Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Summary Precise intrathymic cell migration is important for thymocyte maturation and architecture. The orchestration of thymocyte trafficking, however, is not well understood at a molecular level. Here, we describe highly-regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes which is further affected by T cell receptor/co-receptor engagement. Activation of plexinD1 via one of its ligands, semaphorin3E, represses CCR9/CCL25 signaling in CD69+ thymocytes. In the absence of plexinD1, CD69+ thymocytes remain in the cortex, maturing to form ectopic SP thymocyte clusters in Plxnd1-deficient fetal liver cell-transplanted mice. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions is disrupted and medullary structures form under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients.