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Nuclear Receptors CAR, ERα and RORα Communicate via Phosphorylation to Differentially Regulate the SULT1E1 Gene Expression in Diabetic and Nondiabetic Human Livers
Author(s) -
Fashe Muluneh Markos,
Negishi Masahiko
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05583
Subject(s) - chromatin immunoprecipitation , microbiology and biotechnology , enhancer , biology , electrophoretic mobility shift assay , orphan receptor , reporter gene , gene expression , nuclear receptor , chromatin , gene , promoter , transcription factor , genetics
Studies showed that SULT1E1 is differentially expressed in nondiabetic and diabetic human as well as mouse livers. Here, we intended to investigate the molecular mechanism of the differential expression of SULT1E1 in diabetic and nondiabetic human livers. Utilizing chromatin immunoprecipitation (ChIP) in male human livers and coimmunoprecipitation (CoIP) assays, we report that nuclear receptors CAR, ERα and RORα communicated via phosphorylation of their respective DNA binding domains to differentially regulate the expression of SULT1E1 gene in nondiabetic and diabetic (type 2) male human livers. We studied the functional role of ERα and RORα with respect to SULT1E1 expression in human primary hepatocytes by treating the cells with siRNAs against ERα or RORα and SULT1E1 mRNAs were suppressed along with ERα or RORα mRNAs. Moreover, promoter analysis, electrophoretic mobility shift assays and cell‐based reporter assays in COS‐1 cells identified three DNA elements: ER α response element (h1E1‐ERE) and ROR α response element (h1E1‐RORE) within the enhancer region and a TATA box within the proximal promoter that were critical for the regulation of SULT1E1 gene in human liver cells. ChIP assays using antibodies against ERα, p‐Ser212 ERα, RORα or p‐Ser100 RORα and primers targeting the proximal SULT1E1 promoter revealed that both receptors were phosphorylated and enriched on the proximal region of SULT1E1 gene promoter in nondiabetic but not in diabetic male human livers. Similar observations were also made in ChIP assays performed with antibody against CAR. Moreover, CoIP assay in HepG2 cells overexpressing ERα WT or its ERα S212D mutant and RORα WT, RORα S100A or RORα S100D displayed a stronger interaction between ERα S212D and RORα S100D and this interaction was further enhanced with CAR cotransfection. Altogether, phosphorylation driven interaction between nuclear receptors CAR, p‐Ser212 ERα and p‐Ser100 RORα was critical for a positive expression SULT1E1 gene in human livers and lack thereof might be responsible for the observed downregulation of SULT1E1 in diabetic livers. Support or Funding Information NIEHS ‐ NIH Intramural Research Program