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Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive
1Centre for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Australia.
Abstract:
The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation.
Insights
The c-fms gene, crucial for macrophage development, is regulated by intron 2 sequences. These sequences control transcription elongation, impacting macrophage-specific gene expression and responsiveness to stimuli like LPS and PMA.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- The c-fms protooncogene encodes the receptor for macrophage colony-stimulating factor 1 (CSF-1).
- c-fms is selectively expressed in mononuclear phagocytes and trophoblasts, with exon 1 specific to trophoblasts.
Purpose of the Study:
- To investigate the regulatory mechanisms controlling c-fms gene expression in macrophages.
- To identify sequences responsible for macrophage-specific expression and responsiveness to CSF-1, LPS, and PMA.
Main Methods:
- Isolation and sequencing of murine c-fms genomic DNA flanking exon 2.
- Reverse transcriptase primer extension analysis and RNase protection assays.
- Reporter gene assays using c-fms promoter constructs with and without intron 2.
Main Results:
- A TATA-less promoter with homology to human c-fms was identified, with multiple transcription initiation sites.
- CSF-1, LPS, and PMA acutely down-regulated full-length c-fms mRNA in macrophages, leading to increased unspliced, truncated transcripts.
- Intron 2 sequences conferred macrophage-specific expression and repression by LPS and PMA when incorporated into a c-fms promoter-reporter construct.
Conclusions:
- Macrophage-specific expression of c-fms is regulated by sequences within intron 2.
- These intron 2 sequences appear to control c-fms expression by modulating transcription elongation.