Lmx1b and FoxC Combinatorially Regulate Podocin Expression in Podocytes

Abstract

Podocin is a key protein of the kidney podocyte slit diaphragm protein complex, an important part of the glomerular filtration barrier. Mutations in the human podocin gene NPHS2 cause familial or sporadic forms of renal disease owing to the disruption of filtration barrier integrity. The exclusive expression of NPHS2 in podocytes reflects its unique function and raises interesting questions about its transcriptional regulation. Here, we further define a 2.5-kb zebrafish nphs2 promoter fragment previously described and identify a 49-bp podocyte-specific transcriptional enhancer using Tol2-mediated G 0 transgenesis in zebrafish. Within this enhancer, we identified a cis-acting element composed of two adjacent DNA-binding sites (FLAT-E and forkhead) bound by transcription factors Lmx1b and FoxC. In zebrafish, double knockdown of Lmx1b and FoxC orthologs using morpholino doses that caused no or minimal phenotypic changes upon individual knockdown completely disrupted podocyte development in 40% of injected embryos. Cooverexpression of the two genes potently induced endogenous nphs2 expression in zebrafish podocytes. We found that the NPHS2 promoter also contains a cis-acting Lmx1b-FoxC motif that binds LMX1B and FoxC2. Furthermore, a genome-wide search identified several genes that carry the Lmx1b-FoxC motif in their promoter regions. Among these candidates, motif-driven podocyte enhancer activity of CCNC and MEIS2 was functionally analyzed in vivo. Our results show that podocyte expression of some genes is combinatorially regulated by two transcription factors interacting synergistically with a common enhancer. This finding provides insights into transcriptional mechanisms required for normal and pathologic podocyte functions. Normal glomerular filtration function depends on structural integrity of the filtration barrier. Glomerular podocytes play a key role in establishing and maintaining this unique filtration barrier structure. Mature podocytes are characterized by cell cycle arrest, foot process formation, and the presence of the slit diaphragm, 1 which bridges the gaps between the interdigitating foot processes of neighboring podocytes and functions as a size-selective filtration barrier. 2,3 For their differentiation, as well as for the maintenance of their complex architecture, podocytes require the expression of several specific genes in a correct spatial and temporal fashion. This notion is supported by the identification of many mutations in podocyte-expressed genes as the underlying cause of inherited renal diseases. 4 Moreover, recent studies from genetically modified mice

Glomerular development – Shaping the multi-cellular filtration unit

“…Several podocyte specific target genes appear to be regulated by Foxc2 [158]. A very recent study could demonstrate that Lmx1b and FoxC combinatorially modulate the expression of Podocin and other podocyte genes via binding to a podocyte-specific enhancer carrying a FLAT-E and forkhead DNA binding motif [161].…”

The zebrafish kidney mutant zeppelin reveals that brca2/fancd1 is essential for pronephros development

“…During early development these groups of cells coalesce and migrate toward the midline, recruiting vasculature and forming a fused glomerulus (Drummond et al, 1998). A number of transcription factors and signaling molecules are known to be expressed in podocytes, like lhx1a and mafba , and several have been identified to play important roles in podocyte development, such as wt1a , wt1b, foxc1a, lmx1b.1, osr1 and the Notch pathway effector encoded by rbpj (Bollig et al, 2006; Bollig et al, 2009; Gerlach and Wingert, 2013; Krauss et al, 1991; He et al, 2014; O'Brien et al, 2011; Picker et al, 2002; Swanhart et al, 2010; Tomar et al, 2014; Toyama and Dawid, 1997). Further, these crucial genetic factors are associated with various renal defects and disease states.…”

Genome-Wide Analysis of Wilms’ Tumor 1-Controlled Gene Expression in Podocytes Reveals Key Regulatory Mechanisms

“…Indeed, MAFB/Kreisler, TCF21/POD1, LMX1B, and the FoxC-class TFs have already been shown to be of relevance in podocytes. [15][16][17][18][19] Frequently, networks of TFs cooperatively control gene expression by binding in TF modules to enhancers. These tissuespecific DNA-regulatory elements are located distally from the TSS.…”

“…Furthermore, previously described enhancers conferring podocyte-specific gene expression and the TFs bound to these enhancers were recapitulated by our ChIPseq data and motif analysis (Supplemental Figure 7). 8,16,19 Reanalysis of previously published WT1 ChIPseq data from embryonic kidneys, where WT1 is expressed in nephron best matching motifs from public databases are shown on the right with a similarity E value. Promoters are enriched in motifs generally found close to TSS (Ets-class TF, E box, GC box, CCAAT box), whereas enhancers harbor motifs for TFs with established significance in podocytes (Fox-class TFs, Lmx1b, Mafb).…”