Grand K et al. (2023), HNF1B Alters an Evolutionarily Conserved Nephro...

XB-ART-59533

J Am Soc Nephrol 2023 Mar 01;343:412-432. doi: 10.1681/ASN.2022010076.

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HNF1B Alters an Evolutionarily Conserved Nephrogenic Program of Target Genes.

Grand K , Stoltz M , Rizzo L , Röck R , Kaminski MM , Salinas G , Getwan M , Naert T , Pichler R , Lienkamp SS .

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SIGNIFICANCE STATEMENT: Mutations in hepatocyte nuclear factor-1 β ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis. BACKGROUND: Hepatocyte nuclear factor-1 β (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown. METHODS: Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo . RESULTS: HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b . CONCLUSIONS: HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: a1cf cdh16 cdh6 cdhr5 emx2 esrrb foxc1 fxyd2 gal3st1 gata3 hnf1a hnf1b hnf4a lama5 lhx1 lrp2 nherf4 pax8 pou3f3 ppp1r16b sall1 slc12a1 slc38a3 slc3a1 slc45a2 slc5a1 slc7a9 sord tfap2b vegfa wt1
GO keywords: kidney development [+]
???displayArticle.antibodies??? Atp1a1 Ab3 Hnf1b Ab3
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gRNAs referenced: hnf1b gRNA1 slc45a2 gRNA8 sord gRNA1

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	Figure 1: Renal tissue induction in Xenopus ectodermal explants and mouse embryonic fibroblasts using transcription factors. (A) Schematics of the induction of pronephric tissue from Xenopus animal caps. Induction of pronephric tissue from animal caps treated with retinoic acid (RA) and activin A (ACT) has been previously shown by Moriya et al. (B) Results of induction efficiency using 13 renal organogenesis transcription factors and RA+ACT treatment compared with untreated (uninj.). Explants with a positive signal for the proximal tubule marker (3G8) are illustrated in the images underneath. (C) In situ stainings of distal (slc12a1/Nkcc2) and proximal (slc5a1/Sglt1) renal markers in explants induced by expression of Hnf1a, Hnf1b, Sall1, or all three factors combined compared with RA+ACT and uninjected. (D) Patient-specific mutations and the postnatal renal phenotypes illustrated on the HNF1B gene/protein schematic. (E) Schematics of the reprogramming workflow. Mouse embryonic fibroblasts were directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors (TFs)—Emx2, Hnf4a, Pax8, and HNF1B. To investigate HNF1B and its clinically relevant mutations, reprogramming with mutated HNF1B was conducted. (F) Reprogramming efficiency with patient-specific mutations. (G) Induction of pronephric tissue from animal caps with HNF1B WT and HNF1B R295C mRNA injections. (H) Proximal (3G8), distal, and connecting tubule marker (4A6) stainings in explants and a Xenopus tadpole of the same age. Positive stainings are highlighted with an arrow. Mean percentage of explants showing staining for renal markers in uninjected (n=99), injected with HNF1B WT (n=61), and explant injected with HNF1B R295C (n=91). Error bars, SEM; asterisks indicate significant differences to the positive control (uninj (B), 0TF (F) and uninjected (H)) as assessed by pairwise t-tests with corrections for multiple testing (Bonferroni), biological replicates n = 3–5 (B), n=3 (F), n=7 (H), **P<0.0001, P<0.001, P<0.01, P<0.05. All scale bars are 200 µm.
	Figure 2: Profiles of cells reprogrammed with HNF1B WT and HNF1B R295C. (A) Immunofluorescence stainings of HNF1B WT and R295C iRECs grown in 3D Matrigel for epithelial markers. Merged images consist of iRECs membrane GFP (green), target protein (red), and Hoechst (blue) signals. All scale bars are 50 µm. (B) Heatmap (left) of all the genes with significantly changed expression in at least one of the comparisons (MEFs versus 3TF+HNF1B WT, MEFs versus 3TF+HNF1B R295C, 3TF+HNF1B WT versus 3TF+HNF1B R295C) in mouse fibroblast reprogramming (N=12,742), and heatmap (right) of all the genes significant in at least one of the comparisons (uninjected versus HNF1B WT, uninjected versus HNF1B R295C and HNF1B WT versus HNF1B R295C) in explant inductions (N=16,595). (C) Venn diagrams of significantly differentially expressed (DE) up- and downregulated genes between comparisons in mouse reprogramming (blue) and explant induction (gray). (D) GO over-representation test on DE genes (absolute log2 fold change >1.5) from reprogramming experiments comparing 3TF+HNF1B WT iRECs with MEFs (top) and HNF1B WT pronephric tissue with uninjected animal caps (bottom). GO terms related to renal organogenesis are marked in bold. The same analysis for the R295C mutation condition is showed in Supplemental Figure 2A.
	Figure 3: Transcriptional alterations due to HNF1B patient-specific mutation R295C. (A) Clustering DE genes according to expression patterns. Schematics represent the eight expected expression patterns while line graphs show the expression changes in the mouse transcriptome data. (B) Pie chart of the distribution of the genes in all clusters (4201 genes in total). (C) Heatmaps of top 20 most variable (variance to the mean of the cluster) genes within each cluster. (D) Comparing clusters on the GO biological process level and visualizing top three processes per cluster (full plot in Supplemental Figure 3A). (E) Percentages of genes per cluster with 1, 5, or 10-kilo base pair (K) distance from the calculated transcription starting site (data from ChIP-Seq atlas78).
	Figure 4: Changes in transcription factors and genes related to kidney disease. (A) Expression patterns of transcription factors involved in kidney disease. Line graphs represent mean expression changes in three replicates along the three conditions; the plots were divided to capture the significant changes between HNF1B WT and R295C conditions. (B) Expression profile of disease-associated genes in fibroblasts and reprogrammed tissue. A significant difference in the expression levels comparing 3TF+HNF1B WT versus MEFs are represented in bold; asterisk (*) indicates a significant difference between 3TF+HNF1B R295C versus 3TF+HNF1B WT.
	Figure 5: Species overlap analysis. (A) Overlap of the DE genes in mouse reprogramming and Xenopus renal tissue induction experiments. Blue ellipses indicate reprogramming from fibroblasts (MEF) with 3TF+HNF1B WT or 3TF+HNF1B R295C and the DE comparisons; gray ellipses indicate induction from animal cap (uninjected) with HNF1B WT or HNF1B R295C and the DE comparisons. (B) Heatmap of overlapping DE genes (N=117) in both species, separated on the basis of kidney-specific tissue expression in the top part (N=96). (C) Validated target genes with their function and tissue expression profile in human tissues (expression data from GTEx77).
	Figure 6: In vivo analysis of target genes. (A) MesoSPIM images of the uninjected (left), control slc42a5 knockout (KO) (middle), and hnf1b KO (right) Xenopus tropicalis tadpoles and the kidney area for right (R) uninjected and left (L) manipulated sides. (B) Rescue of the hnf1b KO using WT and R295C HNF1B mRNA compared with control slc45a2 KO and uninjected conditions. Log2 changes of the kidney area from injected to uninjected sides are plotted for three different experiments (dot shape) and pairwise multiple comparisons (ANOVA) show the significance between uninjected, control injections (slc45a2 gRNA) and targeted injections. (C) In situ hybridization combined with kidney-specific antibody (lectin) stainings of six target genes in WT (left) and CRISPR/Cas9 KO (right) Xenopus embryos. (D) In situ hybridization to evaluate sord mRNA expression in CRISPR/Cas9 experiments with a gRNA (red) targeting the sord promoter at the hnf1b binding motive (light gray). Intensity of the staining was scored as weaker or stronger on the injected side or equal on both sides. Significance was calculated with the chi-square test. Scale bar is 100 μm.
	Figure 7: In vitro analysis of target genes. (A) Immunofluorescence staining of HNF1B WT (left) and R295C (right) iRECs for six proteins encoded by Hnf1b-regulated genes. Merged images contain eGFP signals from the iRECs (green), antibody staining, (magenta) and nuclear stain with Hoechst (cyan). Images for all three replicates together with secondary antibodies are presented in Supplemental Figure 6A. (B) Immunofluorescence of six proteins encoded by Hnf1b-regulated genes in mouse kidney cryosections. Merged images contain autofluorescence (autofl.) of the kidney tubules (green), antibody staining (magenta), and nuclear stain with DAPI (cyan). All scale bars are 100 μm.
	Supplementary Figure 1 related to Figure 1 and 2. Inducing renal tissue with HNF1B patient-specific mutation R295C. A. Microtome sections of in situ hybridisation for slc5a1 of animal caps treated with retinoic acid and actin (RA+ACT), extracted from tadpoles co-injected with mRNA of hnf1a, hnf1b and sall1 transcription factors (3TF) or injected with hnf1b mRNA. Sections were stained with Eosin and DAPI. All scale bars are 50µm. B. Western blot analysis of eight HNF1B mutations expressed in 293T cells (top/left). For the Xenopus animal cap induction experiments HNF1B WT or R295C proteins were detected (bottom/right). C. DNA binding assay of HNF1B and HNF1B R295C. The lower band indicates free, unbound DNA. The arrow indicates DNA bound to HNF1B proteins. Proteins were purified from transiently transfected HEK293 cells by immunoprecipitation. DNA sequence of the HNF1B binding motif was labelled with Cy7 which was used for detection. D. Protein stability of HNF1B WT and HNF1B R259C mutant. Changes in protein expression of FLAG-tagged WT and mutant HNF1B upon Cycloheximide (300μg/ul) treatment for indicated lengths of time (3-32h). E. Quantified protein expression of HNF1B WT and HNF1B R295C normalized on Tubulin expression (±SD of minimum n = 3 independent experiments). F. Verifying Cycloheximide activity by analyzing protein expression after 32h of treatment.
	Supplementary Figure 2 related to Figure 2. RNA sequencing of the induced renal tissue. A. First two components from principal component analysis in mouse (left) and Xenopus (right) experiments. Original tissue is marked with the round shape and reprogrammed tissue with a triangle. Colors indicate the sample type - original tissue (green), reprogramming with HNF1B WT (blue) and reprogramming with HNF1B R295C (red). B. Gene Ontology (GO) over-representation test on differentially expressed genes (absolute log2 fold change >1.5) from the reprogramming process comparing 3TF+ HNF1B R295C iRECs (left) and HNF1B R295C pronephric tissue (right) with original material. GO terms related to renal organogenesis are marked in bold. C. Mfuzz soft clustering results from the mouse experiments. Each line represents the expression change between original tissue, 3TF+HNF1B WT and 3TF+HNF1B R295C conditions. Yellow colors indicate low and red high membership values (the similarity of vectors to each other).
	Supplementary Figure 3 related to Figure 3. Transcriptional alterations due to HNF1B patient-specific mutation R295C. A. Full plot of comparing clusters on biological processes, cellular component and molecular function GO levels. B. Distribution of expression changes between 3TF + HNF1B R295C vs 3TF + HNF1B WT in enhanced genes in collecting duct, distal tubular and proximal tubular cells using the single-cell datasets from the Human Protein atlas. Histograms show the genes count (left y-axis) per log2 fold change (FC) and density plot (purple) illustrating distribution of the plot using kernel smoothing (right y-axis). C. The percentage of genes up- (red) or downregulated (blue) in the different renal cell types.
	Supplementary Figure 4 related to Figure 4. Transcriptional stability of the iRECs and genes essential for kidney development in Xenopus organoids. A. Tubular differentiation markers Cdh6, Cdh16, Slc17a1 and Lrp2 in HNF1B WT and HNF1B R295C iRECs of later passages (passage 22) expression levels. Mean and standard deviation of n = 3 biological replicates; differences assessed by ANOVA with Tukey’s correction for multiple comparisons, **P < 0.001, P < 0.05. B. Volcano plot of HNF1B WT vs animal cap tissue DEGs with highly confident (p-value 5e-15) genes essential for kidney development (GO:0001822 and GO:0072006). Cutoff lines for log2foldchange are -1 and 1, for p-value 0.05.
	Supplementary Figure 5 related to Figure 5. In vivo analysis of hnf1b and candidate genes. A. Expression of the 36 candidate genes from the species overlap analysis in Xenopus tropicalis. In situ hybridization was conducted at three developmental stages - neurala, tailbud and tadpole. B. Hnf1b expression in Xenopus laevis at stages 22, 33 and 38. C. In situ hybridization combined with kidney specific LE-lectin stainings of 13 target genes in wild type and CRISPR-Cas9 hnf1b KO Xenopus tropicalis embryos.
	Supplementary Figure 6 related to Figure 6 and 7. Analysis of HNF1B targets in iRECs and in vivo. A. Immunofluorescence stainings of three biological replicates of the reprogrammed iRECs (HNF1B WT and R295C) for six target genes. Merged images consist of iRECs cdh16-eGFP (green), target protein (magenta) and Hoechst (cyan) signals. B. Morphological changes of direct reprogramming using HNF1B WT (up) or HNF1B R295C (down) as seen in differential interference contrast images. C. Genomic sequence showing the hnf1b guide RNA genome targeted site (underlined) and sequencing chromatograms from KO and uninjected embryos. The bargraph represents the percentages of indels occurring at the hnf1b locus in the CRISPR/Cas9 KO experiments. KO and ICE scores based on three technical and five biological replicates.

References [+] :

Aboudehen, Transcription Factor Hepatocyte Nuclear Factor-1β Regulates Renal Cholesterol Metabolism. 2016, Pubmed