Sarafova Lab 2007

Walker Shaw '10 and Matthew Surdel '10

Projects

Identifying a CD4 cell specific transcription factor that binds to the CD4 promoter

During T cell development in the thymus CD4-helper and CD8-killer T cells arise from a common thymocyte precursor. Cd4 gene expression is essential for the development of CD4-helper T cells and for their subsequent ability to mount an effective immune response to foreign antigens. At the same time Cd4 expression is extinguished on thymocytes that develop into CD8-killer T cells. This complex pattern of Cd4 regulation is primarily controlled by the activity of the Cd4 silencer, a cis-regulatory element for Cd4 expression, that silences Cd4 expression in CD8-killer T cells. In the absence of the CD4 silencer CD4 is inappropriately expressed in CD8-killer cells, but it never reaches the high level of CD4 expression normally present on CD4-helper cells. This indicates that there are additional differences in regulation of Cd4 expression between CD4 and CD8 cells that may elucidate how “killer” vs. “helper” transcriptional regulatory programs are established and maintained. In previous studies a Cd4 promoter binding activity was observed that was present in nuclear extracts of CD4, but not CD8 cells, implying that a CD4-cell specific transcription factor exists. This observation could explain the differences in Cd4 gene expression in the two T cell types. In this project we propose to purify and identify this potential candidate transcription factor using a biochemical approach. Nuclear extracts from CD4 and CD8 T cells will be made. After an initial enrichment for DNA-binding proteins by column chromatography, oligo-precipitation will be performed using the wild type vs. mutant binding sequence from the Cd4 promoter. The molecular weight of the unique protein(s) present in the CD4 cell nuclear extract will  be identified by SDS-PAGE and silver staining. In collaboration with the Chemistry department, further characterization and identification of the protein(s) can be done by tandem mass spectrometry.

Progress: We have partially purified proteins with a binding activity specific for the CD4 promoter sequence and are now in the process of identifying them. For details see Matt Surdel's poster

Does CD4 transcription differ in the immature (developing) versus mature CD4-helper T cells?

CD4 is a protein expressed on certain cells in the immune system, predominantly T- lymphocytes. These T-cells develop by entering the thymus as a Double Negative T-Cell Progenitor (no CD4 or CD8 expressed on their cell surface). In the thymus they develop into Double Positive T- Cells (both CD4 and CD8 expressed on cell surface), before differentiating into Single Positive CD4 (T-helper) or CD8 (T-cytotoxic) cells in the immune system. Throughout T-cell development, CD4 expression is regulated by several different known transcriptional elements, including a silencer, a promoter, a DP enhancer, a distal enchancer, and a proximal enhancer. In addition a second promoter for CD4 has been identified in the human and is proposed to be responsiblle for CD4 expression in macrophages and some dendritic cells, but its function during T cell development has not been investigated so far. In the moouse, it has been demonstrated that the DP enhancer is necessary for the expression of CD4 on the cell membrane. We speculate that there may be a different, unknown promoter region that is “turned on” by this DP enhancer and other transcriptional elements that are different from the promoter used for Cd4 expression in mature T-helper cells in the mouse.

Progress: We have identified a new potential transcription initiation site in the first intron of the Cd4 gene and are now in the process of sequencing the 5' end of the profuct. Whether this transcription ptoduct is only made by DP cells remains to be determined. For details see Walker Shaw's poster

Walker Shaw '10 and Scott Lester '10 at the 2007 Davidson College Summer Research Symposium

CD4-helper cells homeostasis

In most mammals more CD4-helper than CD8-killer T cells are produced and mainatained, making the ratio of CD4/CD8 T cell ratio bigger than 1 and varies between 1.3 and 1.9 in the peripheral blood of commonly used mouse strains, such as C57BL/6 and BALB/c. In some strains of mice the variation is a lot bigger (0.7 to 2) and includes mice with a CD4/CD8 ratiosmaller than one. We propose to investigate the genetic reasons and immunological consequences of this altered ratio.