| Summary: | The transcription factors Runx1 and Runx3 are required for permanent silencing of CD4 in maturing CD8+ thymocytes. Runx binding to consensus sites within the CD4 silencer region is required for CD4 silencing post-positive selection. The Runx nuclear matrix targeting sequence (NMTS) is required for CD4 silencing and is implicated in binding to the histone acetyltransferase (HAT) p300, histone deacetylases (HDAC), and histone methyltransferase (HMT) SUV39H1 proteins. Epigenetic modifications of chromatin or post-translational modifications of Runx itself as a result of Runx association with these enzymes may be important for establishment of long-term CD4 silencing. In this study, we show that treatment of thymocytes with the HDAC inhibitor trichostatin A (TSA) and a resulting increase in histone acetylation in the CD4 silencer region results in an increase in CD4 silencing in a Runx-independent manner. We evaluated the role played by Runx lysines and their potential post-translational modification in CD4 silencing by mutational analysis of nine Runx lysine residues. Disruption of lysines within the Runt DNA binding domain known to reduce Runx DNA-binding activity resulted in CD4 derepression, indicating that Runx DNA-binding is required for CD4 silencing. Mutation of other lysines not involved with DNA-binding and reported to be acetylated or methylated did not affect CD4 silencing by Runx. The transduction of thymocytes with the C-terminally truncated Runx1 or Runx1 lacking the NMTS fused with the p300 HAT domain, HDAC1, or SUV39H1 resulted in CD4 derepression, indicating that enforced association of these individual enzymes with the Runx DNA-binding domain promotes CD4 transactivation rather than CD4 silencing. Profiling chromatin marks present under c-terminally truncated Runx1d.190 treatment conditions revealed H3K9me3/H3K4me3 coenrichment in CD4 promoter and silencer regions suggesting the involvement of a dynamic instruction profile in the establishment of CD4 silencing.
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