The genome wide histone methylation patterns in adult mouse podocytes.

A) Schematic illustration of the podocyte isolation procedure. B) Heatmap across a set of 462 podocyte specific genes (Suppl. Table 4) 39 sorted according to gene size. H3K4me3 enrichment was found at the TSS, gene bodies were enriched for H3K4me1 and depleted for H3K27me3. C) Overlay of peaks at the TSS of podocyte specific genes confirmed a strong enrichment of H3K4me3 at the TSS. D) GREAT analysis for H3K4me3 peaks identified peak enrichment at TSS and enriched GO biological processes related to ‘nucleosome assembly’ and ‘protein folding’. E) H3K27me3 peaks are spread further upstream and downstream of the TSS and showed a strong GO term enrichment for ‘transcription factors’ and ‘developmental abnormalities’. F-H) Examples of methylation tracks across the podocyte specific genes Wt1 (F), Nphs2 (G) and the mesoderm regulator Brachyury (H).

MAFB ChIP-Seq analysis.

A) MEME analysis of the peak centred enriched regions reveals MAFB monomeric and dimeric binding sequences. B) MAFB binding site distribution shows strong enrichment at promoter regions. C) GREAT analysis reveals binding at genes involved in cell-matrix adhesion and podocytes pathological processes. D-E) MAFB (orange), WT1 (violet) and H3K4me3 (green) tracks across the podocyte specific genes Nphs1 and Nphs2 and the conservation of binding sites in the mouse and human genome.

Phenotypic analysis in conditional Mafb knockout mice reveals progressive kidney disease with podocyte effacement.

A) Strategy for the conditional knockout in Mafbcko (Wt1CreERT; Mafbflox/flox; mTmG) mice. B) Confirmation of efficient loss of MAFB protein at 72h after induction (note that non-nuclear staining with the MAFB antibody is background) (scale bar: 50µm). C) Electrophoresis of urine samples showed proteinuria at 8 weeks after induction, accompanied by (D) abnormal footprocesses (SEM, I-II), thickening of the basement membrane (TEM, III-IV) and segmental sclerosis (Sirius RED, V-VI). E) Quantification of foot processes and basement membrane thickness. (n= 3 animals per condition, 20 images per animal, **** p<0.001). F) Expression levels of different podocytes specific genes 3 and 30 days after TAM induction. (n=5, **** p<0.001). Abbreviations: SEM=scanning electron microscope; TEM=Transmission electron microscope. GBM=glomerular basement membrane.

RNAseq analysis in Mafbcko podocytes.

A) Heat map of deregulated genes three days after tamoxifen induction. B) Enrichr analysis of 693 downregulated genes (padj<0.01) reveals GO-term enrichment for ‘podocyte dysfunction’ and ‘VEGFA/VEGFR2 signalling’. C) Go Term enrichment analysis for upregulated genes highlighted ‘Tgfb regulation of extracellular matrix’ and ‘Nephrin interactions’. D-E) GSEA analysis of the deregulated genes indicate a rapid reversion of podocytes to a more undifferentiated state after Mafb inactivation. F) Venn diagram and G) gene list of the intersections between the different datasets (Mafbcko upregulated genes, Mafbcko downregulated genes, MAFB ChIP peaks, Podocyte specific genes) identified direct and indirect MAFB targets. H) GSEA analysis of the RNA-seq data highlighted ‘TCF21/POD1 targets’ to be affected in Mafbcko animals. I) ChIP- Seq tracks reveal peaks for MAFB and WT1 approximately 49.5kb upstream of the Tcf21 promoter. J) Inspection of the corresponding chromosomal region reveals a high degree of evolutionary conservation.

ChIP-qPCR analysis of chromatin conformation in Mafb KO embryonic kidney.

A) Immuno-florescence analysis in E18.5 homozygous Mafb knockout animals (MafbGFP/GFP) shows complete loss of the direct targets NPHS1 and NPHS2, whereas WT1 and synaptopodin (SYNPO) remain expressed (scale bar: 100µm). B) qPCR analysis of replicated immunoprecipitated chromatin with WT1 (yellow bars) and H3K4me3 (green bars) antibodies in E18.5 embryonic kidneys reveals loss of histone methylation and WT1 binding at Nphs1 and Nphs2 promoters. C) Schematic illustration of the role of MAFB in podocyte development. Mafb is a target of WT1, but is also required to permit access of WT1 to other podocyte-specific genes.

Representation of ChIPseq tracks.

A-B) Side-by side comparison of two independent ChIP-seq experiments at the Nphs1 (podocytes specific) and Hoxc cluster showed highly reproducible results. All further analyses were therefore performed with merged datasets. C-G) Examples of actively repressed developmental genes: paraxis, Gata4, Dmrt1, Six2 and Hoxd genes cluster, dotted line in G indicate the separation of active and inactive genes of the cluster. Colour scheme: H3K4m3 (green), H3K4me1 (blue,) H3K27me3 (red) and the Input (raw sonicated chromatin, grey).

WT1 ChIP-seq analysis.

A) Schematic illustration of the glomerular isolation procedure used for ChIP experiments. B) Distribution of WT1 peaks shows a large proportion (42.29%) of peaks to map to promoter regions (<1kb). C) De novo MEME analysis of the top 1000 enriched regions identified a core sequence highly similar to the previously identified consensus sequence for WT1 binding. D) Overlap between newly identified and previously published WT1 binding regions. E-F) GREAT analysis identifies ‘focal segmental glomerulosclerosis’ and ‘abnormal renal glomerulus basement membrane thickness’ as highly enriched GO terms.

Validation of the RNA-seq analysis.

A) qPCR analysis showed absence of Mafb and significant reduction of Nphs2 expression 48hours after tamoxifen induction (n=5, ****p<0.001, **p<0.01). B) Pearson correlation and C) hierarchical clustering of samples. D) Volcano plot revealing the spread of down-and up-regulated genes in Mafbcko glomeruli.

MAFB expression pattern during podocyte maturation.

A) Immunofluorescence staining shows that MAFB is not detectable at the early stage of nephrogenesis (vesicle stage, arrowhead), but it appears at the very first steps of podocyte differentiation (comma shaped bodies/precapillary loop stage, asterisk) and remains expressed in adult podocytes. Scale bar: 100µm. B) Single cell analysis in developing human kidneys. Dotted lines identify the early expression of MAFB in proximal S shaped bodies, comparable with the one of WT1 and slightly earlier than TCF21 compared with the appearance of late podocytes markers NPHS1 and NPHS1