Lmx1b and FoxC combinatorially regulate podocin expression in podocytes.
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 G0 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. Co-overexpression 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.
Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, and Bing.He@ki.se Karl.Tryggvason@ki.se.
Bing
B
He
Department of Medical Biochemistry and Biophysics, Division of Vascular Biology, and Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden;
Lwaki
L
Ebarasi
Ludwig Institute for Cancer Research, Oxford University, Oxford, United Kingdom; and.
Zhe
Z
Zhao
Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, and.
Jing
J
Guo
Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, and.
Juha R M
JR
Ojala
Department of Laboratory Medicine, Division of Clinical Research Centre, Karolinska Institute, Stockholm, Sweden;
Kjell
K
Hultenby
Ludwig Institute for Cancer Research, Oxford University, Oxford, United Kingdom; and.
Sarah
S
De Val
Department of Medical Biochemistry and Biophysics, Division of Vascular Biology, and Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden;
Christer
C
Betsholtz
Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, and Cardiovascular & Metabolic Disorders Program, Duke-NUS, Singapore Bing.He@ki.se Karl.Tryggvason@ki.se.
Karl
K
Tryggvason
D020816:Amino Acid Motifs; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D004742:Enhancer Elements, Genetic; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D016296:Mutagenesis; D010641:Phenotype; D050199:Podocytes / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000378:metabolism*
D051858:Forkhead Transcription Factors; C112890:Foxc1 protein, mouse; C426857:Foxc1a protein, zebrafish; D047908:Intracellular Signaling Peptides and Proteins; C112149:LIM homeobox transcription factor 1 beta; D060850:LIM-Homeodomain Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D014157:Transcription Factors; D029961:Zebrafish Proteins; C543330:lmx1bb protein, zebrafish
Combinatorial regulation; Lmx1b and FoxC; NPHS2 expression; podocyte; transgenic zebrafish