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Homeodomain proteins (homeoproteins) have long been recognized as powerful transcriptional regulators. Inappropriate expression of these transcription factors often leads to major developmental malformations or malignant transformation. The in vitro DNA binding sites of homeoproteins are short sequences that are widely distributed throughout the genome and some canonical binding sites have been shown to be functionally important at distances >20 kb away from the nearest transcription start site. In addition to DNA-binding activity, several homeoproteins have been demonstrated to interact with chromatin-modifying enzymes. For example, we and others have reported that the TLX1 homeoprotein of T-cell acute lymphoblastic leukemia (T-ALL) inhibits the PP1/PP2A serine/threonine phosphatases (I. Riz and R.G. Hawley, Oncogene 24: 5561–5575, 2005) and more recently have found that TLX1 modulates histone/transcription factor acetyltransferase CBP activity (I. Riz et al., Oncogene 26: 4115–4123, 2007). PP1/PP2A and CBP are complex molecular machines integrating diverse regulatory pathways that impact on cell survival, proliferation and differentiation outcomes. Organogenesis and malignant transformation - despite obvious differences - share a common requirement for high-order cooperativity of transcription factors and transcriptional cofactors in regulating the expression of multiple sets of genes executing cell fate shifts. Targeting key regulatory nodes in order to coordinately regulate multiple genes is a common strategy of virus induced cell-transformation: accordingly, PP1/PP2A and CBP are targeted by transforming viral proteins. The Groucho/TLE (transducin-like Enhancer-of-split) family of corepressors are another example of master regulators of cell fate; for instance, it was reported that triggering the MAPK signaling cascade inactivates TLE corepressors leading to coordinated derepression of a large number of genes involved in cell proliferation. We now demonstrate that TLX1 interferes with TLE1 repressive function. By streptavidin affinity-based precipitation of biotinylated recombinant TLX1 protein (TLX1 fused to a biotinylation peptide) we show in vivo interaction of TLX1 and TLE1 in several different cell types, including human T-ALL and neuroblastoma cells. Interaction of TLX1 with TLE1 occurs via an Engrailed homology 1 (Eh1)-like domain as documented by GST pull-down assays and laser scanning confocal microscopy. Transient transfection experiments indicate that TLX1 prevents TLE1-mediated repression of reporter genes. Furthermore, in the context of endogenous chromatin structure, TLX1 derepresses the bHLH transcription factor gene, ACSL1(HASH1), a well characterized target of the HES1/TLE1 repressor complex. The process requires direct interaction of TLX1 with TLE1 and binding of TLX1 to DNA, since a point mutation in the Eh1-like motif or deletion of the third helix of the TLX1 homeodomain abrogated the effect. Additional data to be presented suggest a long-range mechanism of transcriptional regulation by TLX1: we propose that “transcriptional activation” by TLX1 (and, by analogy, other homeoproteins that interact with TLE corepressors) results in part from the chaperoned redistribution of TLE corepressors from proximal promoter regions of target genes to distal chromatin regulatory sites.