Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor.
OBJECTIVE
Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT).


METHODS
We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10(-7)M aldosterone for 1h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10(-7)M aldosterone for 3h by microarray.


RESULTS
3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10(-9)M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated.


CONCLUSIONS
We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.
Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan.
Kohei
K
Ueda
Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
Katsunori
K
Fujiki
Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.
Katsuhiko
K
Shirahige
Endocrine Section, G.V. (Sonny) Montgomery VA Medical Center, MS, USA; Endocrinology, University of Mississippi Medical Center, MS, USA.
Celso E
CE
Gomez-Sanchez
Division of Clinical Epigenetics, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
Toshiro
T
Fujita
Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan.
Masaomi
M
Nangaku
Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan; Department of Anatomy and Life Structure, School of Medicine Juntendo University, Tokyo, Japan. Electronic address: mnagase-tky@umin.ac.jp.
Miki
M
Nagase
D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D020869:Gene Expression Profiling; D016678:Genome / Q000235:genetics*; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000235:genetics; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D050199:Podocytes; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022021:Tacrolimus Binding Proteins / Q000235:genetics; D000070876:Tensins; D014157:Transcription Factors / Q000235:genetics; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099159:Tacrolimus Binding Protein 5
D000450:Aldosterone; D017874:Immediate-Early Proteins; D008840:Microfilament Proteins; D020559:Monomeric GTP-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D018161:Receptors, Mineralocorticoid; D022021:Tacrolimus Binding Proteins; D000070876:Tensins; D014157:Transcription Factors; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099159:Tacrolimus Binding Protein 5; C498608:Dsip1 protein, mouse; C000603369:TNS1 protein, human; C000603373:Tns1 protein, mouse; C588124:RASL12 protein, mouse
Aldosterone; ChIP-seq; Distal convoluted tubules; Mineralocorticoid receptor