, 1994;Silverman and Maniatis, 2001).
Our study is a novel report showing an intricate regulatory mechanism which cell relies on to ensure proper development in Drosophila. It also adds a new dimension in various roles played by the nucleoporin ELYS. dElys depletion showed constant activation of NF-B pathway in Drosophila, but how upstream signaling is continued its signals to this pathway, requires further detailed analysis. We speculate that various checkpoint like molecules present in the cell might be sensors for NPC and nuclear lamina assembly defects and activates regulatory pathways in accordance. We anticipate greater roles of nucleoporins in cellular homeostasis maintenance rather than being a mere structural component of NPC, which could be an intriguing question for further research.

In silico analysis:
An orthologue of ELYS in Drosophila was identified by using mouse ELYS as the reference sequence. PSI-BLAST with three iterations using mouse ELYS and its AThook motif sequence was used against the non-redundant protein database at NCBI with default parameters. ELYS orthologues in other organisms were identified by similar search in the NCBI database. T-coffee, multiple sequence alignment server was used for alignment (Notredame et al., 2000). The aligned sequence was then used for visualization in Jalview and highlighted with clustal X color scheme. The phylogenetic tree was inferred from this analysis using Jalview (Waterhouse et al., 2009). Protein sequence motifs were identified using the SMART database for the representative sequence from each organism (Schultz et al., 1998). IBS was used to draw scaled protein illustration for each protein (Liu et al., 2015). Percentage identity was calculated against mouse ELYS as a reference for each ELYS like molecules. Secondary structure for CG14215 was predicted using PSIPRED v3.3, DisoPRED and DomPRED (Jones, 1999). Predicted secondary structure for N-terminal and central helical domain were compared with the experimentally proven secondary structure of human and mouse ELYS (Bilokapic and Schwartz, 2013). 3D structure of NTD of CG1425 was modeled using a Phyre2 algorithm and mouse ELYS NTD structure as a template (Kelley et al., 2015).

Fly strains and genetics:
All flies were reared at 25°C on standard corn meal-yeast-agar medium. RNA interference crosses were grown at 28°C for better expression of GAL4. RNAi line (KK 103547) for CG14215 was obtained from the Vienna Drosophila Resource Centre (VDRC). Other fly lines used in this study were obtained from the Bloomington Drosophila Stock Centre (BDSC) at Indiana University. Controls used in this are F1 progeny from Driver line crossed with W 1118 flies. Fly lines used in this study are mentioned in table S1. All the combination and recombination are made with standard fly genetics. Tissue-specific knockdown of dElys was achieved by Act5C-GAL4, Ey-GAL4 and Wg-GAL4 drivers obtained from BDSC.

Cloning and Transgenic fly generation:
We obtained the clone LD14710 containing full-length CG14215 coding sequence in pBluescript SK (-) vector from Drosophila Genomics Resource Centre (DGRC). This clone was used as a template for cloning full-length CG14215 into pTVW (Vector. No. Odisha. Antibodies were affinity purified over purified antigen chemically cross-linked to N-hydroxysuccinimidyl-sepharose (NHS) beads (Sigma). Eluted with low pH, neutralized and dialyzed against PBS overnight at 4°C. Antibody against full-length dNup43 was also generated using identical protocol.

Immunostaining:
In vivo localization of dElys was revealed by Immunostaining of Drosophila embryos.
W 1118 staged syncytial blastoderm embryos were collected on apple juice agar plates for 4 hours at 25°C. Embryos were processed as mentioned by (Harel et al., 1989;Johansen and Johansen, 2004). Briefly, embryos were dechorionated in 50 % sodium hypochlorite until appendages from 80% of embryos disappear. Embryos were washed thoroughly in embryo wash buffer containing 0.2% NaCl and 0.05% Triton X-100 and fixed with 4% formaldehyde in heptane for 45 min at room temperature. Embryos were then devitellinized in 100% methanol for 30 min at room temperature. Embryos were blocked in 5% neutralized goat serum (Jackson Laboratories). Processed embryos were then used for Immunostaining with the anti-dElys antibody (1:1000) and mAb414 (1:500, Bio-legend). Embryos collected from GFP-Nup107 expressing fly line were processed identically. Embryos were mounted in vectashield mounting medium (Vector Laboratories).
Drosophila S2 cells were immunostained by immobilizing on 0.25 mg/ml concanavalin A coated coverslips for 2 hours at 25°C. MG-132 arrested cells were fixed by 100% methanol at -20°C, blocked with 5% neutralized goat serum (Jackson laboratories) and Airyscan inverted microscope. Images were processed for super-resolution in the inbuilt mathematical algorithm of Zen.2 software of LSM 800 Airyscan. Images were processed with ImageJ (NIH) or Fiji software and Adobe Photoshop CS6 (Adobe Corporation). Nuclei sizes were measured by using Fiji software and graph were plotted with GraphPad software (Prism).

Acridine Orange staining for apoptosis:
To examine the level of apoptosis in control as well as dElys knockdown salivary glands, dissected glands were incubated with 2 µg/ml acridine orange (Sigma) for 5 min at room temperature. Glands were imaged immediately without fixation with Carl Zeiss LSM 780 up-right confocal microscope (Denton et al., 2008;Sarkissian et al., 2014).
Fluorescence intensities of the nucleus were calculated using Fiji software. ~150 nuclei from three independent experiments were counted and the graph was plotted in GraphPad software (Prism).

Bright field microscopy:
For imaging of eye and wing phenotype of dElys knockdown flies, three days old flies were anesthetized with diethyl ether (Merck) and immobilized on sticky gum for proper orientation of Drosophila eye. At least 10 flies from each knockdown experiment were imaged from three independent experiments for each genotype. Drosophila eyes were imaged using Leica fluorescent stereo-microscope M205 FA using a 123X magnification of particular equipment. For wing imagining in case of wing phenotypes with a dElys knockdown, three-day-old fly wings from each genotype were dissected and placed on a glass slide under a coverslip and imaged directly under Leica upright light microscope DM2500 with 10X magnification (Velentzas et al., 2013). At least 15 pairs of wings from each genotype were examined from three independent crosses.

Scanning electron microscopy (SEM):
Drosophila eyes from eye-specific knockdown of dElys were imaged using a scanning electron microscope for detailed analysis of eye structure perturbations. Three days old flies from each RNAi cross was processed as per (Wolff, 2011). Briefly, flies were anesthetized and fixed with 2.5% glutaraldehyde in phosphate buffered saline for 2 hours at 4°C. Flies were washed thoroughly twice with PBS+ 4% sucrose. Flies were dehydrated through a graded ethanol series and were subjected to critical point drying.
Samples were mounted on aluminum stubs with carbon conductive tape. Flies were coated with gold particles in a sputter coating apparatus. Samples were imaged using Carl Zeiss Gemini II FESEM microscope. At least 10 flies from each genotype were imaged from three independent experiments.

In vitro DNA binding experiment:
To analyze DNA binding activity of AT-hook motifs of dElys, we purified fragment spanning all three AT-hook DNA binding motif by cloning C-terminal 104 amino acid For site-directed mutagenesis of arginine residues, we used Q5 site-directed mutagenesis kit (NEB Bio-labs) with primers coding mutated nucleotides. Primers used for mutagenesis were mentioned in Table S2. The clone described above was used as a template for site-directed mutagenesis. Mutations were confirmed using DNA sequencing (IISER-Bhopal sequencing facility). Clones were transformed into E.coli BL21 (DE3) cells and proteins were purified as mentioned earlier.
In vitro DNA binding experiment was performed by incubating template which contains 50% AT richness and 90% AT richness with purified wild-type and mutant proteins. A known cytosolic protein which does not binds to DNA as a negative control. DNA binding experiment was performed in binding buffer containing 100 mM Tris, 500 mM KCl, 10 mM DTT pH 7.5 for 30 min at room temperature and analyzed on to 0.8% agarose gel and detected by UV transilluminator (UVP). Purified DNA used for the binding experiment is 5 nM and total purified protein was used at 0.5 µM concentrations.
EMSA was performed by using Lightshift Chemiluminescent EMSA Kit (Pierce, Thermo Scientific) by using Sdic DNA oligos as described by (Metcalf and Wassarman, 2006).

Quantitative-PCR:
Total RNA was isolated from Control and dElys knockdown third instar larva head complex using total tissue RNA isolation kit (Favorgen Biotech). One µg of total RNA was used to synthesize cDNA using iScript cDNA synthesis (Bio-Rad). cDNA was diluted 5 times and 1 µl of cDNA from each genotype were used as a template for semiquantitative PCR using dElys specific primers and RpL49 as a control. Real-time PCR on the same cDNA was done in Roche Lightcycler 480 at standard cycling conditions and probed with SYBR-green (Bio-Rad) in real time using dElys and Actin RT primers.
(F) An antibody generated against dElys identifies a band of ~235 kDa in Drosophila third instar larva head lysate in control. RNAi mediated depletion of dElys is reflected as a decrease in band intensity. α-tubulin was used as loading control.