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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4208-4221
Mpl Ligand Enhances the Transcription of the Cyclin D3 Gene: A
Potential Role for Sp1 Transcription Factor
Zhengyu Wang,
Ying Zhang,
Jun Lu,
Shinnshin Sun, and
Katya Ravid
From the Department of Biochemistry and Whitaker Cardiovascular
Institute, Boston University School of Medicine, Boston, MA.
Cyclin D3 plays a major role in the development of polyploidy in
megakaryocytes. The expression of cyclin D3 gene and the level of
cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057
megakaryocytic cell line, as indicated by Northern and Western blot
analyses, and by nuclear run-on assays and transfection experiments
with cyclin D3 promoter constructs. DNase I footprinting of the
promoter region showed protected segments, at  75 to 60 bp and at
134 to 92 bp, which display binding sites for the Sp family of
transcription factors. Gel mobility shift assay and supershifts with
specific antibodies indicate that Sp1 binds to these regions in the
cyclin D3 promoter and that Sp1 binding activity is significantly
increased by Mpl ligand. Mutation of either Sp1 site both decreases the
basal promoter activity and eliminates the induction by Mpl ligand. We
find that the nonphosphorylated form of SP1 has greater affinity for
the cyclin D3 promoter and that the majority of Sp1 in the cells is
nonphosphorylated. Mpl ligand treatment results in increased levels of
Sp1 protein, which also appears as nonphosphorylated. Okadaic acid,
which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a
phosphorylated form, decreases cyclin D3 gene expression and suppresses
Mpl ligand induction. Our data point to the potential of Mpl ligand to
activate at once several Sp1-dependent genes during megakaryopoiesis.
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