Genome-wide analyses reveal the extent of opportunistic STAT5 binding that does not yield transcriptional activation of neighboring genes

Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2012; 40(10):4461-72. DOI: 10.1093/nar/gks056
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ABSTRACT Signal Transducers and Activators of Transcription (STAT) 5A/B regulate cytokine-inducible genes upon binding to GAS motifs. It is not known what percentage of genes with GAS motifs bind to and are regulated by STAT5. Moreover, it is not clear whether genome-wide STAT5 binding is modulated by its concentration. To clarify these issues we established genome-wide STAT5 binding upon growth hormone (GH) stimulation of wild-type (WT) mouse embryonic fibroblasts (MEFs) and MEFs overexpressing STAT5A more than 20-fold. Upon GH stimulation, 23 827 and 111 939 STAT5A binding sites were detected in WT and STAT5A overexpressing MEFs, respectively. 13 278 and 71 561 peaks contained at least one GAS motif. 1586 and 8613 binding sites were located within 2.5 kb of promoter sequences, respectively. Stringent filtering revealed 78 genes in which the promoter/upstream region (-10 kb to +0.5 kb) was recognized by STAT5 both in WT and STAT5 overexpressing MEFs and 347 genes that bound STAT5 only in overexpressing cells. Genome-wide expression analyses identified that the majority of STAT5-bound genes was not under GH control. Up to 40% of STAT5-bound genes were not expressed. For the first time we demonstrate the magnitude of opportunistic genomic STAT5 binding that does not translate into transcriptional activation of neighboring genes.

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Available from: Daeyoup Lee, Sep 29, 2015
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    • "Nevertheless, the sheer number of sites bound by a given TF, often more than 10,000, has made it difficult to gain a global understanding of TF-mediated control of cell type identity. The question has been raised therefore whether a large proportion of binding events are " opportunistic " rather than " functional " (John et al, 2011; Zhu et al, 2012), where " functional " would refer to those binding events that are relevant in terms of transcriptional control processes. Indeed, some high profile studies have restricted their analysis of TF binding sites to the minority of sites located in the vicinity of genes that change expression following external stimulation or TF knock-down (Garber et al, 2012). "
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    ABSTRACT: Despite major advances in the generation of genome-wide binding maps, the mechanisms by which transcription factors (TFs) regulate cell type identity have remained largely obscure. Through comparative analysis of 10 key haematopoietic TFs in both mast cells and blood progenitors, we demonstrate that the largely cell type-specific binding profiles are not opportunistic, but instead contribute to cell type-specific transcriptional control, because (i) mathematical modelling of differential binding of shared TFs can explain differential gene expression, (ii) consensus binding sites are important for cell type-specific binding and (iii) knock-down of blood stem cell regulators in mast cells reveals mast cell-specific genes as direct targets. Finally, we show that the known mast cell regulators Mitf and c-fos likely contribute to the global reorganisation of TF binding profiles. Taken together therefore, our study elucidates how key regulatory TFs contribute to transcriptional programmes in several distinct mammalian cell types.
    The EMBO Journal 06/2014; 33(11). DOI:10.1002/embj.201386825 · 10.43 Impact Factor
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    • "Additionally, the magnitude of opportunistic genomic STAT5 binding does not necessarily translate into transcriptional activation of neighboring genes. For example, Zhu et al. demonstrated that STAT5 binding to promoter upstream sequences does not automatically convey STAT5 control over those genes [24]. Yamaji et al. also reported that STAT5 binding to promoter sequences was not necessarily an indicator for the overall expression of the respective genes [25]. "
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    ABSTRACT: Signal transducer and activator of transcription (STAT) comprises a family of universal transcription factors that help cells sense and respond to environmental signals. STAT5 refers to two highly related proteins, STAT5A and STAT5B, with critical function: their complete deficiency is lethal in mice; in humans, STAT5B deficiency alone leads to endocrine and immunological problems, while STAT5A deficiency has not been reported. STAT5A and STAT5B show peptide sequence similarities greater than 90%, but subtle structural differences suggest possible non-redundant roles in gene regulation. However, these roles remain unclear in humans. We applied chromatin immunoprecipitation followed by DNA sequencing using human CD4(+) T cells to detect candidate genes regulated by STAT5A and/or STAT5B, and quantitative-PCR in STAT5A or STAT5B knock-down (KD) human CD4(+) T cells to validate the findings. Our data show STAT5A and STAT5B play redundant roles in cell proliferation and apoptosis via SGK1 interaction. Interestingly, we found a novel, unique role for STAT5A in binding to genes involved in neural development and function (NDRG1, DNAJC6, and SSH2), while STAT5B appears to play a distinct role in T cell development and function via DOCK8, SNX9, FOXP3 and IL2RA binding. Our results also suggest that one or more co-activators for STAT5A and/or STAT5B may play important roles in establishing different binding abilities and gene regulation behaviors. The new identification of these genes regulated by STAT5A and/or STAT5B has major implications for understanding the pathophysiology of cancer progression, neural disorders, and immune abnormalities.
    PLoS ONE 01/2014; 9(1):e86790. DOI:10.1371/journal.pone.0086790 · 3.23 Impact Factor
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    • "A total of 823 of these gene/ BR matches, corresponding to 445 individual genes, were significantly different in both categories (Figure 8). If STAT3 binding did not influence DLBCL gene expression, we would expect the nature of STAT3 binding near a gene to have no correlation with its expression level (Zhu et al. 2012). Instead, we observe that an increase in STAT3 binding is correlated with an increase in expression in most of these pairs (83%, n = 686), an association that is highly significant at P = 2.3 · 10 228 (Table 4). "
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    ABSTRACT: STAT3 is a signal transducer that, when dysregulated, becomes a powerful oncogene found in many human cancers, including diffuse large B cell lymphoma. Diffuse large B cell lymphoma is the most common form of non-Hodgkin's lymphoma and has two major subtypes: germinal center B-cell-like and activated B-cell-like. When compared to the germinal center B-cell-like form, activated B-cell-like lymphomas respond much more poorly to current therapies and often exhibit overexpression or overactivation of STAT3. To investigate how STAT3 might contribute to this aggressive phenotype, we have integrated genome-wide studies of STAT3 DNA binding using ChIP-sequencing with whole-transcriptome profiling using RNA-sequencing. STAT3 binding sites are present near almost a third of all genes that differ in expression between the two subtypes, and examining the affected genes identifies previously undetected and clinically significant pathways downstream of STAT3 that drive oncogenesis. Novel treatments aimed at these pathways may increase the survivability of activated B-cell-like diffuse large B cell lymphoma.
    G3-Genes Genomes Genetics 10/2013; 3(12). DOI:10.1534/g3.113.007674 · 3.20 Impact Factor
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