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Identification of an upstream enhancer containing an AML1 site in the human myeloperoxidase (MPO) gene
G E Austin1, W G Zhao, A Regmi
1Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, Decatur, GA 30033, USA. austin.garth@atlanta.va.gov
Abstract:
Myeloperoxidase (MPO) is an important antibacterial enzyme found only in granulocytes and monocytes. The human MPO gene is transcribed early during myelogenesis but MPO RNA synthesis ceases at the end of the promyelocyte stage of myeloid maturation. We recently identified a basal MPO promoter and several adjacent cis-elements in the proximal 5'-flanking region of this gene. Transfection studies using constructs containing several kb of 5'-flanking MPO DNA revealed the presence of a DNA segment located between bp (base pair) -4200 and bp -3800 with enhancer activity for the endogenous basal MPO promoter. Deletion studies revealed the core enhancer activity to lie between bp -4100 and bp -3844. The percentage enhancement of promoter activity is greater in MPO-expressing myeloid cells than in MPO-non-expressing myeloid cells or non-myeloid cells. Furthermore. the enhancer confers TPA- or DMSO-responsiveness upon either endogenous or exogenous promoters. DNase I footprinting and transfection experiments identified an AML1 site as a functionally important element within the enhancer. Gelshift competition and supershift experiments demonstrated the binding of the alpha subunit of the transcription factor AML1 to this site in HL-60 cells. This distal enhancer appears likely to play an important role in the control of MPO transcription during myeloid differentiation.
Insights
Researchers discovered a novel DNA enhancer region that controls the transcription of the myeloperoxidase (MPO) gene, crucial for myeloid differentiation and antibacterial functions.
Area of Science:
- Molecular Biology
- Hematology
- Gene Regulation
Background:
- Myeloperoxidase (MPO) is a key antibacterial enzyme produced by granulocytes and monocytes.
- MPO gene transcription occurs early in myeloid development and ceases at the promyelocyte stage.
Purpose of the Study:
- To identify and characterize regulatory elements controlling MPO gene transcription.
- To investigate the role of a distal enhancer in MPO gene expression during myeloid differentiation.
Main Methods:
- Gene transfection studies using MPO 5'-flanking DNA constructs.
- Deletion analysis to pinpoint core enhancer regions.
- DNase I footprinting, gelshift, and supershift assays to identify transcription factor binding sites.
Main Results:
- A distal enhancer with core activity between base pairs -4100 and -3844 was identified.
- This enhancer significantly boosts MPO promoter activity, especially in MPO-expressing myeloid cells.
- The enhancer confers responsiveness to TPA or DMSO and binds the AML1 transcription factor subunit.
Conclusions:
- A distal enhancer plays a critical role in regulating MPO transcription during myeloid differentiation.
- The AML1 transcription factor binding site within the enhancer is functionally important.
- Understanding these regulatory mechanisms can provide insights into myeloid cell development and function.