A SEQUENCE OF THE CIS GENE PROMOTER INTERACTS PREFERENTIALLY WITH 2 ASSOCIATED STAT5A DIMERS - A DISTINCT BIOCHEMICAL DIFFERENCE BETWEEN STAT5A AND STAT5B

Two distinct genes encode the closely related signal transducer and ac tivator of transcription proteins STAT5A and STAT5B. The molecular mec hanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 targ et genes, among them the CTS (cytokine-inducible SH2-containing protei n) gene, have been identified. We cloned the human CIS gene and studie d the human CIS gene promoter. This promoter contains four STAT bindin g elements organized in two pairs. By electrophoretic mobility shift a ssay studies using nuclear extracts of UT7 cells stimulated with eryth ropoietin, we showed that these four sequences bound to STAT5-containi ng complexes that exhibited different patterns and affinities: the thr ee upstream STAT binding sequences bound to two distinct STAT5-contain ing complexes (C0 and C1) and the downstream STAT box bound only to th e slower-migrating C1 band, Using nuclear extracts from COS-7 cells tr ansfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 t etramer and was dependent on the presence of STAT5A, STAT5B lacked thi s property and bound with a stronger affinity than did STAT5A to the f our STAT sequences as a homodimer (C0 complex), This distinct biochemi cal difference between STAT5A and STAT5B was confirmed with purified a ctivated STAT5 recombinant proteins. Moreover, me showed that the pres ence on the same side of the DNA helix of a second STAT sequence incre ased 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 e xplain the transcriptional regulation diversity of STAT5.