1. dKeap1 and Lamin co-localize and form protein complexes in the nucleus
Lamin Dm0 (also named as “Lamin” in Flybase, stated as “Lamin” in this article) is the major type of lamin protein that forms the nuclear lamina in most of the Drosophila tissues. To explore the molecular function of dKeap1 in heterochromatin architecture, we tested whether dKeap1 associates with Lamin. We first visualized the subcellular localization of dKeap1 and Lamin in salivary gland cells via co-immunostaining (Fig. 1A). Although most of the dKeap1 immunosignals were in the nucleoplasm, co-localization of dKeap1 and Lamin signals were detected at the nuclear lamina, indicating that some dKeap1 proteins localize to the nuclear lamina.
We next visualized the potential formation of dKeap1-Lamin complexes in the Drosophila S2 cell line using the bimolecular fluorescence complementation (BiFC) assay (Hu et al, 2002). In the BiFC assay, the N-terminus and C-terminus of YFP (YN and YC) were fused to dKeap1 and Lamin, respectively (Fig. 1B). Fluorescence signals representing BiFC complexes were detected in nuclei of S2 cells that co-expressed YN-dKeap1 and YC-Lamin (Fig. 1B). As a negative control, no BiFC signal was detected in S2 cells that expressed BiFC fusions for dKeap1 and a cardiac transcription factor Tinman (Liu et al. , 2009). Therefore, dKeap1 and Lamin can form specific complexes in the nucleus.
2. Ectopic dKeap1 re-distributes Lamin and heterochromatin
dKeap1-Lamin BiFC complexes were detected mainly in the nucleoplasm of S2 cells (Fig. 1B). This is in contrast with the peripheral localization of endogenous Lamin in the nucleus (Fig. 1A). We hypothesized that the mis-localization of dKeap1-Lamin BiFC complex was caused by the overexpression of dKeap1 fusion proteins. To test this possibility, we overexpressed the UAS-controlled dKeap1 full length (FL) fusion protein (Fig. 2A) in salivary gland cells using Sgs3-GAL4 (Cherbas et al. , 2003). Significant amounts of Lamin proteins were detected in the nucleoplasm, with a pattern partially overlapping with dKeap1 fusion proteins (Fig. 2B). The levels of Lamin proteins were not altered upon dKeap1 overexpression (Fig. 2C). Therefore, overexpression of dKeap1 proteins caused a redistribution of Lamin proteins from the original peripheral sites to the center area of the nucleus.
A similar Lamin redistribution phenotype was also observed in salivary gland cells that overexpressed YFP-dKeap1-∆Kelch, a dKeap1 truncation lacking the CncC-interacting Kelch domain (Fig. 2A,B). The signals of YFP-dKeap1-∆Kelch were mainly detected in the nucleus. As this truncation induced the same effects as YFP-dKeap1-FL did, we concluded that the dKeap1-CncC interaction is not involved in the relocation of lamina by ectopic dKeap1. Expressing dKeap1 N-terminal deletion (YFP-dKeap1-∆NTD) or C-terminal deletion (YFP-dKeap1-∆CTD) had no or only moderate effect on Lamin distribution. YFP-dKeap1-∆CTD localized almost exclusively to the cytoplasm (Fig. 2B) (Carlson et al., 2022). YFP-dKeap1-∆NTD localized to both the cytoplasm and nucleus, and the ratio of different portions varied in different cells (Fig. 2B). The nuclear accumulation of dKeap1-∆NTD had no significant effect to the Lamin distribution, indicating that the N-terminal domain of dKeap1 is required for the relocation of Lamin. The expression levels of all the fusion proteins were comparable and none of them altered Lamin protein levels (Fig. 2C; Fig. 3B), indicating that ectopic dKeap1 proteins induced Lamin re-distribution rather than altering Lamin protein levels.
Intra-nuclear distributions of lamin proteins have been found in embryonic stem cells and some adult stem cells, presumably associated with a distinct global chromatin architecture in these cells (Dorland et al. , 2019, Meshorer and Misteli, 2006). Intra-nuclear lamins and abnormal nuclear lamina are also revealed in aging cells (Scaffidi and Misteli, 2006). Overexpression of dKeap1 fusion proteins cannot fully rescue the viability and fertility of the dKeap1 null mutants (Carlson et al., 2022). We hypothesize that this developmental defect is associated with the dKeap1-induced relocation of Lamin and the consequent mis-organization of heterochromatin.
To test this hypothesis, we investigated the localization of heterochromatin marker histone H3K9me2 in salivary gland cells. In wildtype cells, H3K9me2 is found at the chromocenter, the pericentric heterochromatin region on the polytene chromosome (Fig. 2D) (Zhang et al. , 2006). Significant relocations of H3K9me2 immuno-signals to loci outside of the chromocenter were detected in nuclei with dKeap1 overexpression (Fig. 2D, Fig. S1A). dKeap1 overexpression had no effect on the level of H3K9me2 (Fig. 2E), suggesting that dKeap1 overexpression caused a spreading of H3K9me2 from the pericentric region to chromosome arms. The relocation of H3K9me2 is likely caused by ectopic dKeap1 and Lamin accumulation in the center area of the nucleus. On the other hand, dKeap1 knockout reduced the level of H3K9me2 (Fig. 2E), consistent with our previous finding (Carlson et al., 2019). All these results support a positive role of dKeap1 in heterochromatin formation and/or maintenance.
3. Mis-regulated dKeap1 proteins disrupt nuclear lamina
Severely defected nuclear lamina morphologies were observed when the dKeap1 fusion proteins were expressed in a dKeap1 null background. All the truncations, when expressed in the dKeap1 null background, caused dramatic Lamin redistribution to the nucleoplasm (Fig. 3A). Interestingly, expression of dKeap1-FL, ∆NTD or ∆CTD altered the morphologies of nuclear laminas and the shapes of nuclei (Fig. 3A), regardless of whether the fusion proteins were primarily in the nucleus or cytoplasm. In around 30% of cells that expressed ∆CTD, nuclear shapes and organizations were the most severely affected. These cells showed partial nuclear fragmentation and potential breakdown of the nuclear envelope as indicated by the invading of ∆CTD fusion proteins from the cytoplasm into the nucleus (Fig. 2B; Fig. 3A). The re-distributed Lamin and defected nuclear lamina were also seen in other cell types such as the diploid follicle cells and polyploid nurse cells in ovaries (Fig. S1B).
The lack of native dKeap1 proteins in the dKeap1 null background should account for the severe Lamin defects induced by ectopic dKeap1 fusions. However, no significant Lamin defect was detected in cells of the dKeap1 null larvae (Fig. S1C). Intrinsic dKeap1 proteins localized to both the nucleoplasm and the nuclear lamina (Fig. 1A). However, none of the dKeap1 fusion proteins showed localization to the nuclear lamina (Fig. 2B), indicating that the YFP-dKeap1 fusions cannot function the same as intrinsic dKeap1 proteins in the nuclear lamina. In support of this, overexpression of YFP-dKeap1-FL largely but cannot fully rescue the viability and fertility of the dKeap1 null mutant (Carlson et al., 2022). It is possible that the YFP tag interferes with the interaction of dKeap1 and Lamin. Taken together, we concluded that the disrupted Lamin morphology is a combinatory effect of both the ectopic expression dKeap1 fusion proteins and the lack of endogenous dKeap1 proteins. Since that the dKeap1-∆CTD induced the worst nuclear lamina disruption, the C-terminal domains of dKeap1 may play the most significant role in the maintenance of a normal nuclear lamina shape.
The lamin defects induced by mis-regulated dKeap1 is comparable but different to several other lamin phenotypes that have been reported. In Drosophila, reduction of a WAS family protein wash in salivary gland cells results in patternless strands of Lamin in the nucleoplasm (Verboon et al. , 2020). Overexpression of Lamin or an inner nuclear membrane protein Kugelkern result in “blebs”, “invagination” and “lobulation” of nuclear envelopes in different cell types (Brandt et al. , 2008, Polychronidou et al. , 2010, Uchino et al. , 2017). In mammalian cells, mutations in Lamin A or Lamin B1 lead to “blebs”, “donuts”, and “honeycomb” defects of nuclear lamina (Jung et al. , 2013, van Tienen et al. , 2019). Compared to these phenotypes, the honeycomb-like lamina defect seen in our study shows much larger “holes” throughout the entire nucleus. We concluded that dKeap1 is directly involved in the maintenance of nuclear lamina morphology.
4. Molecular interaction between dKeap1 and Lamin
We examined the molecular interaction of dKeap1 and Lamin using the co-immunoprecipitation assay. Lamin immunoblotting signal was detected in the anti-GFP precipitation from embryos that expressed YFP-dKeap1 (Fig. 4A), indicating that the overexpressed dKeap1 fusion proteins interacts with Lamin. The antiserum against dKeap1 also co-precipitated Lamin proteins from the lysate of wildtype embryos (Fig. 4B), suggesting that endogenous dKeap1 and Lamin form protein complexes in vivo.
To examine whether dKeap1 and Lamin directly interact with each other, the in vitro GST-pull down assay was conducted (Fig. 4C). GST-dKeap1 and GST-Lamin were expressed in and purified from E. coli. dKeap1 and Lamin proteins were generated by removal of the GST tag using protease. Both GST-dKeap1 and GST-Lamin were able to pull down Lamin and dKeap1, respectively (Fig. 4C). Therefore, dKeap1 and Lamin physically interact with each other.
The mechanism by which dKeap1 modulates chromatin structure in cooperation with Lamin remains to be elucidated. Lamin proteins can directly bind nucleosomes and can also regulate chromatin through interactions with other proteins (Shevelyov and Ulianov, 2019, Towbin et al., 2013). dKeap1 can bind to the euchromatin polytene chromosome arms using the C-terminal tail (Carlson et al., 2022). It is possible that dKeap1 interacts with heterochromatin through Lamin. It is also possible that Lamin controls chromatin structure through dKeap1 chromatin binding. We have found that CncC, the key interaction partner of dKeap1, also facilitates heterochromatin formation (Carlson et al., 2019). The molecular and biological functions of CncC in the dKeap1-Lamin complex and pathway remain to be explored.
5. dKeap1 functions downstream of Lamin in the genetic pathway
Given that dKeap1 molecularly interacts with Lamin and regulates Lamin localization, we hypothesized that dKeap1 and Lamin coregulate transcription. To determine if dKeap1 and Lamin regulate gene expression in the same developmental pathway, we explored potential genetic interactions between dKeap1 and Lamin. Lamin overexpression causes early lethality in Drosophila (Munoz-Alarcon et al. , 2007). In our experiments, larvae that overexpressed Lamin driven by tub-GAL4 died at L1 or early L2 larval stage (Fig. 5A). Double mutants which contained overexpressed Lamin and a heterozygous dKeap1 null allele survived to late L2 or early L3 stage (Fig. 5A), indicating that reduction of dKeap1 was able to partially rescue the lethality caused by Lamin overexpression. Excessive Lamin proteins could cause lethality via mis-regulation of developmental transcription. These results suggest that dKeap1 can act down-stream of Lamin in the regulation of gene expression during Drosophila development.
Mutations in lamin Dm0 show reduced viability, abnormal tissue differentiation, and defects in fertility, ovary size, ventriculus, and locomotion (Lenz-Bohme et al. , 1997, Munoz-Alarcon et al., 2007, Osouda et al., 2005). A dKeap1 mutant with disrupted dKeap1 chromatin binding also shows reduced viability and fertility (Carlson et al., 2022). It would be interesting to determine the developmental genes and programs that are coregulated by dKeap1 and Lamin in the future.