Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene

• Published in: Biochimica et biophysica acta Vol. 1829; no. 2; pp. 231 - 238
• Main Authors: Yuan, Yuan, Chow, Billy K.C., Lee, Vien H.Y., Lee, Leo T.O.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-02-2013
• Subjects: Animals; Gene Expression Regulation; Humans; Neuron restrictive silencer factor; PC12 Cells; Promoter Regions, Genetic; Protein Binding; Rats; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Gastrointestinal Hormone - genetics; Receptors, Gastrointestinal Hormone - metabolism; Repressor Proteins - genetics; Repressor Proteins - metabolism; Secretin receptor; Sp-protein; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp3 Transcription Factor - genetics; Sp3 Transcription Factor - metabolism; Transcriptional Activation; Transcriptional regulation; Secretin receptor; NRSE; NRSF; TSA; shRNA; hSCTR; PANC-1; Sp-protein; ChIP; Transcriptional regulation; Neuron restrictive silencer factor
• ISSN: 1874-9399; 0006-3002; 1876-4320
• DOI: 10.1016/j.bbagrm.2012.11.002

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (−83 to −67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.
► The binding of NRSF to hSCTR was investigated by gel shift and ChIP assays. ► The interactions of NRSF with Sp1 and Sp3 were studied by co-immunoprecipitation. ► The Sp1 to Sp3 ratio mediates the silencing effects of NRSF on hSCTR. ► Treatment of TSA relieves”” that HDAC is key to the silencer activity of NRSF.