Poly C binding protein, a single-stranded DNA binding protein, regulates mouse mu-opioid receptor gene expression

Previously, a single-stranded (ss) DNA element, polypyrimidine (PPy) element, was found to be important for the proximal promoter activity of mouse micro-opioid receptor (MOR) gene in a neuronal cell model. In this study, we identified the presence of unknown ssDNA binding proteins specifically bound to MOR ssPPy element in the mouse brain, implicating the physiological significance of these proteins. To identify the ssDNA binding proteins, yeast one-hybrid system with PPy element as the bait was used to screen a mouse brain cDNA library. The clone encoding poly C binding protein (PCBP) was obtained. Its full-length cDNA sequence and protein with molecular weight approximately 38 kDa were confirmed. Electrophoretic mobility shift analysis (EMSA) revealed that PCBP bound to ssPPy element, but not doubled-stranded, in a sequence-specific manner. EMSA with anti-PCBP antibody demonstrated the involvement of PCBP in MOR ssPPy/proteins complexes of mouse brain and MOR expressing neuroblastoma NMB cells. Functional analysis showed that PCBP trans-activated MOR promoter as well as a heterologous promoter containing MOR PPy element. Importantly, ectopic expression of PCBP in NMB cells up-regulated the expression level of endogenous MOR gene in vivo in a dose-dependent manner. Collectively, above results suggest that PCBP participates in neuronal MOR gene expression.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.     

Sp1 and Sp3 regulate basal transcription of the survivin gene

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. In this study, we cloned and identified the proximal 269 bp promoter of survivin gene, which exhibited strong promoter activity in HeLa cells. The TATA-less, GC-rich promoter contains 7 putative binding sites for Sp1, two of which (one at position -148 to -153, the other at position -127 to -140) are essential in regulating basal survivin promoter activity. Not only Sp1 but also Sp3 can activate the survivin promoter, which were proven by EMSA, blocking Sp1 or Sp3 using RNAi or mithramycin treatment of HeLa cells, and overexpression of Sp1 or Sp3. Our results collectively suggest that Sp1 cooperates with Sp3 to regulate survivin promoter activity.     

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression.