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The transcription factor (TF) yes‐associated protein 1 (YAP1) is a major effector of the tumor suppressive Hippo signaling pathway and is also necessary to maintain pluripotency in embryonic stem cells. Elevated levels of YAP1 expression antagonize the tumor suppressive effects of the Hippo pathway that normally represses YAP1 function. High YAP1 expression is observed in several types of human cancers and is particularly prominent in cancer stem cells (CSCs). The stem cell TF Sox2, which marks and maintains CSCs in osteosarcomas (OSs), promotes YAP1 expression by binding to an intronic enhancer element and YAP1 expression is also crucial for the maintainance of OS stem cells. To further understand the regulation of YAP1 expression in OSs, we subjected the  YAP1  intronic enhancer to scanning mutagenesis to identify all DNA cis‐elements critical for enhancer function. Through this approach, we identified two novel TFs, GA binding protein (GABP) and myeloid zinc finger 1 (MZF1), which are essential for basal  YAP1  transcription. These factors are highly expressed in OSs and bind to distinct sites in the  YAP1  enhancer. Depletion of either factor leads to drastically reduced YAP1 expression and thus a reversal of stem cell properties. We also found that YAP1 can regulate the expression of Sox2 by binding to two distinct DNA binding sites upstream and downstream of the Sox2 gene. Thus, Sox2 and YAP1 reinforce each others expression to maintain stemness and tumorigenicity in OSs, but the activity of MZF1 and GABP is essential for  YAP1  transcription. S tem  C ells   2017;35:2340–2350

Myeloid Zinc Finger 1 and GA Binding Protein Co‐Operate with Sox2 in Regulating the Expression of Yes‐Associated Protein 1 in Cancer Cells