Bicc1 Forms a Complex with Polycystin-1 and Polycystin-2.

Full-length and deletion myc-tagged constructs of Bicc1 were co-expressed with either full-length HA-tagged PC1 or PC2 in HEK-293 cells and tested for their ability to interact by co-IP. (a) Schematic diagram of the constructs used in this experiment. (b) Western blot analysis following co-IP experiments, using GST tagged constructs as bait, identified protein interactions between PC1 or PC2 domains and Bicc1. pcDNA3 was included as a negative control. CT = C-terminus, NT = N-terminus, GST = Glutathione S-Transferase. Co-IP experiments (n=3) were quantified in (c). (d) Western blot analysis following in-vitro pulldown experiments, using purified GST tagged constructs as bait, identified direct protein interactions between PC1 or PC2 domains and in vitro translated myc-Bicc1. In-vitro binding experiments (n=3) were quantified in (e). (f) Western blot analysis following co-IP experiments, using a rabbit PC1 antibody (2b7) as bait, identified protein interactions between endogenous PC1 and BICC1 in UCL93 cells. A non-immune rabbit IgG antibody or no antibody was included as a negative control; * denotes a non-specific IgG band. (g) Western blot analysis following co-IP experiments, using an anti-mouse Bicc1 or anti-goat PC2 antibody as bait, identified protein interactions between endogenous PC2 and BICC1 in UCL93 cells. Non-immune goat and mouse IgG was included as a negative control.

Interactions between Bicc1 and Polycystin1/2 Require Different Binding Motifs.

Full-length and deletion myc-tagged constructs of Bicc1 were co-expressed with either full-length HA-tagged PC1 or PC2 in HEK-293 cells and tested for their ability to interact by co-IP. (a) Schematic diagram of the constructs used in this set of experiments with the amino acid positions of full-length Bicc1 or the different deletions indicated. (b,c) Western blot analysis following co-IP experiments, using a PC1-HA-tagged construct as bait, identified protein interactions between PC1 and Bicc1 domains. pcDNA3 was included as a negative control (b). co-IP experiments (n=3) were quantified in (c). (d,e) Western blot analysis following co-IP experiments, using a PC2-HA tagged construct as bait, identified protein interactions between PC2 and Bicc1 domains (d). pcDNA3 was included as a negative control. Quantification of the co-IP experiments (n=3) is shown in (e). (f, g) Western blot analysis following co-IP experiments, using a PC1-HA-tagged construct as bait. The interaction between PC1 and PC2 was not altered in the presence of either full-length Bicc1 or its deletion domains. pcDNA3 was included as a negative control. Asterix represents non-specific interaction with mouse IgG. (f). co-IP experiments (n=3) were quantified in (g). One way ANOVA comparisons were performed to assess significance and P values are indicated. Error bars represent standard error of the mean.

Cooperativity of Bicc1 and PKD Genes in Xenopus.

(a-d) mRNA expression of Pkd1, Pkhd1, Pkd2 and Bicc1 in the Xenopus pronephros at stage 39. (e-i”) Knockdown of Bicc1 (f-f”), Pkd1 (g-g”), Pkd2 (h-h”) and Pkhd1 (i-i”) by antisense morpholino oligomers (MOs) results in a PKD phenotype compared to uninjected control Xenopus embryos (e-e”). The phenotype is characterized by the formation of edema due to kidney dysfunction (e,f,g,h,i; stage 43), the development of dilated renal tubules (e’,f’,g’,h’,i’; stage 43) and the loss of Nbc1 in the late distal tubule by whole mount in situ hybridizations (arrowheads in e”,f”,g”,h”,i”; stage 39). (j,k) To examine cooperativity, Xenopus embryos were injected with suboptimal amounts of the MOs, either alone or in combination, and analyzed for edema formation at stage 43 (j) and the expression of Nbc1 at stage 39 (k). More than three independent experiments were analyzed. In (k), gray bars show reduced and black bars show absent expression of Nbc1 in the late distal tubule. Data are the accumulation of multiple independent fertilizations with N>30 for each condition.

Cooperativity of Bicc1 and Pkd1 and Pkd2 in Mouse.

(a-c) Bicc1 and Pkd2 interact genetically. Offspring from Bicc1;Pkd2 compound mice at postnatal day P4 and P14 are compared by outside kidney morphology at postnatal day P14 (a), and kidney to body weight ratio (KW/BW) at P14 (b) and P4 (c). (d-g) Bicc1 and Pkd1 interact genetically. Bicc1;Pkd1 compound mice are compared by outside kidney morphology at P14 (d), estimation plot of KW/BW ratio comparing littermates at P14 with a P-value = 0.092 (e), and cystic index of proximal tubules (PT) and collecting ducts (CD) at P7 (f) and P14 (g). Two-sided paired t-tests were performed to assess significance and the P-values are indicated; error bars represent standard deviation. (h-k) qRT-PCR analysis for Bicc1, Pkd1, and Pkd2 expression (h-j) and quantification of the PC2 expression levels by Western blot (k) in kidneys at P4 before the onset of a strong cystic kidney phenotype. Data were analyzed by t-test and the P-values are indicated.

Identification of Human BICC1 Variants.

(a-c) BICC1 p.G821E/PKD2 p.C632R case with pedigree and the electropherograms (a), the structural analysis of the PKD2 showing the local structure around the cysteine at position 632 (indicated in red) and its putative impact in the variant including the REVEL score (b) as well as its location within the global PC2 structure highlighting the potential of the variant impacting the PC2 ion channel function (c). (d,e) Western blot analysis for PC2 comparing wildtype HEK293T, BICC1 G821E, BICC1 S240P and BICC1 KO HEK293T and quantification thereof. γ-Tubulin was used as loading control. (f-i) BICC1 I1179+1G>T/PKD1 PKD1 p.Ala3981Val case with pedigree and the electropherograms (f), the ultrasound analysis of the left and right kidneys (g,h) and the structural analysis of the PC1 showing the local structure around the alanine at position 3981 (indicated in red) and its putative impact in the variant including the REVEL score (i).

The Homozygous BICC1 S240P Mutation is a Hypomorphic Cystic Disease-Causing Variant.

(a-e) Consanguineous multiplex pedigree with two siblings affected by VEO-ADPKD identified the homozygous BICC1 missense variant c.718T>C (p.S240P) absent from gnomAD and other internal and public databases. Electropherogram is shown in (a). The affected girl presented at a few months of age with renal failure and enlarged polycystic kidneys that lacked corticomedullary differentiation (c). Histology after bilateral nephrectomy showed polycystic kidneys more suggestive of ADPKD than ARPKD without any dysplastic element. Her younger brother exhibited enlarged hyperechogenic polycystic kidneys prenatally by ultrasound (b). In addition, in his early infancy arterial hypertension and a Dandy-Walker malformation with a low-pressure communicating hydrocephalus were noted (d,e). (f) Ribbon diagram and schematic diagram of BICC1 contains KH, KHL and SAM domains showing the variants discovered. (g) Solid boxes correspond to local impacts of p.S240P on BICC1 structure, interactions are labeled as dashed lines (pseudobonds). GXXG motifs colored in magenta, representative missense variant residues colored in red and residues adjacent to selected variant (<5Å) colored in tan. (h) Rescue experiments of Xenopus embryos lacking BicC1 by co-injections with the wild type or mutant constructs. Embryos were scored for the re-expression of Nbc1 in the late distal tubule by whole mount in situ hybridizations. Quantification of at least 3 independent experiments is shown. (i,j) HEK293T cells were transfected with Flag-tagged constructs of wild type or mutant Bicc1 and the subcellular localization of Bicc1 was visualized (red). Nuclei were counterstained with DAPI (blue). (k) Protein stability analysis using tetracycline-inducible HEK293T cells comparing the expression levels of Bicc1 and Bicc1(S240P) 24 hours after removal of tetracycline and addition of cycloheximide. γ-Tubulin was used as loading control. The percentage of protein destabilization because of protein synthesis inhibition by cycloheximide is indicated. (l) Western Blot analysis of wildtype HEK293T, cells lacking BICC1 (BICC1-/-) and isogenic cells with the BICC1 S240P mutation for PC2 expression. GAPDH was used as loading control. (m,n) Bar graph of the mRNA-seq transcriptomic analysis comparing BICC1 wildtype, knockout and S240P isogenic HEK293T cells showing the eight most significantly upregulated transcripts (based on their Padj levels) in the BICC1 KO cells (m). For each gene, the normalized expression levels from each of the 6 samples (2 wildtype, KO and 240P each) is shown. (n) GSEA plot showing the enrichment of the Hallmark Epithelial_Mesenchymal_Transition data set in the BICC1 KO cells vs. the BICC1 S240P cells.