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The gene encoding the myeloid-related protein 14 (MRP14), a calcium-binding protein expressed in granulocytes and
H Melkonyan1, H A Hofmann, W Nacken
1Institute of Experimental Dermatology, University of Münster, von Esmarchstr. 56, D-48149 Münster, Germany.
Abstract:
Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are two Ca2+-binding proteins of the S-100 family highly abundant in myelomonocytic cells. The expression is not only dependent on the developmental status of the cell but also on the inflammatory situation in the tissue. In order to identify regulatory elements responsible for the high expression of MRP14 in myeloid cells, reporter gene constructs have been transfected into HL-60 cells, Mono Mac 6 cells, and L132 cells. We demonstrated that a DNA element in the first intron (positions 153-361) enhances the transcriptional activity of the homologous promoter and of the heterologous herpes simplex virus thymidine kinase promoter up to 37-fold. To further identify the functional site, the region between positions 153 and 192 was analyzed functionally using the thymidine kinase promoter. The region increased the expression in the same magnitude as the complete intron. This enhancer is highly conserved in the human and murine MRP genes, indicative of its involvement in the transcription of MRPs. Protein binding to the region is demonstrated using EMSA, DNA cross-linking, Southwestern blotting, and affinity purification. Affinity purification confirms that four proteins bind to the enhancer element.
Insights
Researchers identified a key DNA enhancer element in the first intron of the MRP14 gene. This element significantly boosts gene expression in myeloid cells and binds multiple proteins, suggesting its crucial role in regulating MRP14 transcription.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Myeloid-related proteins 8 and 14 (MRP8 and MRP14) are calcium-binding proteins of the S-100 family.
- These proteins are highly expressed in myelomonocytic cells and their expression is linked to cellular development and inflammation.
Purpose of the Study:
- To identify regulatory DNA elements responsible for high MRP14 expression in myeloid cells.
- To characterize the function and protein interactions of these regulatory elements.
Main Methods:
- Reporter gene assays using transfected HL-60, Mono Mac 6, and L132 cells.
- Functional analysis of specific DNA regions within the MRP14 gene intron.
- Electrophoretic mobility shift assay (EMSA), DNA cross-linking, Southwestern blotting, and affinity purification to study protein binding.
Main Results:
- A DNA element located in the first intron (positions 153-361) of the MRP14 gene enhances transcriptional activity up to 37-fold.
- A smaller region within this intron (positions 153-192) demonstrated similar enhancer activity.
- This enhancer region is conserved across human and murine MRP genes.
- Protein binding studies confirmed that four proteins interact with this enhancer element.
Conclusions:
- A conserved intronic enhancer element significantly drives MRP14 gene transcription in myeloid cells.
- This enhancer region is a critical regulatory site for MRP14 expression.
- The identified enhancer element and its associated proteins are important for understanding myeloid cell gene regulation.