Identification and analysis of Il-2 induced Stat5 Target genes in Human CD4 and CD8 T cells

Signal transducers and activators of transcription (STAT) 5a and 5b, are key signalling<br/>proteins activated by the cytokine interleukin-2 (IL-2), and therefore critically regulates<br/>important immunological processes such as T cell homeostasis and immune-regulation.<br/>While the biological functions of STAT5 are well established from murine genetic<br/>studies, the downstream mediators of these proteins are poorly understood. In this study,<br/>an improved chromatin immunoprecipitation (ChIP)-cloning method, using magnetic<br/>microbeads for immunocapture of chromatin was developed to identify in-vivo STAT5a<br/>and STAT5b binding sites in fresh and activated primary human CD4 and CD8<br/>peripheral T cells.<br/>Six libraries were generated, which identified 329 STAT5a and/or STAT5b-specific<br/>binding sites of which 87% contained canonical GAS motifs, TTCN3GAA and/or<br/>TTN5AA. Genomic mapping of sites revealed the striking observation that the majority<br/>of STAT5-binding sites identified here mapped to intergenic (&gt;50kb upstream) or<br/>intronic, rather than promoter proximal regions. Bioinformatic analyses, using Gene<br/>Ontology programmes to annotate and functionally classify the genes associated with<br/>binding sites, predicted novel functions for STAT5 such as transport and metabolism, in<br/>addition to previously known functions such as cell differentiation, proliferation, signal<br/>transduction, apoptosis and development. Additionally, several target-genes were<br/>identified, whose aberrant functions are associated with malignant transformation of<br/>cells, consistent with the frequent dysregulation of STAT5 noted in various cancers.<br/>ChIP-PCR validation studies on a subset of sites from each library, demonstrated that<br/>98% were bonafide STAT5 binding sites. Kinetic gene expression analyses performed<br/>on 31 annotated genes, by qRT-PCR revealed 17 novel target-genes that were<br/>upregulated (76%) or downregulated (24%) following IL-2 stimulation, and included<br/>two lineage-specification factors, c-MAF and RORA. Given the importance of IL-2 in<br/>facilitating CD4 Th2 cell differentiation, the regulation of c-MAF by STAT5 was<br/>functionally characterised further using biochemical and molecular techniques. These<br/>studies revealed that the epigenetic regulation of c-MAF is dependent on STAT5 and<br/>IL-2 signalling. In conclusion, this study has identified a number of novel STAT5<br/>regulated genes downstream of IL-2 activation of T cells, and provides an insight into<br/>the various cellular functions regulated by these proteins.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2010

Author dc:creator

• Rani, Aradhana

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/efe24c7c-eb52-44f8-8e58-86b6ca0dfdfa

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/efe24c7c-eb52-44f8-8e58-86b6ca0dfdfa