Biomedical subjects

Jane L Ko

Publications and source records attributed to Jane L Ko.

6 recordsLinked to original sources

PubMed2006

Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells.

Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

Animals↗

PubMed2006

Molecular basis underlying the poly C binding protein 1 as a regulator of the proximal promoter of mouse mu-opioid receptor gene.

Previous studies showed poly C binding protein 1 (PCBP) participating in the mu-opioid receptor (MOR) gene regulation via binding to a single-stranded (ss) DNA element. In this report, we therefore investigate the molecular basis of PCBP regulating the MOR gene expression. Various truncated PCBPs, including one domain (KH1, KH2, variable or KH3), two- (K12, K2v or Kv3) or three-sequential domains (K12v or K2v3), were constructed. The MOR ssDNA binding abilities of these truncated PCBPs were examined using electrophoretic mobility shift assay (EMSA). KH1 domain possessed a strong MOR ssDNA binding activity. Variable domain displayed no binding, and KH2 or KH3 domain possessed a weak MOR ssDNA binding activity. Binding of two-domain PCBPs indicated an additive effect of two-domain combinations. Interestingly, K2v3, a three-domain PCBP, displayed as strong ssDNA binding as that of K12v, suggesting synergism of KH2, KH3 and variable domains for the binding activity. Functional analysis demonstrated one-domain PCBPs exhibiting no transactivation on the MOR proximal promoter. Two-domain PCBPs displayed approximately 20% activity, while three-domain PCBPs displayed 70%-85% of full-length PCBP activity. Taken together, these results suggested that no single domain possessed sufficient functional activity to serve as an independent transactivation domain, and the combination of three sequential domains was necessary for its optimal activity to activate the MOR proximal promoter. In summary, our data suggested that cooperativity of three sequential domains is essential for PCBP functioning as a MOR gene regulator. Various ways in which this cooperativity could occur are discussed.

Animals↗

PubMed2006

Interplay of Sps and poly(C) binding protein 1 on the mu-opioid receptor gene expression.

The proximal promoter of mouse mu-opioid receptor (MOR) gene is the dominant promoter for directing MOR-1 gene expression in brain. Sp1/Sp3 (Sps) and poly(C) binding protein 1 (PCBP) bind to a cis-element of MOR proximal promoter. Functional interaction between Sps and PCBP and their individual roles on MOR proximal core promoter were investigated using SL2 cells, devoid of Sps and PCBP. Each factor contributed differentially to the promoter, with a rank order of activity Sp1>Sp3>PCBP. Functional analysis suggested the interplay of Sps and PCBP in an additive manner. The in vivo binding of individual Sps or PCBP to MOR proximal promoter was demonstrated using chromatin immunoprecipitation (ChIP). Re-ChIP assays further suggested simultaneous bindings of Sps and PCBP to the proximal promoter, indicating physiologically relevant communication between Sps and PCBP. Collectively, results documented that a functional coordination between Sps and PCBP contributed to cell-specific MOR gene expression.

Animals↗

PubMed2005

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.

Animals↗

PubMed2003

Role of an AP-2-like element in transcriptional regulation of mouse mu-opioid receptor gene.

Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse mu-opioid receptor (MOR) gene. In this study, we defined another functional element located the in -450 to -400 bp (translational start site designated as +1) region of the proximal promoter, which is also essential for the full promoter activity. It is designated as the morAP-2-like element for its sequence homologous to the consensus AP-2 element. Surprisingly, electrophoretic mobility shift analysis (EMSA) revealed that Sp1 and Sp3, but not AP-2 proteins, were specifically bound to the morAP-2-like element. Mutation of the morAP-2-like element, resulting in a loss of Sp binding, led to an approximately 35% decrease in activity, further confirming the positive role of the morAP-2-like element in MOR gene expression. Dephosphorylation of Sp proteins with alkaline phosphatase also decreased Sp binding to the morAP-2-like element in EMSA, suggesting phosphorylation of Sp is essential for its binding to this element. However, direct or indirect activation of PKA, a classical G-protein coupled signaling pathway, resulted in no significant change of Sp binding to the morAP-2-like element, nor of the promoter activity the SH-SY5Y cells, MOR expressing cells, suggesting that phosphorylation of Sp does not involve PKA. These results suggest that the binding of different phosphorylated forms of Sp proteins to the morAP-2-like element may contribute to the fine tuning of MOR expression in different cells.

Animals↗

PubMed2002

Differential promoter usage of mouse mu-opioid receptor gene during development.

Previously, we demonstrated that mouse mu-opioid receptor (MOR) gene expression is regulated by both distal and proximal promoters, with the latter playing a major role in controlling MOR transcription in the adult mouse brain. Here, we report studies of the relative usages of the mouse MOR dual promoters during murine development. We used the reverse transcription-polymerase chain reaction (RT-PCR) method, which gave results similar to those using binding assays or in situ hybridization. However, due to the greater sensitivity of RT-PCR method, we were able to detect the emergence of MOR as early as at embryonic day 8.5 (E8.5). We found that both proximal and distal promoters were active at E8.5. The proximal promoter initiated approximately two-thirds of total MOR transcripts at E8.5, with the distal promoter directing transcription of the remaining one-third. This is the greatest relative contribution of the distal promoter to MOR transcription we have observed during any time in development. Thereafter, the percentage of transcripts directed by the distal promoter gradually declined, and remained at a low but detectable level (approximately 5% of total MOR transcripts) throughout development and adulthood. Conversely, a progressive increase of the contribution of the proximal promoter to MOR transcription was observed during development, reaching its maximum in the adult. In summary, our results demonstrated the pivotal role of the proximal promoter in directing MOR transcription during murine development.

Aging↗