Thesis Description

The Role of c-Myb During T Cell Activation

The proto oncogene c-myb is a transcription factor which regulates growth and differentiation of haemopoietic cells. c-myb is not expressed in resting peripheral T cells but it is upregulated in response to interleukin-2 stimulation. Previous studies using a T cell specific dominant negative myb allele, MEnT, showed that c-Myb is necessary for T cell activation. However, little is known about the genes c-Myb regulates. Furthermore, post-translational modifications of c-Myb mean that its expression cannot be directly correlated to its activity. This project aims to address both of these questions and hence gain an understanding of the role of c-Myb in T cell activation.

A tamoxifen inducible version of the interfering c-myb allele, MERT, was previously used to make a subtracted library of transcripts regulated by c-Myb in the murine haemopoietic progenitor cell line FDCP-mix. In order to identify transcripts regulated by c-Myb in T cells, the library was screened with cDNAs made from activated T cells. Also, MERT expressing FDCP-mix and EL4 cells were used to prepare probes for hybridisation to gene arrays. A list of putative target genes was generated and the expression of a small subset of these genes has been characterised in T cells.

MERT, under the control of the human CD2 promoter and LCR or the H-2K promoter, was used to examine of c-Myb activity in primary peripheral T cells. Resting MERT T cells have normal morphology. When activated in vitro they show reduced proliferation and increased apoptosis, but only in the presence of 4-OHT. The MERT T cells may also be used to validate putative Myb target genes. In support of this, CD43 and CD53, isolated as Myb targets in the FDCP-mix subtraction screen, are both downregulated in MERT T cells in the presence of 4-OHT.

To examine c-Myb activity in vivo, a transgene consisting of a modified green fluorescent protein (GFP) under the control of a promoter containing Myb binding sites was used to determine whether GFP expression correlated with c-Myb activity.