Background: Nuclear factor of activated T cells 1 (NFAT1 or NFATc2) is proposed to plays a role in Graft versus host disease (GVHD) because it regulates the transcription of many cytokines and surface regulatory proteins including: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), IL-13, IFN-γ, CD40L, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α. NFAT1 regulates the expression of these genes through direct interaction with the leucine zipper region of AP-1 (Fos/Jun) and through cooperative binding to a 15-bp DNA sequence that contains both NFAT1 and AP-1 sites. The highly conserved basic regions of AP-1 (Fos/Jun heterodimer) bind to the TGTTTCA consensus sequence in DNA. Human Bach2 is also a basic-leucine zipper (bZip) protein. The amino acid residues in contact with the DNA in the basic regions of c-Jun and c-Fos are also identical in Bach2 (human and mouse). The high conservation of DNA-contacting amino acid residues between Bach2 and AP-1 strongly suggests that Bach2 (homodimers) and AP-1 (Fos/Jun heterodimers) can bind to the same DNA site, which may block the formation of NFAT1/AP-1/DNA complex and the subsequent expression of cytokine genes. Clinical studies have determined that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD, compared to Bone Marrow (BM), due in part to reduced donor T-cell cytokine production.

Methods: We compared the RNA and protein levels of NFAT1, c-Jun, c-Fos, and Bach2 in human UCB CD4+ T-cells with Adult Blood (AB) CD4+ T-cells by Quantitative RT-PCR analyses and Western blots. This comparison was done in both stimulated and non-stimulated conditions. Our ongoing interest is to test whether Bach2 binds the same DNA site as AP-1, and to test the potential of Bach2 and AP-1 competing for the same DNA site using Electrophoretic mobility shift assay (EMSA) with purified Protein of Bach2, c-Jun, and c-Fos from E. coli.

Results: Our data showed a high expression of Bach2 RNA in both non-stimulated and stimulated UCB CD4+T-cells. In both the non-stimulated comparison and stimulated comparison samples, we detected no significant changes in RNA levels of NFAT1, c-Jun, or c-Fos.

Conclusions: Our data support the hypothesis that Bach2 acts as a transcriptional repressor of cytokine genes due to the fact that highly expressed Bach2 in UCB competes with AP-1 for same DNA binding site.

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