Abstract
Signaling initiated by the BCR-ABL1 kinase causes chronic myelogenous leukemia (CML). Recently, we reported that expression of Fyn, a Src kinase, is heightened in CML cells and patient specimens and confers in vitro and in vivo proliferative advantages. Fyn is regulated by redox, and because BCR-ABL1 raises intracellular oxidant levels, which have been implicated in CML progression, we explored the molecular regulation of Fyn. Here we identify the transcription factors that drive redox- and BCR-ABL1-dependent Fyn expression. Promoter deletion analysis in 293T, BaF3, BaF3-p210, and K562 cells identified the region essential for basal transcriptional activity. Mutation of Sp1 and Egr1 binding sites within the essential region diminished Fyn promoter activity and identified Egr1 as conferring redox sensitivity. Gel shift and chromatin immunoprecipitation assays confirmed the binding of Sp1 and Egr1 to the promoter fragments. Importantly, knockdown of Sp1 or Egr1 with small interference RNA or inhibition of Sp1 binding by mithramycin A repressed Fyn protein expression. Our work is the first to define transcription factors that are responsible for endogenous, oxidative stress-dependent and BCR-ABL1-dependent Fyn expression.
Highlights
A less studied but intriguing feature of the BCR-ABL1 kinase is its ability to raise levels of intracellular reactive oxygen species (ROS) (6 – 8)
In keeping with the oxidant dependence of Fyn described in other cell types [15], K562 cells expressing BCR-ABL1 pulsed with 1 mM hydrogen peroxide for 1 h showed increased Fyn protein expression by ϳ1.4fold as measured by Western blotting and densitometric analysis (Fig. 1A)
We report that ROS are responsible for up-regulation of Fyn mRNA and protein levels by inducing expression of Egr1, a transcription factor that interacts with the Fyn promoter in an oxidant-dependent fashion
Summary
A less studied but intriguing feature of the BCR-ABL1 kinase is its ability to raise levels of intracellular reactive oxygen species (ROS) (6 – 8). ROS-dependent Transcription of Fyn Is within the Promoter Region Spanning Ϫ200 to Ϫ100 bp—Our previous studies, as well as data in Fig. 1 (B and C), show that the expression of BCR-ABL1 promotes Fyn mRNA and protein expression.
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