Dynamic transcriptome changes during adipose tissue energy expenditure reveal critical roles for long noncoding RNA regulators
Fig 5
Transcription of brown adipose tissue–enriched lncRNA 10 (lncBATE10) is controlled by cAMP-cAMP response element-binding protein (Creb) signaling pathway.
(A) Real-time PCR analysis of the expression of lncBATE10 in primary brown adipocytes culture treated with cAMP and norepinephrine (NE) for 4 hours. n = 3 (B) Potential transcriptional factor binding sites in the lncBATE10 promoter region. Predicted by online program MatInspector (www.genomatix.de). The arrow indicates the transcriptional orientation. (C) LncBATE10 Promoter reporter assay. Promoter regions upstream of the transcriptional start site of lncBATE10 with different truncations were cloned into pGL3-Basic vector. Reporters were transfected into 293T cells. Thirty-six hours after transfection, cells were further treated with 1 uM forskolin for 2 hours and subjected to luciferase assay. Error bars are mean ± SEM, n = 3, *P < 0.05 (Student t test). (D) Four site-specific mutations were made in the functional Creb binding site to construct the mutant promoter. (E) 293T cells transfected with wide-type or mutant reporter were treated with a different dose of forskolin, followed by luciferase assay. Data were normalized by Renilla activities of a cotransfected pRL-CMV plasmid. Error bars are mean ± SEM, n = 3, *P < 0.05 (1-way ANOVA). (F) Chromatin immunoprecipitation (ChIP)-PCR with primers detecting the CREB binding site and a control region 3,000 bp upstream the promoter before and after forskolin treatment. The individual numerical values that underlie the summary data can be found in S13 Data.