A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

Abstract

Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.STAT proteins are latent transcription factors containing a Src homology 2 domain (SH2 domain) that become activated by tyrosine phosphorylation. The binding of the STAT SH2 domains to the phosphorylated cytokine receptors allows their tyrosine phosphorylation by Jak kinases. After dimerization and nuclear translocation, STAT dimers bind to specific DNA sequences, thereby allowing downstream gene regulation.

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line

“…In STAT5A7/7 mice, proliferation of macrophages in response to GM-CSF was shown to be a ected, and in mice where STAT5A and STAT5B genes were simultaneously inactivated, consistent reduction in the size and in the number of hematopoietic progenitor colonies induced by IL-3, GM-CSF and IL-5 were observed (Feldman et al, 1997;Teglund et al, 1998). Despite these disparate biological e ects, a few STAT5 regulated genes have been identi®ed in hematopoietic cells: CD25, oncostatin (OSM) and CIS genes that contain in their promoter region one or several STAT5 binding sites (Lecine et al, 1996;Matsumoto et al, 1997;Yoshimura et al, 1996;Verdier et al, 1998).…”

The role of Stat5a and Stat5b in signaling by IL-2 family cytokines

“…So far, eight members of this family of proteins have been identi®ed (Hilton et al, 1998;Kovanen and Leonard, 1999). Expression of CIS is regulated at least in part by cytokine-induced STAT proteins (Matsumoto et al, 1997;Verdier et al, 1998). The biological importance of this family of proteins in modulating cytokine signals was ®rst implicated by those in vitro studies and then further underscored by analyses of CIS transgenic mice and mice de®cient in SOCS1 and SOCS3.…”

The role of STATs in myeloid differentiation and leukemia

“…One of the ®rst STAT target genes to be identi®ed was the cytokine-inducible SH2-containing protein (CIS), subsequently found to be part of a large family of regulatory molecules (Yoshimura, 1998). CIS was found to be induced by a wide variety of cytokines including GM-CSF, IL-3 and EPO, and there are STAT-binding sites present in the CIS promoter (Matsumoto et al, 1997; Verdier et al, 1998). In bone marrow derived macrophages from STAT5a (7/7) mice, GM-CSF induced expression of CIS was greatly reduced demonstrating a critical role for STAT5a in its transcriptional regulation (Feldman et al, 1997).…”