Abstract
Signal transducers and activators of transcription (STAT) 5a and 5b, are key signallingproteins 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.
| Date of Award | Apr 2010 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Sponsors | MRC Medical Research Council & GSK GlaxoSmithKline Research & Development Ltd |