2 research outputs found
Functional and Structural Profiling of the Human Thrombopoietin Gene Promoter*S⃞
Human thrombopoietin (TPO) is involved in cardiovascular disease as it
regulates megakaryocyte development and enhances platelet
adhesion/aggregation. The THPO promoter structure is still
controversial. By reverse transcription-PCR, we confirm that THPO
transcription is cell line-dependently initiated at two alternative promoters,
which we newly designated P1a and P1. We subsequently electrophoretically
scanned and resequenced these portions in 95 and 57 patients with
cardiovascular disease, respectively, and identified seven variants
(–1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A,
G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in
pGL3-basic vector, five molecular haplotypes (MolHaps1–5) were observed:
[A–622-C–413-C+5-G+115;
wild type (wt)],
[A–622-T–413-C+5-G+115],
[G–622-T–413-C+5-G+115],
[A–622-C–413-A+5-G+115],
[A–622-C–413-C+5-A+115],
and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4,
and 5 were significantly more active than wt (all p values ≤0.01)
in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity
(p < 0.0001 in HEK293T and p < 0.01 in HepG2, compared
with wt). Electrophoretic mobility shift assays revealed that A-622G and
C-413T individually differed from MolHaps in their DNA-protein interaction
patterns. Supershift and chromatin immunoprecipitation assays identified
CCAAT/enhancer-binding protein δ as the binding protein exclusively for
the –622A allelic portion