On subcellular distribution of the zinc finger 469 protein (ZNF469) and observed discrepancy in the localization of endogenous and overexpressed ZNF469

Abstract

The zinc finger 469 gene (ZNF469) is a single-exon gene predicted to encode a protein of 3953 amino acids. Despite pathogenic ZNF469 variants being associated with Brittle Cornea Syndrome (BCS), relatively little is known about ZNF469 beyond its participation in regulating the expression of genes encoding extracellular matrix proteins. In this study, we examined the expression and intracellular localization of ZNF469 in different cell lines. The level of ZNF469 mRNA varied from low levels in HEK293 cells to high levels in HeLa cells and primary fibroblasts. Antibodies against ZNF469 reacted among others with a protein of approximately 400 kDa in immunoblot analysis, which was mainly present in the insoluble fraction of the cytoplasm. Immunofluorescence analysis of interphase cells showed small cytoplasmic puncta and weak nuclear staining. In dividing HeLa cells, the antibodies recognized foci that also stained for proteasomes. In transfected cells, ZNF469 was observed mainly in foci resembling nuclear speckles in interphase and at the midbody during mitosis. The nuclear foci showed overlapping staining with proteasomes. In live cell imaging, liquid-like properties of the nuclear foci were recorded as they changed shape and position and occasionally fused with each other. During stress granule formation, cytoplasmic foci showed overlapping staining with G3BP1. Finally, in silico analysis revealed large intrinsically disordered regions with multiple low complexity domains in ZNF469. Our data indicate that ZNF469 forms aggregates possibly as biomolecular condensates when overexpressed. However, care must be taken when analyzing the intracellular distribution of ZNF469 due to the discrepancy in the localization of endogenous ZNF469 and overexpressed ZNF469 in transfected cells.

Keywords: biomolecular condensates; intrinsically disordered regions; midbody; nuclear speckles; proteasomes; stress granules.

Fig. 1

ZNF469 RNA and protein expression levels were measured by quantitative PCR (A) and immunoblot analysis (B) of ZNF469 in different cell lines. (A) After amplification with TaqMan probes, quantitative PCR results were analyzed using the ∆∆Ct method, normalizing the ZNF469 levels to the reference gene HPRT1. Results were obtained from a total of 9 replicates for each cell line. Error bars indicate SD. (B) The HiMark molecular weight standard is shown to the left. Representative blots from three biological replicates are presented.

Fig. 2

(A–D) HEK293 cells transfected with plasmids expressing a ZNF469‐AcGFP fusion protein (green) were labeled with antibodies against ZNF469 (red). Nuclei are shown in blue. An overlay with double labeled nuclear speckles in shown in yellow. (E) HEK293 cells transfected with a plasmid expressing ZNF469 without the AcGFP tag incubated with antibodies against ZNF469 (red). The experiments were performed in three biological replicates with similar results. Scale bar: 5 μm.

Fig. 3

Intracellular localization of ZNF469 in interphase cells. (A) Subcellular fractionation of HeLa cells. Immunoblot showing endogenously expressed proteins detected by anti‐ZNF469 antibodies. C, soluble cytosolic fraction; M, insoluble cytosolic fraction; N, nuclear fraction; WCL, whole cell lysates. HP1α was used as marker for the nuclear fraction while β‐tubulin and Na⁺/K⁺‐ATPase were used as markers for the soluble and insoluble cytosolic fraction, respectively. The blots represent examples from three biological replicates. (B–D) Immunofluorescence analysis using anti‐ZNF469 antibodies (green) and antibodies against β‐tubulin (red) in primary fibroblasts from a healthy individual (B), HEK293 cells (C), and HeLa cells (D). Similar results were obtained in three biological replicates. Scale bar: 5 μm.

Fig. 4

Double labeling immunofluorescence analysis of dividing HeLa cells (A–E). HeLa cells were incubated with antibodies against ZNF469 (green) and 19 S proteasomes (red) (A–D) or β‐tubulin (red) (E). (A and B) Metaphase. (C–E) Telophase (abscission). (D) Enlarged image from (C). Arrows in (B) and yellow‐white staining in (C and D) indicate overlapping staining for ZNF469 and 19 S proteasomes. Example images from three biological replicates are shown. Scale bar: 5 μm.

Fig. 5

HeLa cells transfected with plasmids expressing the ZNF469‐AcGFP fusion protein (green) and labeled with antibodies against 19 S proteasome (red). Nuclei are shown in blue. White signal indicates overlapping staining of ZNF469‐AcGFP and 19 S proteasomes. The experiment was repeated twice, each in three technical replicates. Scale bar: 5 μm.

Fig. 6

Expression of ZNF469 during stress granule formation in wt U2OS cells transfected with ZNF469‐AcGFP (green). Anti‐G3BP1 antibodies were used to detect the stress granule marker G3BP1 (red). Stress modes included sodium arsenite (SA), clotrimazole (CZ), MG132, and sorbitol. Arrows indicate stress granules with overlapping staining of ZNF469 and G3BP1. All experiments were conducted in three technical replicates. Scale bar: 5 μm.