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Title: Identification and analysis of Il-2 induced Stat5 Target genes in Human CD4 and CD8 T cells
Author: Rani, Aradhana
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2010
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Signal transducers and activators of transcription (STAT) 5a and 5b, are key signalling proteins activated by the cytokine interleukin-2 (IL-2), and therefore critically regulates important immunological processes such as T cell homeostasis and immune-regulation. While the biological functions of STAT5 are well established from murine genetic studies, the downstream mediators of these proteins are poorly understood. In this study, an improved chromatin immunoprecipitation (ChIP)-cloning method, using magnetic microbeads for immunocapture of chromatin was developed to identify in-vivo STAT5a and STAT5b binding sites in fresh and activated primary human CD4 and CD8 peripheral T cells. Six libraries were generated, which identified 329 STAT5a and/or STAT5b-specific binding sites of which 87% contained canonical GAS motifs, TTCN3GAA and/or TTN5AA. Genomic mapping of sites revealed the striking observation that the majority of STAT5-binding sites identified here mapped to intergenic (>50kb upstream) or intronic, rather than promoter proximal regions. Bioinformatic analyses, using Gene Ontology programmes to annotate and functionally classify the genes associated with binding sites, predicted novel functions for STAT5 such as transport and metabolism, in addition to previously known functions such as cell differentiation, proliferation, signal transduction, apoptosis and development. Additionally, several target-genes were identified, whose aberrant functions are associated with malignant transformation of cells, consistent with the frequent dysregulation of STAT5 noted in various cancers. ChIP-PCR validation studies on a subset of sites from each library, demonstrated that 98% were bonafide STAT5 binding sites. Kinetic gene expression analyses performed on 31 annotated genes, by qRT-PCR revealed 17 novel target-genes that were upregulated (76%) or downregulated (24%) following IL-2 stimulation, and included two lineage-specification factors, c-MAF and RORA. Given the importance of IL-2 in facilitating CD4 Th2 cell differentiation, the regulation of c-MAF by STAT5 was functionally characterised further using biochemical and molecular techniques. These studies revealed that the epigenetic regulation of c-MAF is dependent on STAT5 and IL-2 signalling. In conclusion, this study has identified a number of novel STAT5 regulated genes downstream of IL-2 activation of T cells, and provides an insight into the various cellular functions regulated by these proteins.
Supervisor: Not available Sponsor: MRC ; GSK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available