TRANSCRIPTIONAL ACTIVATION BY MEIS1A IN RESPONSE TO PKA SIGNALLING REQUIRES THE TORC FAMILY OF CREB CO-ACTIVATORS* Siew-Lee Goh 1,2** , Yvonne Looi 2,** , Hui Shen 2 , Jun Fang 1 , Caroline Bodner 1 , Martin Houle 1 , Andy Cheuk-Him Ng 3 , Robert A. Screaton 3 , and Mark Featherstone 2 From McGill Cancer Centre 1 , McGill University, Montreal, Quebec, Canada H3G 1Y6; School of Biological Sciences 2 , Nanyang Technological University, Singapore, 637551; Apoptosis Research Centre 3 , Children’s Hospital of Eastern Ontario, Department of Pediatrics and Department of Biochemistry, Immunology and Microbiology, University of Ottawa University of Ottawa, Ottawa, Canada K1H 8L1 Running head: PKA-inducible transactivation by MEIS1A requires TORCs Address correspondence to: Mark Featherstone, School of Biological Sciences, Nanyang Technological University, Room 03n-41, 60 Nanyang Drive, Singapore 637551; Ph: +65 6514 1007; Fax: +65 6791 3856; E-mail: msfeatherstone@ntu.edu.sg; or Robert Screaton, Apoptosis Research Centre, Children’s Hospital of Eastern Ontario, University of Ottawa, Room R3103, 401 Smyth Road, Ottawa, ON, Canada K1H 8L1, Ph: +1 613) 738 4180, Fax: +1 613 738 4833, E-mail: rob@mgcheo.med.uottawa.ca **These authors contributed equally to this work. The transcription factor encoded by the murine ecotropic integration site 1 gene, MEIS1, is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C-terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element binding protein (CREB), CREB binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators Transducers of Regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C- terminus, while chromatin immunoprecipitation (ChIP) revealed PKA- inducible recruitment of MEIS1, PBX1 and TORC2 on the MEIS1 target genes Hoxb1, Hoxb2 and Meis1. The MEIS1-interaction domain on TORC1 was mapped to the N- terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA- inducible transactivation through the MEIS1A C-terminus, suggesting a concerted action in developmental and oncogenic processes. The homeodomain is a DNA-binding structure shared by numerous transcription factors throughout eukaryotes, and is most commonly 60 amino acids in length (1). The three amino acid loop extension (TALE) class of homeoproteins is so named for an extra 3 residues in the loop between helices 1 and 2 in the typical homeodomain (2). Members of the TALE class in mammals include the MEIS, PREP and PBX families which participate in relatively complex interactions between themselves and with the products of another group of homeodomain-containing proteins, the HOX family (3). PBX proteins form cooperative DNA-binding heterodimers with MEIS, PREP or HOX proteins, and coordinate the formation of higher order heterotrimeric complexes of PBX, HOX and MEIS or PREP (3,4). DNA-binding PBX homodimers have also been noted, further extending the possible permutations for these partners (5). Targets of PBX•MEIS heterodimers include the bovine CYP17 gene (6), while those of PBX•MEIS•HOX trimers include the Hoxb1 autoregulatory element (ARE) and Hoxb2 rhombomere 4 (r4) enhancer (7-9). The Meis1 gene was identified near a site of frequent retroviral insertion leading to acute myeloid leukemia (AML) in BXH-2 mice (10). It has been further associated with human and mouse leukemias through frequent coordinated upregulation in these cancers, and http://www.jbc.org/cgi/doi/10.1074/jbc.M109.005090 The latest version is at JBC Papers in Press. Published on May 27, 2009 as Manuscript M109.005090 Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on April 21, 2016 http://www.jbc.org/ Downloaded from