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Single-stranded DNA-binding complex involved in transcriptional regulation of mouse mu-opioid receptor gene
1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. koxxx001@maroon.tc.umn.edu
Abstract:
Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is approximately 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.
Insights
Mouse mu-opioid receptor (mor) gene transcription is regulated by both single-stranded (ss) and double-stranded (ds) DNA binding factors. A novel protein, mor polypyrimidine-binding protein (mPy), interacts with ss DNA and is crucial for mor gene activation.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- The mouse mu-opioid receptor (mor) gene has dual promoters (proximal and distal).
- Proximal promoter activity is critical for mor transcription in the mouse brain.
- Sp factors binding to double-stranded (ds) cis-regulatory elements are known to be essential for proximal promoter activity.
Purpose of the Study:
- To investigate the role of single-stranded (ss) cis-regulatory elements and associated protein factors in proximal mor promoter activity.
- To identify novel protein factors involved in the regulation of mouse mor gene transcription.
Main Methods:
- S1 nuclease sensitivity assays to detect ss DNA conformation in the mor polypyrimidine/polypurine (PPy/u) region.
- Electrophoretic mobility shift assays (EMSA) using nuclear extracts from SH-SY5Y cells.
- Southwestern blot analysis to determine the size of interacting proteins.
- Functional analysis of promoter activity.
Main Results:
- A 26-bp mor PPy/u region can adopt an ss DNA conformation.
- A novel nuclear protein, mor polypyrimidine-binding protein (mPy), specifically binds to the sense strand of the PPy/u region.
- mPy protein (approximately 25 kDa) is distinct from Sp factors.
- mPy protein can trans-activate both the mor promoter and a heterologous promoter.
- Combinatorial binding of ss (mPy) and ds (Sp) DNA binding factors to overlapping regions is necessary for proximal promoter activation.
Conclusions:
- Mouse mor gene transcription is regulated by a complex interplay between ss and ds DNA binding factors.
- mPy represents a novel trans-acting factor crucial for proximal mor promoter activation.
- The findings reveal a new layer of gene regulation involving ss DNA binding proteins in opioid receptor gene expression.