An anti-C/EBP antibody was used in this ChIP experiment, and a rabbit immunoglobulin G (rIgG) was used as a negative control. binding, resulting in myeloid differentiation and loss of leukemia maintenance GSK2838232A (5). We previously found that, in leukemic cells, AML1-ETO is usually stabilized and functions through the AML1-ETO-containing transcription factor complex (AETFC), which contains multiple transcription (co)factors that include AML1-ETO, CBF, E proteins HEB and E2A, hematopoietic bHLH transcription factor LYL1, LIM domain name protein LMO2 and its binding partner LDB1 (6). These AETFC components mutually stabilize each other and cooperatively bind and regulate target genes, and AETFC integrity and proper GSK2838232A conformation are essential for leukemogenesis (6). Thus, destabilization of AETFC provides a strategy to target AML1-ETO. Notably, it has been generally proposed that the stability of a protein complex can be reflected by its sensitivity to overexpression versus depletion of individual components (7). First, many complexes can be destabilized by overexpression of individual components that, in a dosage-dependent manner, make promiscuous interactions that switch the topology of the complex and thereby destabilize it. This mechanism, known as dosage sensitivity, is widely applicable to the regulation of protein functions in organisms ranging from yeast to human (8), including the interplay among the key transcription factors in hematopoiesis and leukemogenesis (9). Second, other complexes show a lack of sensitivity (termed robustness) to component overexpression, likely because they possess strong multivalent interactions that cannot be altered by dosage increase, but can be perturbed by depletion, of individual components (10). In this study, we investigated a means to destabilize AETFC, as well as the underlying mechanism. Following the principle explained above, we first examined whether overexpression of AETFC components could impact the stability of the complex. In addition, several known interacting partners of AETFC components, including C/EBP, TAL1 and ID1, were also analyzed. We transduced Kasumi-1 cells with retroviruses expressing HEB, E2A, E2-2, LDB1, LYL1, LMO2, C/EBP, TAL1 or ID1 (Supplementary Physique S1a), and decided the protein levels of each AETFC component by immunoblot. The results showed that overexpression of the AETFC components failed to destabilize the complex (Physique 1a). Thus, this result, in combination GSK2838232A with our previous observation that knockdown of AETFC components in Kasumi-1 cells prospects to degradation of the complex (6), displays the robustness of AETFC. This result is also consistent with the extremely strong biochemical stability of AETFC that we previously established (6). Open in a separate window Physique 1. Destabilization of AETFC by overexpression of C/EBP and its role in cell differentiation and leukemogenesis.(a) Immunoblot analysis of AETFC components in Kasumi-1 cells upon overexpression of indicated proteins. Note that overexpression of C/EBP, but not the AETFC components, prospects to a decrease of AETFC components. Overexpression of TAL1 or ID1 only decreases LYL1, suggesting different mechanism(s) relative to C/EBP. Asterisks denote the larger sizes of exogenous tagged proteins relative to the endogenous ones. (b) RNA-seq and GSEA (panel, data are offered as mean standard deviation (SD) of three impartial experiments with triplicates each time. (c) Myeloid differentiation of the AML1-ETO9a-expressing mouse leukemic cells ((panel, shown are Kaplan-Meier survival curves of indicated numbers of mice transplanted with 10 000 or 5 000 leukemic cells; values are calculated by the log rank test. Unexpectedly, overexpression of C/EBP dramatically decreased the protein levels of all AETFC components (Physique 1a) and led to an accompanying inhibition of Kasumi-1 cell growth (Supplementary Physique S1b). To verify the loss-of-function of AETFC, we performed RNA-seq of the cells. Gene set enrichment analysis (GSEA) revealed that previously recognized (6) Rabbit Polyclonal to Glucokinase Regulator effects of AETFC-loss on both the up- and downregulated target genes tend to be mimicked by GSK2838232A C/EBP overexpression; this was.