Nuclear translocation of vitellogenin in the honey bee (Apis mellifera)

Abstract

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used cell fractionation, immunohistology, and cell culture to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggest a new fundamental regulatory role for this ubiquitous protein.

Supplementary information: The online version contains supplementary material available at 10.1007/s13592-022-00914-9.

Keywords: DNA binding; Vitellogenin; nuclear protein.

Figure 1.

Western blot of cytosolic and nuclear fractions of honey bee fat body tissue. St. size standard. The full-length (180 kDa) Vg is dominating the cytosolic fraction of the fat body proteins, whereas the 40-kDa N-sheet mostly localizes in the nuclear fraction. Also, other fragments of approximately 70 and 25 kDa were visible in the nuclear fraction. Three individuals were pooled for each lane. N = 3.

Figure 2.

Localization of V signals in the honey bee fat body using an antibody targeting the Vg N-sheet. A–C Confocal images of fat body cells representing six biological samples. The left panel depicts the nuclear stain DAPI, the middle panel depicts Vg N-sheet signal via binding of the secondary antibody (Alexa 568), and the right panel depicts the superposition of DAPI (cyan) and Vg N-sheet signal (red). A Vg N-sheet signal co-localizes with DAPI in cell nuclei. B Zoom-in of a single cell showing Vg N-sheet nuclear translocation. C Cells that do not show Vg N-sheet co-localization with DAPI. Instead, Vg signal is found in granules in the cytosol. The scalebar = 10 µm, and magnification = 40 × objective. Control staining images (no primary antibody) are displayed in S2.

Figure 3.

Insect ovarian HighFive cells cultured with honey bee Vg. HighFive cells were incubated with purified honey bee Vg labelled with an Alexa 488 fluorophore. After 1 h incubation, Vg was observed both in the cytosol as bright granules and in the nucleus as a haze, indicating uptake of Vg into the nucleus. Scalebar = 5 µm, and magnification 63 × objective. Representative negative controls (samples not incubated with Vg) are displayed in S3.

Figure 4.

Genomic distribution of observed Vg N-sheet-DNA binding sites compared with a null distribution. The null distribution was created by randomly shuffling 782 non-overlapping ChIP-seq peaks for 1000 iterations. We found more Vg N-sheet-DNA binding sites than expected by chance in promotor regions (χ² = 175.18, P = 5.47E − 40), transcription termination sites (χ² = 91.68, P = 2.09E − 24), and intergenic regions (χ² = 46.14, P = 1.10E − 11), and fewer than expected by chance in exons (χ² = 3.75, P = 0.05) and introns (χ² = 244.55, P = 4.01E − 55).

Figure 5.

Logo representations for the top motif predictions. A Top motif prediction from analysis with “large” motif setting (35.75% enrichment). B Top motif prediction from analysis with “medium” motif setting (34.5% enrichment). C Top motif prediction from analysis with “xl” motif setting (33.1% enrichment).

Figure 6.

The 3D reconstruction of the honey bee Vg reveals the exposure of the cutting site. A Different views of the 3D reconstruction of the Vg from honey bee. The four on the left are orthogonal side views of the volume, whereas the two on the right correspond to the two end-on views of the 3D reconstruction. Bar indicates 100 Å. B The same views with the atomic structure of lipovitellin from lamprey (pdb 1lsh) docked into the EM 3D reconstruction. The N-sheet domain, which protrudes from the main body of the structure, is highlighted in orange. The domain colored red points to the linker that connects the N-sheet domain to the lipid cavity (yellow mass).