Chromatin alterations during transformation of B cells by a constitutively active mutant of STAT5

The transcription factor signal transducer and activator of transcription 5 (STAT5) is activated conditionally and transiently by external stimuli. Thereupon, STAT5 modulates the transcription of its target genes, promoting cell survival and growth. Constitutive STAT5 activity has been shown to be oncogenic in hematopoietic cells. This correlates with the acquisition of cancer hallmarks, such as ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’. Chromatin dynamics is of pivotal importance for the regulation of transcriptional activity and DNA damage repair. Accordingly, cancer hallmarks are not only effected by oncogenic ‘driver’ alterations at the DNA level, but also at the chromatin level. Sustained DNA binding of constitutively active STAT5 might have distinct effects on chromatin, which might lead to ‘driver’ chromatin alterations and underlie its oncogenicity. The main goal of the present study was to identify ‘driver’ chromatin alterations and other ‘driver’ events during the oncogenesis process induced by constitutively active STAT5. The constitutively active STAT5 mutant STAT5A-1*6 was previously shown to induce oncogenesis in the IL-3-dependent pro-B cell line Ba/F3 by enabling cytokine-independent survival and growth. Specific aims of this study were to characterize the effects of STAT5A-1*6 expression on (i) cell survival and growth, (ii) expression of selected STAT5 target genes and (iii) chromatin rearrangements. To monitor the oncogenesis process, a stable Ba/F3 cell line – inducibly expressing STAT5A-1*6 upon doxycycline administration (Tet-on expression system) – was generated and validated. Short- and long-term STAT5A-1*6 induction experiments were conducted and STAT5A-1*6 protein levels and activation (Western blot), and cell phenotype in terms of survival, growth and genome stability (cell counting, flow cytometric analysis of cellular DNA content/cell cycle states) were analyzed. Expression of STAT5 target genes including Cis, Osm, Spi2.1, c-Myc, Pim-1, Id-1 and TNFRSF13b was investigated in parallel using RT-qPCR. As expected, STAT5A-1*6 expression enabled cytokine-independent survival and growth of Ba/F3 cells. Cell viability and proliferation rates increased gradually during the initial phase of induction. Interestingly, after 4–5 weeks of induction cell survival and growth no longer depended on STAT5A-1*6 expression. In addition, in one out of four experiments, STAT5A-1*6-expressing cells accumulated chromosomal aberrations. The correlation patterns of STAT5A-1*6 and STAT5 target gene expression suggested dose-dependent STAT5A-1*6-mediated transcriptional activation of STAT5 target genes, at least within the first few weeks of induction. Later on however, sustained expression of the STAT5 target genes c-Myc and Pim-1 became independent of STAT5A-1*6. Altogether, these observations suggest the acquisition of the ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’ cancer hallmarks, possibly due to continually accumulating ‘driver’ alterations upon sustained expression of STAT5A-1*6. Interestingly, using chromatin immunoprecipitation STAT5 DNA binding to Cis, Osm, Spi2.1, Id-1 and TNFRSF13b was correlated with a strong decrease in histone H3 occupancy, likely reflecting a loss in nucleosomes. This histone H3 loss was particularly prominent at the STAT5 binding sites, regardless of (i) their location within the gene locus, of (ii) transcriptional activation and of (iii) cytokine supplementation. In addition, sustained STAT5A-1*6 DNA binding patterns were associated with broadened histone H3 loss in regions distant from STAT5 binding sites. Taken together, these data strongly suggest that DNA binding of STAT5 causes a local nucleosome loss, and possibly a global nucleosome loss along its target genes. Accordingly, I propose a general STAT5-mediated chromatin decondensation mechanism leading to a nucleosome loss around STAT5 binding sites, at a step preceding transcriptional activation. The broadened STAT5A-1*6-associated histone H3 loss patterns also raise the possibility of distinct STAT5A-1*6-mediated ‘driver’ chromatin alterations, which might misregulate chromatin dynamics and, in turn, promote the acquisition of ‘driver’ DNA alterations (i.e. the ‘genomic instability’ cancer hallmark). Together, these DNA and chromatin alteration events might underlie the oncogenicity of constitutively active STAT5. Further characterization of these events might contribute to a better understanding of the mechanism of STAT5-mediated oncogenesis and possibly to the identification of novel molecular targets for the development of drugs against STAT5-associated cancers.