Molecular analysis of the human thrombin-activatable fibrinolysis inhibitor gene promoter
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Published on: February 13, 2013
Mathieu Garand1, Nazareth Bastajian, Michael E Nesheim
1Department of Biochemistry, Queen's University, Kingston, ON, Canada.
Abstract:
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. Little is presently known of the factors that regulate expression of CPB2, the gene encoding TAFI. This study identified 10 potential transcription factor binding sites (denoted A-J) within the proximal promoter region of CPB2, spanning nucleotides -425 to +21; two of these represent previously-described binding sites for CCAAT/enhancer binding protein and glucocorticoid receptor. We identified additional transcription factors that bind within the proximal CPB2 promoter, namely, nuclear factor-Y (NF-Y) and hepatocyte nuclear factor-1alpha (HNF-1alpha). Binding of NF-Y to the region between nucleotides -76 to -59 (Site B) is important for basal CPB2 promoter activity; NF-Y may be a key factor for the recruitment of the transcriptional machinery to the TAFI gene promoter. HNF-1alpha binds at the interface between Sites C and B. Transient transfections of CPB2 promoter-reporter constructs showed that HNF-1alpha binding is essential for the activity of this promoter in HepG2 cells, indicating that HNF-1alpha is involved in the liver-specific expression of CPB2.
Insights
This study identifies key transcription factors, nuclear factor-Y (NF-Y) and hepatocyte nuclear factor-1alpha (HNF-1alpha), that regulate the CPB2 gene, which encodes thrombin-activatable fibrinolysis inhibitor (TAFI). These factors are crucial for TAFI gene expression and liver-specific activity.
Area of Science:
- Molecular Biology
- Gene Regulation
- Hemostasis and Thrombosis
Background:
- Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-enzyme that inhibits fibrinolysis upon activation.
- The regulatory mechanisms governing the expression of the CPB2 gene, which encodes TAFI, are not well understood.
Purpose of the Study:
- To identify transcription factors that bind to and regulate the proximal promoter region of the CPB2 gene.
- To elucidate the role of identified transcription factors in the basal and liver-specific expression of TAFI.
Main Methods:
- Bioinformatic analysis to identify potential transcription factor binding sites within the CPB2 promoter (-425 to +21).
- Electrophoretic mobility shift assays (EMSA) to confirm binding of nuclear factors.
- Transient transfection assays using CPB2 promoter-reporter constructs in HepG2 cells to assess promoter activity.
Main Results:
- Ten potential transcription factor binding sites (A-J) were identified in the CPB2 proximal promoter.
- Nuclear factor-Y (NF-Y) binds to Site B (-76 to -59) and is essential for basal CPB2 promoter activity.
- Hepatocyte nuclear factor-1alpha (HNF-1alpha) binds near Site B and is critical for CPB2 promoter activity in HepG2 cells, indicating a role in liver-specific expression.
Conclusions:
- NF-Y is a key regulator of basal CPB2 gene transcription, likely involved in recruiting the transcriptional machinery.
- HNF-1alpha plays a crucial role in mediating the liver-specific expression of the CPB2 gene.
- These findings provide new insights into the transcriptional regulation of TAFI.
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