Felix Kliewe^a     Andreas W. Kuss^b     Florian Siegerist^a     Sindy Schröder^a     Sandra Schordan^a     Nadine Artelt^a     Frances Kindt^a     Kerstin Amann^c     Maja T. Lindenmeyer^d     Karlhans Endlich^a     Nicole Endlich^a    

^aDepartment of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany, ^bHuman Molecular Genetics Group, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany, ^cDepartment of Nephropathology, University Medicine Erlangen, Erlangen, Germany, ^dDepartment of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Key Words
Tcf21 • Parietal epithelial cells • ChIP-Seq • Differentiation • Podocyte • Transcription factors

Abstract
Background/Aims: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. Methods: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. Results: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin‑1, β-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. Conclusion: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.  

Introduction

Over the last years, many studies have demonstrated that the de-differentiation and loss of podocytes are the major cause of chronic kidney disease (CKD) [1–3]. It would be therefore worthwhile to find a strategy to replace lost and damaged podocytes as it is already described for the transdifferentiation of fibroblasts into several different cell types, e.g. neurons. This transdifferentiation was induced by the expression of a small number of specific transcription factors. Whether there are progenitor cells which are able to differentiate into podocytes is still an ongoing and controversial debate. One hypothesis is that parietal epithelial cells (PECs) are able to transdifferentiate into podocytes to some extent and under specific circumstances [4–12]. Since in the literature it is described that several transcription factors are crucial for podocyte differentiation like Wt1, Lmx1b, Foxc2, and MafB [13–16], we focused our attention on a transcription factor of the basic-loop-helix family, Tcf21 which was already shown to be essential for the proper development of glomeruli [17, 18].
Tcf21, also known as Pod1, capsulin, or epicardin, is not only crucial for the differentiation of immature podocytes but also for a number of different cell types during embryogenesis of the heart, lung, kidney and spleen [17–22]. By the generation of Tcf21 null mutant mice that die at the perinatal period by major cardiopulmonary defects, it was nicely demonstrated that Tcf21 is essential for development [18]. Additionally, it was shown that the kidneys of the Tcf21 knockout mice were severely hypoplastic due to a delay of nephrogenesis and abnormal branching of the ureteric bud. The few glomeruli found in kidneys of Tcf21 knockout mice appeared to arrest at the capillary loop stage of development [18]. Moreover, podocyte-specific Tcf21 knockout mice developed massive proteinuria at 3-5 weeks of age with prominent glomerular lesions which is similar to human FSGS [23].
Because Tcf21 is so important for proper kidney development and function, we studied the influence of Tcf21 for the differentiation of PECs into podocytes in vitro and looked for target genes by Chromatin ImmunoPrecipitation followed by DNA-Sequencing (ChIP-Seq).


Materials and Methods

Cell culture

PECs were cultivated in EBM medium (Lonza, Basel, Switzerland) supplemented with EGM-MV singlequots T75 (Lonza) as reported [24]. PECs were passaged every 3-4 days. Transfection of PECs was done with 1 µg/ml GFP- and the Tcf21-GFP plasmids (OriGene Technologies, Rockville, MD, USA) by Lipofectamine^® 2000 transfection (Invitrogen, Carlsbad, CA, USA) in serum-free RPMI medium (Lonza) according to the manufacturer’s instructions. Cells were used 48 hours after transfection for immuncytochemistry, for protein- and RNA-isolation.

In Situ Cell Death Detection (TUNEL assay)

The In Situ Cell Death Detection Kit, TMR red (Sigma-Aldrich, St. Louis, MO, USA) is based on the detection of single- and double-stranded DNA breaks that occur at the early stages of apoptosis. Apoptotic cells were fixed and permeabilized. Subsequently, the cells are incubated with the TUNEL reaction. After washing, the label incorporated at the damaged sites of the DNA is visualized by fluorescence microscopy.

BrdU Labeling and Detection

Cell proliferation was measured with the thymidine analog BrdU (5-bromo-2’-deoxyuridine) following its incorporation into newly synthesized DNA and its subsequent detection with an anti-BrdU antibody by using “5-Bromo-2′-deoxy-uridine Labeling and Detection Kit I” (Roche, Basel, Switzerland)

Histology

For paraffin sections of mouse kidneys and human biopsies, samples were dehydrated and embedded into paraffin by standard procedures. Paraffin sections (5 µm) were done on a Leica SM 2000R (Leica Microsystems, Wetzlar, Germany). After rehydration, sections were unmasked in citrate buffer (0.1 M, pH 6.0) by heating for 5 min in a pressure cooker. The sections were stained with 1 mg/100 ml Hoechst 33342 (Sigma-Aldrich) for 30 min. For the immunofluorescence double-staining, samples were incubated with an antibody against synaptopodin (1:50; mouse; Progen Biotechnik GmbH, Heidelberg, Germany) and Tcf21 (rabbit; Sigma-Aldrich, HPA013189, 1:100) overnight. Samples were washed with 1x PBS for 3x 5 min and incubated with Cy2- and Cy3-conjugated anti-mouse/-rabbit secondary antibodies (1:250; Jackson ImmunoResearch Laboratories, West Grove, USA) for 1 hour. After additional washing, the samples were mounted in Mowiol (Carl Roth, Karlsruhe, Germany) for fluorescence microscopy. Additionally, paraffin sections were stained using the Vectastain kit (Vector Laboratories, Burlingame, CA, USA) following manufacturer’s instructions. Visualization was performed with DAB substrate kit (SK-4100; Vector Laboratories) followed by nuclear staining with hematoxylin and mounting in Eukitt (Sigma-Aldrich). In controls, 1x PBS was used instead of primary antibody. Photographs were taken on an Olympus BX50 microscope equipped with an Olympus DP10 digital camera (Tokyo, Japan).

Kidney biopsies

Kidney biopsies were archived at the Department of Nephropathology, Institute of Pathology, University Hospital Erlangen, Germany. The use of remnant kidney biopsy material was approved by the Ethics Committee of the Friedrich-Alexander-University of Erlangen-Nürnberg, waiving the need for retrospective consent for the use of archived rest material.

Immunocytochemistry

PECs were fixed with 2% paraformaldehyde (PFA) for 10 min, permeabilized by 0.3% Triton-X (Sigma-Aldrich) for 4 min and blocked for 1 hour with blocking solution (2% FBS, 2% BSA and 0.2% fish gelatin in PBS). Primary antibodies were diluted in blocking solution and incubated for 1 hour on cells. Antibodies used in this study for immunocytochemistry can be viewed in Supplementary Table 1 (for all supplementary material see www.cellphysiolbiochem.com). Secondary antibodies were diluted in blocking solution and incubated with the cells for 30 min: anti-mouse-Cy3 (Jackson ImmunoResearch Laboratories, 1:300) and anti-rabbit-Cy3 (Jackson Immuno Research, 1:300). For the visualization of the actin cytoskeleton, cells were stained with Alexa Fluor 546 phalloidin (1:100; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. For nuclei staining DAPI (1:150; Sigma-Aldrich) was used for 5 min. All samples were mounted in Mowiol (Carl Roth, Karlsruhe, Germany) and used for laser scanning microscopy (LSM).
Fluorescence mRNA in situ hybridization (FISH). For mRNA FISH, we used the RNAscope 2.5 HD Assay Red (Advanced Cell Diagnostics, Newark, CA, USA). Protocol was applied according to manufacturer’s instructions with following changes: Tissue was digested for 15 instead of 30 min to yield better glomerular morphology. After FastRed detection, slides were collected in 1x PBS, blocked with the aforementioned blocking solution for 1 hour at room temperature. A premixed antibody solution containing a rabbit anti-human podocin antibody (1:150, IBL, Cat No. 29040) and an Alexa Fluor 488-conjugated dual monoclonal alpaka anti-rabbit VHH nanobody (1:1000, ChromoTek GmbH, Planegg-Martinsried, Germany) was incubated at 4°C overnight. After several washes in PBS, cell nuclei were counterstained with 0.1 mg/ml DAPI and sections mounted in Mowiol for microscopy.
Microarrays on human kidney biopsies. Human renal biopsy specimens and Affymetrix microarray expression data were procured within the framework of the European Renal cDNA Bank - Kröner-Fresenius Biopsy Bank [25, 26]. Biopsies were obtained from patients after informed consent and with approval of the local ethics committees. Following renal biopsy, the tissue was transferred to RNase inhibitor and microdissected into glomeruli and tubulointerstitium. Total RNA was isolated from microdissected glomeruli, reverse transcribed, and linearly amplified according to a protocol previously reported [27].
The published microarray expression data used in this study came from individual patients with DN and FSGS. Pre-transplantation kidney biopsies from living donors were used as control renal tissue (GSE99340, GSE32591, GSE37463). Fragmentation, hybridization, staining, and imaging were performed with the Affymetrix HG-U 133plus 2.0 system (Affymetrix, Santa Clara, CA) according to the Affymetrix Expression Analysis Technical Manual. CEL file normalization was performed with the Robust Multichip Average method using RMAExpress (Version 1.0.5) and the human Entrez-Gene custom CDF annotation from Brain Array version 18. The log-transformed dataset was corrected for batch effect using ComBat from the GenePattern pipeline (http://www.broadinstitute.org/cancer/software/genepattern/). To identify differentially expressed genes the SAM (Significance Analysis of Microarrays) method was applied using TiGR (MeV, Version 4.8.1) [28]. A q-value below 0.05 was considered to be statistically significant.

Laser scanning microscopy

Images were captured either with a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany), 40x/63x oil immersion objectives in the Leica Application Suite software (Leica Microsystems, Version 2.6.0).

RNA extraction, cDNA synthesis, qRT-PCR

Samples from cells/tissues were processed in Tri-Reagent (Sigma-Aldrich, St. Louis, MO, USA) according manufactures instructions. For cDNA synthesis, 1 µg of the isolated total RNA was transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The quantitative real-time PCR (qRT-PCR) analysis was performed on a LightCycler Nano (Roche Diagnostics GmbH, Mannheim, Germany) by using the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with Actb and Rpl32 as reference genes. Relative quantifications of the mRNA levels were done by the efficiency corrected calculation model by Pfaffl [29] and are shown with standard deviations from three biological replicates. Primers used for mouse samples can be viewed in Supplementary Table 2.
Expression level of mRNA corresponding to Tcf21 (ENSMUSG00000045680) was also determined by RNA sequencing of samples isolated from murine glomeruli. Sequencing was carried out on a SOLiD 5500xl sequencing platform (Life Technologies, Carlsbad CA, USA) as previously described [30]. Sequence analysis was based on GRCm38/mm10. Benjamini-Hochberg adjusted Wald test p-values (p_adj) were determined to identify significant differences in gene expression between samples.

Western blot analysis

Cells were trypsinized, washed twice in PBS and dissolved in RIPA lysis buffer (Thermo Fisher Scientific). Protein homogenates were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). After addition of SDS-PAGE sample buffer (final concentrations: 32 mM Tris-HCl, 1% SDS, 5% glycerol, 0.05% bromphenol blue, 3.25% 2‑mercaptoethanol, pH 6.8), the cell lysates were heated at 95°C, 5 min and separated on a 4-20% gradient Mini-Protean TGX Gel (Bio-Rad). Furthermore, separated proteins were blotted on nitrocellulose membranes using the Trans-Blot Turbo RTA Transfer Kit (Bio-Rad) and the Trans-Blot Turbo Transfer System (Bio-Rad) at 1.3 A/25 V for 10 min. Blots were washed in 1x TBST (50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 1 mM MgCl₂) supplemented with Tween-20 0.1%; AppliChem) and blocked with 5% milk powder for 1 hour at room temperature. Moreover, primary antibodies were diluted in 2.5% milk powder (in 1x TBST) and incubated over night by 4°C. After intensive washing in 1x TBST blots were incubated with secondary antibodies for 60 min, washed again, developed with the Clarity™ Western ECL Substrate (Bio-Rad) and visualized on X-ray films (Fujifilm Super RX, FUJIFILM, Tokyo, Japan). Antibodies used in this study for Western blot can be viewed in Supplementary Table 3. For the relative quantification, developed x-ray films were scanned and analyzed using ImageJ (version 1.49m; NIH, Bethesda, MD, USA).

ChIP-Seq and bioinformatic analysis

PECs were transfected with Tcf21-myc and myc empty vector (control plasmid) for 48 h. Cells were treated with 1% formaldehyde for 10 min at room temperature. Formaldehyde was inactivated by adding glycine (final concentration 0.125M) to the cells, followed by two times washing with cold PBS. Cells were lysed for 10 min on ice (20mM HEPES pH 7.4, 1mM EDTA, 150mM NaCl, 1% SDS, protease inhibitor). Nuclei were collected by centrifugation (1700g for 5min) and resuspended for sonication in shearing buffer (20mM HEPES pH 7.4, 1mM EDTA, 150mM NaCl, 0.4% SDS, protease inhibitor). Crosslinked chromatin was sheared for 10 min (duty cycle 5% by sonication using a Covaris S220 Focused-ultrasonicator (Covaris, Woburn, MA 01801, USA) and pre-cleared with protein A/G agarose (Santa Cruz Biotechnology). ChIP was performed overnight in IP dilution buffer (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X100, 0.01% SDS), using 5 μg anti-TCF21 (H‑75; sc‑32914; Santa Cruz), anti-myc antibody (9E10; sc‑40; Santa Cruz), control IgG or anti-myc (9E10) magnetic beads (OriGene Technologies, Rockville, MD, USA). IP complexes were washed twice with wash buffer 1 (100 mM Tris pH 8.0, 500 mM LiCl, 1% NP-40 and 1% deoxycholate), two times with high salt wash buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and twice with TE buffer. Bound chromatin was eluted from the protein A/G agarose or beads respectively twice with elution buffer (1% SDS, 0.1 M NaHCO₃). After reverse-crosslinking, RNaseA and proteinase K digestion, chromatin was cleaned up and the DNA concentration measured using Qubit 2.0 fluorometric quantitation. The DNA fragments were then prepared for sequencing on a 5500xl SOLiD system (Life Technologies, Carlsbad, CA, USA) using recommended protocols and equipment (Life Technologies, Carlsbad, CA, USA). In short: The DNA fragments were end-polished, size-selected using Ampure XP Beads (Beckman Coulter, Brea, Ca, USA) and subject to A-tailing, adapter ligation and barcoding. The library was purified and the concentration and size distribution was determined using a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA). The DNA library was then diluted and attached to beads during clonal emulsion PCR amplification using an Applied Biosystems SOLiD EZ Bead Amplifier (Life Technologies, Carlsbad, CA, USA). After enrichment and 3’ modification the library was immobilized onto a flowchip and sequenced (75 bases in the forward and 35 bases in the reverse direction) using a 5500xl SOLiD system (Life Technologies, Carlsbad, CA, USA). The Lifescope Genomic Analysis Software (Life Technologies, Carlsbad, CA, USA) was used to call the bases and generate BAM files by alignment of the reads to the mouse genome (GRCm38/mm10). To identify putative Tcf21-myc binding sites the BAM data files were analysed using MACS1.4 or the GENOMATIX software suite.
De novo motif discovery was performed by using MEMESuite [31], more precisely DREME (Discriminative Regular Expression Motif Elicitation) motif comparison [32] was performed against a database of pooled TF motifs (JASPAR database) [33]. Functional annotation of high confidence binding regions was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) [34]. Gene enrichment analysis was performed by ToppGene Suite [35]. For visual inspection of Tcf21 binding to specific genomic regions, normalized read densities were uploaded to the Integrative Genomics Viewer (IGV) [36]. Identical viewing range for both Tcf21 and control samples set to the maximum peak within the window. To create density distributions (heat maps) around TSSs of Tcf21 peaks/targets, Seqminer v.1.3.3 was used [37].

Statistical analysis

All data are given as means ±SD or ±SEM, analyzed by unpaired t test with repeated measurements (n). Differences were determined significant at a p-value <0.05.

Results

Tcf21 is down-regulated during the de-differentiation of podocytes

As shown in Fig. 1A, Tcf21 is strongly expressed in kidney sections. Co-localization studies using the podocyte-specific protein synaptopodin confirmed that Tcf21 is strongly expressed in the nuclei of mouse and human podocytes (Fig. 1A). In contrast, parietal epithelial cells (PECs) which are still under debate regarding their role as progenitor cells for podocytes, weakly expressed Tcf21 in vivo (Fig. 1A). This was confirmed by single mRNA in situ hybridization (RNAscope^®) combined with podocin immunofluorescence of human kidneys (Fig. 1B).

To study the expression of Tcf21 in dependence on the differentiation of podocytes, we performed comparative gene expression analysis of freshly isolated mouse podocytes, cultured primary mouse podocytes as well as cultured primary mouse PECs. We have found that Tcf21 was one of the strongest expressed genes in freshly isolated podocytes (27-fold) compared to podocytes and PECs in vitro (Fig. 1C). Furthermore, to study the expression of Tcf21 during the de-differentiation of podocytes, we used our established GlomAssay [38].
This assay allows us to study podocytes directly in situ. After isolation, glomeruli were cultured under standard conditions and RNA was isolated after 3, 6 and 9 days followed by RNA_Seq analysis [38]. We have found a continuous decrease of Tcf21 mRNA until day 9 (fold change 3d: 0.55; p_adj=0.025; 6d: 0.41; p_adj=0.0045; 9d: 0.36; p_adj=0.0004) (Fig. 1D) indicating a strong downregulation during the de-differentiation of podocytes.

Tcf21 regulates the expression of specific PEC and podocyte markers

Since it is still under debate that PECs might be a type of progenitor cells that could differentiate into podocytes, we studied the effect of Tcf21 on PECs. Therefore, we transfected PECs with a plasmid encoding for Tcf21 coupled to GFP (PEC-Tcf21). As a control, PECs were transfected with a plasmid encoding for GFP alone (PEC-Ctrl). The expression levels of transfected Tcf21 were quantified by Western blots (Fig. 2C and Supplementary Fig. 1). Using an antibody specific for Tcf21, we verified that PEC-Tcf21 expresses Tcf21-GFP (Fig. 2A). We found that Tcf21-GFP was exclusively localized in the nuclei of PEC-Tcf21 (Fig. 2A). Further, we observed no significant change of the cell morphology and the actin cytoskeleton, respectively, as shown in Fig. 2B.
To find out whether PEC-Tcf21 differentiate into podocyte-like cells, we determined the expression of specific PEC as well as podocytes markers by qRT-PCR and Western blot. Western blot analysis showed that specific PEC markers became regulated by Tcf21: caveolin‑1 (-53±7%), β-catenin (-27±3%) and Pax2 (-27±4%) (Fig. 2C and D). The expression of the podocyte-specific markers synaptopodin (Synpo) and Wt1 did not change due to the transfection of Tcf21, in contrast to the slit membrane protein Cd2ap that was significantly upregulated (58±11%) in PEC‑Tcf21 compared to the control. Similar results were received by qRT-PCR (Cav1: ‑49±7%; Ctnnb1: ‑44±6%; Pax2: ‑38±13%, Cd2ap: 55±17%) (Fig. 2E). Further­more, the RNA expression of synaptopodin as well as Wt1 was not affected by Tcf21 transfection.

Tcf21 induces multilobulation, budding and micronuclei formation (MBM) in PECs

Interestingly, we observed that the expression of Tcf21 in PECs affected the nuclei of PEC-Tcf21. Three typical characteristics, like multilobulation, budding of nuclei as well as the formation of micronuclei (MBM) were found 48 hours after transfection. To identify whether these changes are caused by an induction of apoptosis in PECs, we performed a TUNEL assay that detects single- and double-stranded DNA breaks as a sign of apoptosis. However, we have not found any indication for apoptosis (Fig. 2F). Surprisingly, we observed DNA synthesis (Fig. 2G and H) verified by BrdU incorporation (Fig. 2G) as well as by the detection of phosphorylated histone H3 (Fig. 2H). These results suggest that Tcf21 induces mitosis in PEC cells.

Tcf21 induced MBM is accompanied by multiple incomplete mitoses

Since we observed DNA synthesis together with MBM in PEC-Tcf21, we studied mitosis by staining for α-tubulin as one monomer of microtubules and γ-tubulin, the major tubulin of centrosomes. In contrast to the control-transfected PEC, we found that MBM (Fig. 3A) was accompanied by a multiplication of centrosomes (Fig. 3B). Furthermore, we have found that this multilobulation was reversible after the addition of the microtubule inhibitors nocodazole as well as taxol (Fig. 3C). Incubation with cytochalasin D, an inhibitor of the polymerization of actin, did not affect the number of cells with multilobulated nuclei (Fig. 3C).
Additionally, we have found that Tcf21-transfected PECs developed 3-4 or sometimes more than 5 centrosomes. By the staining for α-tubulin, we identified that Tcf21-transfected cells developed multiple spindle apparatuses which were arrested in the anaphase (Fig. 3D). The percentage of cells with such a multiple and incomplete mitosis was up to 28±5% compared to the control (2±3%) (Fig. 3E). Moreover, 14±2% of the Tcf21-transfected PECs showed a significant increase of tetraploidy in the metaphase preparation compared to the control (Fig. 3F, G).

ChIP-Seq analysis identifies genome wide Tcf21-binding sites

Since Tcf21 seems to play a central role for the maintenance of the glomerulus in vivo, we wanted to identify important and novel binding partners of TCF21. Therefore, we performed a chromatin immunoprecipitation followed by DNA-Sequencing (ChIP-Seq) of chromatin isolated from Tcf21-transfected PECs. The data were compared to the control cells that were transfected with an empty vector.
Our ChIP-Seq analysis identified 9125 peaks (putative Tcf21 target genes). To create density distributions around transcription start sites (TSSs), heat maps were created using SeqMINER [37] (Fig. 4A). We next mapped these Tcf21-binding sites and found about 54% of Tcf21-binding sites within ±5 kbp of any TSS (Fig. 4B). 76% of these binding sites were approximately ±1 kbp around the TSSs. The availability of sequence information across a large number of TF binding sites allowed the identification of the canonical binding sequence of Tcf21.
By de novo TF motif enrichment within the Tcf21 peaks, we identified the nucleotide sequence CAGCTG (23.6% of peaks, P = 4.8e-042) (Fig 4C). We found that this sequence matched the CANNTG sequence. This is a common E-box binding motif used by bHLH factors. Moreover, this sequence is identical to the E-box motif that is known to mediate the binding of bHLH partners of Tcf21, including Tcf12 and Tcf3 (Fig. 4C) [39].
Interestingly, many of these Tcf21 target genes are involved in the regulation of the cell cycle, cell division, microtubule-based processes and chromosome segregation (Supplementary Table 4).
Additionally, DAVID was used to perform functional annotation clustering on peaks in relation to TSSs [34]. Peak-associated genes showed also a clear enrichment of cell cycle genes (Fig. 4D). We found several Tcf21 peaks in the promoter regions of cell cycle genes like Mapt, Mdm2 or Cdc45 (peaks highlighted with gray boxes in Fig. 4E). By qRT-PCR, we showed that Mapt was highly up-regulated in the presence of Tcf21 (92±12%) (Fig. 4F). In contrast, Cdc45 which decreased by 52±8% as well as Mdm2 by 55±10%, a protein know to induce the mitotic catastrophy (Fig. 4F). The downregulation of Mdm2 due to Tcf21 transfection was also confirmed by immunostaining (Fig. 4G). Furthermore, we found Tcf21 bindings sites in the promoter regions of p53 (Trp53; NM_001127233; Tcf21 peak 43bp from TSS) and p21 (Cdkn1a; NM_001111099; Tcf21 peak -38bp from TSS), which are important regulators of the cell cycle.

Tcf21 regulates the expression of specific cyclins

Since Tcf21 induced incomplete mitosis, we studied the expression of specific cyclins like D1 and D2 after the transfection with Tcf21 (Fig. 5A). By immunofluorescence staining we have found a downregulation of cyclin D1 and an upregulation of cyclin D2.
By qRT-PCR, we demonstrated that cyclin D1 and B1 are significantly reduced by 49±7% and 58±8%, respectively, due to the expression of Tcf21 in PECs. In contrast, the expression of cyclin D2 and E1 was significantly increased by 153±20% and 150±13% (Fig. 5B).

YY1 rescues the Tcf21-induced MBM

Since it is known that YY1, an important transcriptional repressor protein, is able to regulate de-/differentiation, we studied the expression of YY1 in dependence on Tcf21. We have found by qRT-PCR that YY1 is significantly downregulated (-52±7%) in Tcf21-PECs (Fig. 6B) which was confirmed by immunofluorescence staining (Fig. 4A) and Western blot (-48±8%) (Fig. 6C, D).
Moreover, we found that the co-expression of YY1 in Tcf21-PECs rescued the MBM. We determined a significant reduction of multilobulation, budding as well as tetraploidy (by 62 %) shown in Fig. 6E and F. These results demonstrated that YY1 and Tcf21 are important regulators of the cell cycle.

TCF21 expression is down-regulated in patients suffering from diabetic nephropathy

An analysis of microarray data of the Nephroseq database [40–42] has shown that the expression of TCF21 in glomeruli was 3.4 times higher compared to the expression in the tubules of human kidneys (Fig. 7A). However, the expression of Tcf21 mRNA was significantly decreased in mice (2.6 times) and human patients (6.3 times) suffering from diabetic nephropathy (DN) (Fig. 7B).
These results were verified by single mRNA in situ hybridization (Fig. 7C) of human biopsies of patients suffering from DN and FSGS compared to healthy control. The results suggest that podocytes in patients with DN tend to have a lower expression of Tcf21, which could then influence the maintenance of the podocytes. Furthermore, we measured the mRNA expression of TCF21 in microdissected glomeruli originated from renal biopsies of patients suffering from DN and FSGS and compared to mRNA levels of healthy living donors (Fig. 7D) (European Renal cDNA Bank) [25, 26]. We observed that patients with DN showed a significant decrease of TCF21 mRNA compared to the control (log fold change: -0.44). In contrast, patients suffering from focal segmental glomerulosclerosis (FSGS), showed a slight, but significant increase of TCF21 mRNA expression (log fold change: 0.19).

Discussion

Dedifferentiation and loss of podocytes are major causes for the development of severe kidney diseases like FSGS and DN. Since podocytes are growth-restricted, the loss of podocytes cannot be compensated, resulting finally in the development of end-stage renal disease (ESRD). Since several years, there is an ongoing debate whether podocyte progenitor cells exists in or nearby the glomerulus. The groups of Moeller, Romagnani and Shankland have demonstrated that PECs, flat epithelial cells covering the inner side of Bowman’s capsule, function as progenitor cells under specific circumstances [4, 6, 11, 43–45]. Therefore, it is of great interest to reveal the mechanisms involved in a possible transformation of PECs into podocyte-like cells as it was described for other cell types like neurons and cardiomyocytes. Parmar and colleagues showed that fibroblasts could directly differentiate into neurons after transfection with three specific transcription factors [46]. Recently, we found that the expression of the transcription factor Dach1 switched PECs into podocyte-like cells which expressed the podocyte specific protein synaptopodin [47].
Several publications have shown that the transcription factor Tcf21 is important for the development of a number of cell types during embryogenesis of the heart, lung, kidney and spleen [17–22]. As a potential candidate for the transdifferentiation of PECs into podocyte-like cells, Tcf21 which is weakly expressed in PECs in vivo, was investigated.
Our study revealed that the expression of Tcf21 in PECs resulted in a significant downregulation of the PEC-specific proteins caveolin‑1, β‑catenin and Pax2 suggesting that PECs de-differentiation has started. Additionally, we found an upregulation of CD2AP, a podocytes-specific as well as essential slit membrane protein indicating a transition of Tcf21-transfected PECs into podocyte-like cells.
To study which genes might be activated by Tcf21, we performed ChIP-Seq analysis of Tcf21-transfected PECs. The analysis revealed that Tcf21 binds to specific promoter regions of e. g. cell cycle-regulating genes as well as microtubule-associated proteins. Therefore, we studied the expression of cyclin D1 and cyclin D2 which are important for cell cycle and cell division. It has been already shown that cyclin D1, which is essential for the induction of DNA synthesis as well as for cell proliferation [48], controls the proliferation of PECs during development and is constitutively expressed in PECs [49, 50]. However, since podocytes are postmitotic, cyclin D1 is not expressed in podocytes in vivo [49]. Our data have shown that cyclin D1 is downregulated after the expression of Tcf21 in PECs which seems to reflect the in vivo situation of podocytes. Interestingly, we observed that cyclin D2 which is responsible for the transition of the cell cycle from G1 to S phase, is upregulated after the expression of Tcf21. Whether cyclin D2 could replace cyclin D1 function is not clear.
Beside this, we identified the promoter region of the cell division cycle protein 45 (Cdc45), a protein that is essential for the replication of the DNA, as a binding site for Tcf21 by ChIP-Seq analysis. We found that Cdc45 is downregulated in PECs after the transfection with Tcf21. Since Cdc45 is responsible for proper replication, it makes sense that Cdc45 became downregulated after Tcf21 transfection.
The promoter region of the microtubule-associated protein Mapt, which is only expressed in podocytes and not in PECs in vivo [51, 52], was also identified as a binding sites for Tcf21. We revealed that the expression of Mapt is significantly upregulated in Tcf21-transfected PECs. These data support our hypothesis that Tcf21 induces a transition of PECs into podocyte-like cells.
Interestingly, we observed that the Tcf21 expression in PECs induced multilobulation, budding of the nuclei and the formation of micronuclei (MBM). Further, Tcf21-induced MBM was often accompanied by a multiplication of centrosomes and tetraploidy. In combination with the regulation of cyclins, these data suggest that Tcf21 regulate the cell cycle as well as the mitosis in cultured PECs similar to the findings in adrenocortical and hepatocarcinoma tumor cells [53]. Since we have found that Mdm2, an E3 ubiquitin-protein ligase is responsible for the mitotic catastrophe of podocytes due to an inhibition of the p53-dependent apoptosis pathway [54, 55], we studied whether Tcf21 binds the promoter region of Mdm2. Indeed, we found that Tcf21 binds the promoter region of Mdm2 as well as down-regulates the expression of Mdm2 determined by qRT-PCR and by immunhistological staining. Since Mdm2 inhibits and degrades p53, a protein essential for cell arrest as well as apoptosis [56–59], one might suggest that MBM might be the results of the Mdm2 inhibition due to the expression of Tcf21. Alternatively; Tcf21 could directly regulate the expression of p53 since we identified a binding site of Tcf21 to the promoter region of p53.
These results might explain one function of Tcf21 which is strongly expressed in podocytes in vivo to avoid mitosis because mitosis would change the complex 3-D morphology of differentiated podocytes resulting in a leaky filtration barrier. This hypothesis is supported by the findings that podocyte-specific Tcf21 knockout mice develop massive proteinuria [23].
Since it was reported that the transcription factor YY1 is an negative regulator of p53 as well as of p21 [60–64], another important protein for cell cycle regulation and that the loss of YY1 resulted in polyploidy and chromatid aberrations [65], we wanted to know whether we could rescue the Tcf21-induced MBM by the co-expression of YY1. Indeed, we observed a complete rescue of the MBM suggesting a role of YY1 in the differentiation of podocytes. That YY1 might play an important role in the transition of undifferentiated into differentiated podocytes was nicely shown by Jha and colleagues [66]. Furthermore, it was shown that the expression of YY1 ameliorates podocyte damage in diabetic nephropathy (DN) [67].
To study the expression of Tcf21 in patients with podocytopathy, we stained biopsies of patients suffering from DN. We have found that not only the protein expression was reduced, but especially the expression of Tcf21 mRNA was significantly downregulated in the biopsies of the patients with DN which was determined by RNAscope^® as well as by Nephroseq data analysis. This is in nice accordance with the results found in db/db mice where Tcf21 is significantly downregulated in mice at an age of 7 weeks [68]. Surprisingly, Usui and coworkers have shown a very faint expression of Tcf21 in their control kidneys and a massive increase of Tcf21 expression in biopsies of patients with membranous glomerulonephritis [69]. However, there might be a problem with the control tissue because it is well-known that Tcf21 is strongly expressed in healthy podocytes.


Conclusion

Taken together, we demonstrated that the transcription factor Tcf21 is an important regulator of essential cell cycle genes, MBM, centrosome replication as well as an inductor of tetraploidy. Furthermore, we could show that Tcf21 is able to transdifferentiate PECs into podocyte-like cells and became downregulated during the dedifferentiation of podocytes in patients suffering from DN. These results might be helpful to find therapeutic therapies to avoid podocytopathies.

Acknowledgements

The authors thank Mandy Weise for technical assistance and Robert Weissmann and Lars Jensen for ChIP-Seq bioinformatic analysis. Part of the work was also supported by an Emerging Fields Initiative (EFI) for Cell Cycle in Disease and Regeneration (CYDER) from the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) (Germany). We also thank all participating centers of the European Renal cDNA Bank - Kröner-Fresenius biopsy bank (ERCB-KFB) and their patients for their cooperation. Active members at the time of the study please see (Shved et al., Sci Rep 2017;7(1):8576).

 

Author Contributions

The study was designed by NE, KE and FK; FK and SS contributed to the cell culture experiments; FK performed ChIP-Seq, supported by AWK; biopsies were handled and analyzed by FK, NA, FS and KA; SS and FS performed RNAscope®; all other experiments were performed by FK; experimental data was analyzed by FK; FK, NE and KE wrote the main manuscript text. FK prepared figures. All authors reviewed the manuscript.

 

Funding

This work was supported by a grant of the European Foundation for the Study of Diabetes/ Novo Nordisk to KE and by a grant of the EU (COST action BM0702 “Kidney and Urinary Proteomics – EuroKUP”) and of the Federal Ministry of Education and Research (BMBF, grant 01GM1518B, STOP‐FSGS) to NE as well as by the Südmeyer und Dr. Büchtemann Stiftung. This work was also supported by the Research Network Molecular Medicine of the University Medicine Greifswald. The ERCB-KFB was supported by the Else Kröner-Fresenius Foundation.

 

Disclosure Statement

The authors declare no competing financial interests.

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