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Catfish Oct2 binding affinity and functional preference for octamer motifs, and interaction with OBF-1
D A Ross1, M Lyles, B E Ledford
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
Developmental and Comparative Immunology
|July 13, 1999
Summary
Catfish Oct2 transcription factor binds DNA motifs with varying affinities. Its activity differs between catfish and mouse cells, and it cooperates with OBF-1 for transcriptional activation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Oct2 transcription factor plays a crucial role in regulating gene expression in B and T lymphocytes.
- Understanding the DNA-binding specificity and transcriptional activity of Oct2 across different species is essential for deciphering immune cell development and function.
Purpose of the Study:
- To investigate the DNA-binding affinity and transcriptional activation potential of the catfish Oct2 transcription factor.
- To compare the activity of catfish Oct2 in both homologous (catfish) and heterologous (mouse) cellular contexts.
- To explore the functional interaction between catfish Oct2 and the mammalian co-activator OBF-1.
Main Methods:
- Electromobility shift assays (EMSA) were employed to assess DNA-binding affinity.
- Surface plasmon resonance (SPR) was utilized to quantify the interaction kinetics between Oct2 and DNA motifs.
- Reporter gene assays were performed to measure transcriptional activation in catfish and mouse cell lines.
Main Results:
- Catfish Oct2 exhibited a slightly higher affinity for the consensus octamer motif (ATGCAAAT) compared to a variant motif (ATGtAAAT).
- Catfish Oct2 isoforms (alpha and beta) activated transcription similarly from both motifs in catfish B and T cells.
- In a mouse cell line, catfish Oct2 and mouse Oct2 showed enhanced transcriptional activation from the variant octamer motif.
- Catfish Oct2 demonstrated synergistic transcriptional activation with OBF-1 in catfish T cells, with OBF-1 activity being position-dependent (promoter vs. enhancer).
Conclusions:
- The DNA-binding specificity of catfish Oct2 is motif-dependent, with functional implications varying across species.
- Catfish Oct2 can interact with mammalian co-activators like OBF-1, suggesting conserved regulatory mechanisms.
- The localization of octamer motifs significantly influences OBF-1-mediated transcriptional activity in catfish cells.