STAT5 outcompetes STAT3 to regulate the expression of the oncogenic transcriptional modulator BCL6

Abstract

Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.

Fig 1

STAT5 and STAT3 oppositely regulate BCL6 expression. (A) Lysates from SK-BR-3 cells that were untreated (−) or stimulated with prolactin (Prl) or LIF were immunoprecipitated (IP) for STAT5 or STAT3 and analyzed by immunoblotting for the phosphorylated form of each STAT. (B) RNA from SK-BR-3 cells stimulated with prolactin or LIF for 90 min was analyzed by quantitative reverse transcription (qRT)-PCR for BCL6 expression normalized to GAPDH (n = 3; *, P < 0.05). (C) Nuclear extracts from SK-BR-3 cells treated with prolactin for the indicated times were analyzed by immunoblotting for BCL6 expression. (D) (Top) Schematic map of the first two exons of the BCL6 gene and the locations of the regions (and primer pairs) analyzed in ChIP experiments. (Bottom) SK-BR-3 cells were stimulated with prolactin or LIF, and ChIP was performed using the indicated antibodies. Binding to the indicated sites was analyzed by qPCR relative to a negative-control binding region in the rhodopsin gene (n = 3; *, P < 0.05; **, P < 0.01). untx, untreated. The error bars indicate standard deviations.

Fig 2

STAT5 and STAT3 oppositely modulate RNA Pol II initiation. (A) SK-BR-3 cells stimulated with prolactin or LIF were analyzed by ChIP-qPCR using the indicated antibodies. Binding to the indicated sites was analyzed by qPCR relative to a negative-control binding region in the rhodopsin gene. (B) SK-BR-3 cells were stimulated with prolactin or LIF, and binding of BCL6 was analyzed by ChIP. (C) SK-BR-3 cells were stimulated with prolactin at the indicated time points, and BCL6, STAT5, and P-Pol 5 binding was analyzed by ChIP. The error bars indicate standard errors of the means.

Fig 3

Histone acetylation and PARP are not involved in STAT5-mediated repression of BCL6. (A) SK-BR-3 cells stimulated with prolactin or LIF were analyzed by ChIP using antibodies directed toward acetylated histone 4 (A-H4). (B) SK-BR-3 cells were pretreated with TSA or depsipeptide for 2 h and stimulated with prolactin for 90 min. BCL6 mRNA expression was analyzed by qRT-PCR relative to GAPDH. (C) SK-BR-3 cells were treated for 4 h with the PARP inhibitor 3-aba prior to prolactin stimulation. BCL6 mRNA expression was analyzed by qPCR relative to GAPDH (n = 2; *, P < 0.05). The error bars indicate standard errors of the means.

Fig 4

C/EBPβ is coordinately recruited by STAT5 and STAT3 to regulate BCL6 expression, whereas FoxA1 is oppositely recruited. (A) SK-BR-3 cells stimulated with prolactin or LIF were analyzed by ChIP using antibodies directed toward C/EBPβ (n = 2; **, P < 0.01). (B) SK-BR-3 cells were transfected with siC/EBPβ, and STAT phosphorylation (top) and BCL6 mRNA expression (bottom) were analyzed (n = 2). (C) SK-BR-3 cells stimulated with prolactin or LIF were analyzed by ChIP for FoxA1 binding to the indicated regions (n = 2; *, P < 0.05; **, P < 0.01). (D) SK-BR-3 cells transfected with siFoxA1 were analyzed by immunoblotting for the indicated proteins (left) or by qRT-PCR for BCL6 mRNA expression (right) (n = 2). The error bars indicate standard deviations.

Fig 5

STAT5 inhibits STAT3 binding to region B. (A) SK-BR-3 cells were stimulated with prolactin, LIF, or the combination for the indicated times, and BCL6 mRNA expression was analyzed. The error bars indicate standard errors of the means. (B) SK-BR-3 cells were stimulated as described above, and the nuclear and cytoplasmic locations of the indicated proteins were analyzed by immunoblotting. (C) SK-BR-3 cells were transfected with the STAT5/STAT3-responsive region B luciferase construct (TBR-Luc) or with the STAT3-responsive region A luciferase construct (A-Luc) and were then stimulated with prolactin, LIF, or the combination for 24 h and analyzed for luciferase activity (n = 2; *, P < 0.05). (D) Cells stimulated with prolactin, LIF, or the combination were analyzed for STAT5 and STAT3 binding to the indicated regions by ChIP. The error bars (C and D) indicate standard deviations.

Fig 6

STAT5 inhibits the effects of STAT3 on regulation of BCL6. SK-BR-3 cells stimulated with prolactin, LIF, or the combination were analyzed by ChIP using the indicated antibodies for binding to regions B and A and the intervening control region. The error bars indicate standard errors of the means.

Fig 7

STAT5 inhibition is dominant over STAT3 activation of BCL6. (A to C) SK-BR-3 cells were stimulated with LIF and increasing doses of prolactin. (A) Phosphorylation of STAT3 and STAT5 was measured by immunoblotting. (B) BCL6 mRNA expression was measured by qRT-PCR (n = 2; *, P < 0.05). (C) STAT5 and STAT3 binding was measured by ChIP. STAT3 binding with LIF only and STAT5 binding with prolactin only in region B were normalized to 100 (n = 2; *, P < 0.05). (D) ChIP was performed for binding of the indicated proteins to region B in MDA-MB-468 cells stimulated with prolactin (top) or the paired MDA-MB-468 sublines stably transfected with constitutively active STAT5a1*6 or empty vector (bottom). The error bars indicate standard deviations.