Identification of a minimal cis-element and cognate trans-factor(s) required for induction of Rac2 gene expression...

Rac2 is a Rho family GTPase that is widely expressed in hematopoietic cells and plays a critical role in host defense. This study investigates the mechanisms responsible for increased Rac2 gene expression during myeloid cell differentiation. Treatment of K562 chronic myelogenous leukemia cells with phorbol-12-myristate-13-acetate (PMA) induces megakaryocytic differentiation and Rac2 gene transcription following a lag of 6-12 h. Promoter/luciferase reporter gene assays reveal that a 135 bp cis-element located between -4223 and -4008 bp upstream of the Rac2 transcription start site is necessary and sufficient for PMA-induced gene expression. The AP1 transcription factor binds to three cis-elements within the 135 bp Rac2 gene regulatory region both in vitro and in vivo following PMA treatment, and mutagenesis of the AP1 binding sites ablates the PMA responsiveness of the 135 bp Rac2 gene regulatory region. Over-expression of AP1 is sufficient to induce expression of a transiently transfected Rac2 promoter/luciferase plasmid, but not the endogenous Rac2 gene. Induction of AP1 in vitro DNA-binding activity is apparent within 1 h of PMA stimulation. However, AP1 binding to the endogenous Rac2 promoter exhibits a lag of 5-9 h, which correlates with reduced histone H3-Lys9 methylation, increased histone H3 acetylation, and increased nuclease accessibility within the 135 bp Rac2 gene regulatory region. These results demonstrate that PMA induction of Rac2 expression during terminal myeloid differentiation requires the coordinate induction of transcription factors and remodeling of Rac2 gene chromatin structure.