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The transcription factor ccaat/enhancer binding protein β (C/EBPβ) and miR-27a regulate the expression of porcine Dickkopf2 (DKK2).

ABSTRACT: Using Affymetrix porcine Gene-Chip analyses, we found that Dickkopf2 (DKK2), a WNT antagonist, is differentially expressed in pre-ovulatory follicles between Large White and Chinese Taihu sows. This study aims to identify the regulatory factors responsible for DKK2 expression. Deletion fragment and mutation analyses identified DKK2-D3 as the porcine DKK2 core promoter. There were four C/EBPβ binding sites within the DKK2 core promoter. The C allele that results from a spontaneous alteration (DKK2 c.-1130 T > C) in the core promoter was associated with a higher total number born (TNB) and a higher number born alive (NBA) in all parities in a synthetic pig population. This was possibly the result of a change in C/EBPβ binding ability, which was confirmed using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). Moreover, C/EBPβ specifically bound to and activated the DKK2 promoter, as revealed by mutation analysis, overexpression and RNA interference (RNAi) experiments. We also confirmed that miR-27a is a negative regulator of the DKK2 gene using miR-27a overexpression and inhibition experiments and mutation analyses. RTCA xCELLigence experiments showed that miR-27a suppressed Chinese hamster ovary (CHO) cell proliferation by down-regulating DKK2 gene expression. Taken together, our findings suggest that C/EBPβ and miR-27a control DKK2 transcription.

SUBMITTER: Tao H

PROVIDER: S-EPMC4675968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells.MethodsTo identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling.ResultsWe identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3.ConclusionsThese findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.

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The transcription factor ccaat/enhancer binding protein β (C/EBPβ) and miR-27a regulate the expression of porcine Dickkopf2 (DKK2).

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