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

Abstract

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.

Introduction

Secretin (SCT), a brain-gut peptide belonging to the secretin/vasoactive intestinal peptide/glucagon peptide family, functions to stimulate secretion of bicarbonate, electrolytes and water from pancreatic ductal epithelial cells [1], while its effects on other gastrointestinal tissues including intestine [2], stomach [3] and liver [4] were also suggested. Recently, the neuroactive functions of secretin were studied. It was suggested that in the cerebellum, secretin acts as a retrograde messenger to facilitate GABA release from the presynaptic basket cells, either directly or indirectly via an unknown glutamate source, resulting in potentiating evoked inhibitory postsynaptic currents (IPSCs) in Purkinje cells [5], [6]. Recent evidences have suggested that SCT is potentially a neurohypophysial factor [7]. SCT and its receptor (SCTR) overlap with the functions of angiotensin II (ANGII), and more importantly, are needed in mediating the central actions of ANGII-induced responses [8].

The activities of secretin are mediated via a class II G protein-coupled receptor, secretin receptor (SCTR). Using human pancreatic ductal carcinoma (PANC-1) and bovine pancreatic ductal (BPD-1) cells as models, we have previously identified a 106-bp core promoter element (− 263 to − 158, relative to the ATG start codon) in the 5′ flanking region of the human secretin receptor (hSCTR) gene [9]. This core promoter is controlled by the competitive binding of specificity protein 1 and 3 (Sp1 and Sp3) with two functional GC boxes (− 240 to − 226 and − 203 to − 194). In addition, the methylation status of CpG dinucleotides in the CpG island which overlaps with the core promoter was also found to be a critical factor to mediate the cell-specific expression of the hSCTR [10].

To understand further the spatial and temporal expression of hSCTR, in this report, we sought to investigate the functions of a putative neuron restrictive silencer element (NRSE) located downstream (− 83 to − 67, relative to ATG) of the hSCTR core promoter. NRSE, also known as repressor element-1 (RE-1) with a consensus sequence “NTYAGMRCCNNRGMSAG” [11], was initially identified to regulate a number of neuron-specific genes by repressing their expressions in non-neural tissues [12]. Recently, NRSE is regarded a common repressor element as it can suppress an increasing number of non-neuronal genes. A genome-wide search indicated the presence of about 1800 putative NRSE sites in both human and mouse genomes [11]. The protein factor that interacts with NRSE is neuron-restrictive silencer factor (NRSF), which is a member of the zinc-finger GLi-Krüppel family [13]. There are three domains in NRSF: a Krüppel-type zinc-finger for binding NRSE, an N-terminal repressor domain for interacting with SIN3 transcription regulator (Sin3), and a C-terminal repressor domain for recruiting the corepressor element 1 silencing transcription factor (CoREST). Both Sin3 and CoREST interact with histone deacetylase containing complex to deacetylate core histone proteins, and as a consequence, they work together to silence target genes by forming condensed chromatin structures and mediates developmental stage-specific gene expression [14], [15], [16]. An in silico analysis indicates that all members of the secretin receptor family contain at least one putative NRSE-like motif in their 5’ flanking regions [17]. With the knowledge that most of NRSF-regulated genes also possess GC-boxes in their promoter regions [18], [19], for this reason, the functional relationships between Sp-protein(s) and NRSF in controlling hSCTR expression was investigated. Findings reported here not only provide crucial information regarding the cell-specific expression of hSCTR, but also are applicable to the understanding of other GC-box/NRSF co-regulated genes, including several members of the secretin receptor family.