Induction of BATF gene expression by Stat3: Potential role in the differentiation of a myeloid leukemia cell line
Abstract
Previous studies from our laboratory have demonstrated that the Stat3-dependent induction of macrophage differentiation initiated by the treatment of M1 myeloid leukemia cells with leukemia inhibitory factor involves a dramatic up-regulation of BATF mRNA and protein. BATF, a basic leucine zipper (bZIP) transcription factor, is an inhibitor of AP-1 transcriptional activity and biological activity in mammalian cells. BATF transcription is controlled by a TATA-less promoter and we identified a single LIF-responsive transcription start point located 159, 137 and 66 bp downstream of 3 consensus binding sites for Stat3. EMSA using BATF promoter fragments containing site-directed mutations in these Stat3 consensus sites have demonstrated that only the -66 site binds Stat3 in response to LIF. A series of BATF promoter-luciferase reporter genes were constructed and were used to confirm the significant dependence of LIF-induced BATF transcription on the integrity of the -66 Stat3 binding site. Mutational analysis also revealed a partial dependence of LIF-induced BATF transcription on the integrity of a consensus AP-1 binding site located at position -82 just 5' to the identified Stat3 binding site. Subsequent ChIP analysis demonstrated direct binding of c-Jun as well as Stat3 to the BATF promoter. AP-1 reporter gene assays also revealed that AP-1 activity in general also is induced upon LIF treatment. This undoubtedly reflects the induction of the Jun family members (c-Jun, JunB and JunD) which are known to be rapidly induced and to remain elevated upon LIF treatment. The AP-1 reporter gene assays also demonstrated that AP-1 activity is modulated in a pattern that mirrors BATF protein expression. This modulation suggests a role for BATF in transiently regulating AP-1 target genes whose orderly transcription is critical to the control of macrophage differentiation. To test this hypothesis, RNA interference was used to knockdown BATF expression in M1 cells. Upon LIF treatment, M1/BATF-RNAi cells failed to display the morphological changes, growth arrest, and acquisition of phagocytic activity associated with the entry of M1 cells into the macrophage differentiation program. Finally, examination of gene expression revealed that induced expression of the cyclin dependent kinase inhibitor p21CIP1 is undetectable in M1/BATF-RNAi cells.
Degree
Ph.D.
Advisors
Taparowsky, Purdue University.
Subject Area
Molecular biology|Cellular biology
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