Past visits present: TCF/LEFs partner with ATFs for β-catenin-independent activity.

Perspective Past Visits Present: TCF/LEFs Partner with ATFs for b- Catenin–Independent Activity Stephanie Sprowl, Marian L. Waterman* Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California, United States of America TCF/LEF transcription factors are best known for their role as mediators of Wnt signaling, helping Wnt direct devel- opmental transitions of stem cells in tissues or driving cell transformation and cancer when Wnt is aberrantly active. These factors possess a High Mobility Group DNA-binding domain that recognizes a motif called the Wnt Response Element (WRE: 59-CTTTGWW-39) and an N- terminal domain that binds b-catenin (Figure 1A). b-catenin is the cytoplasmic- nuclear mediator that communicates Wnt signals from the plasma membrane to TCF/LEFs for transcription activation (Figure 1C). The vast majority of pub- lished studies about TCF/LEFs focus on their recruitment of this mediator to Wnt target genes. This implies that ‘‘life’’ for TCF/LEFs began in 1996 when yeast two hybrid screens identified their mutual, strong interaction [1–3]. In fact, discovery of TCF/LEFs had nothing to do with Wnt and b-catenin. TCF/LEFs were first described as DNA-binding proteins that regulated transcription of lymphocyte- specific genes such as the T-Cell Receptor complex, and they did so by cooperating with transcription factors bound to juxta- posed elements in enhancers [4–7]. The original TCF1 and LEF1 were each characterized as a set of protein isoforms, differing by the presence or absence of N- terminal (b-catenin) and C-terminal do- mains, none of which were needed for enhancer activity [8–12]. In this issue, Grumolato et al. [13] report that TCF1 and LEF1 have constitutive, elevated activity in leukemia and lymphoma cells. They report that this activity is indepen- dent of b-catenin and instead involves direct recruitment of ATF2 and related family members (Figure 1B). Grumolato et al. describe how the TOPflash reporter for Wnt signaling, a luciferase gene driven by a minimal promoter with multimers of WREs, has elevated constitutive activity in leukemia cell lines. One might assume that this activity derives from TCF/LEF recruit- ment of b-catenin. However, very little stabilized b-catenin could be detected and reporter activity was recapitulated using PLOS Genetics | www.plosgenetics.org truncated forms of TCF1 missing the N- terminal b-catenin–binding domain (iso- forms labelled dominant negatives, or dnTCF/dnLEF [Figure 1A]). In another twist, family member TCF4 could not substitute even though it has a b-catenin– binding domain. This meant that selective action by LEF1 and TCF1 occurred through recruitment of other transcription factors via domains distinct from the b- catenin–binding domain. Using a candi- date approach, the authors tested for functional interactions with proteins that bind AP1 sites. AP1 factors are homo- and heterodimerizing leucine zipper proteins of the Jun, Fos, ATF, and JDP families [14]. Grumolato et al. report that ATF family members (especially ATF2) bind directly to TCF1 and LEF1, not TCF4, and that interactions primarily require the Context Dependent Regulatory domain (CRD; Figure 1A, B). That TCF1/LEF1- ATF2 interactions are detected in multiple types of hematopoietic tumor cell lines suggests that ATF recruitment might account for a significant portion of the ‘‘Wnt reporter activity’’ in these cell types. Knockdown of ATF2 reduced cell growth and lowered expression of TCF1 and LEF1 target genes, similar to effects from overexpression of a dominant negative form of TCF4. Observations such as these suggest that ATF2 is integral to the regulatory role that TCF1/LEF1 play in lymphocytes. These discoveries highlight how TCF1/ LEF1 are closely intertwined with ATF proteins. Indeed, one of the first interac- tions for LEF1 and, later, TCF1 was with proteins that bind an ATF/CREB ele- ment in the T-Cell Receptor alpha chain enhancer (Figure 1D; [4,5]); interestingly, ATF4 was first discovered on the basis of its binding to this element (reviewed in [15]). Additional lymphocyte-specific en- hancers were discovered as collections of ATF/CREB, TCF/LEF, and ETS elements [15]), and functional studies showed that TCF1 and LEF1 cooperated with these proteins bound to neighboring elements to create strong enhancers. Importantly, the b-catenin–binding do- main was entirely dispensable, its deletion enabling even greater activity in some assays [9,12]. Instead, it was the CRD and a strong DNA-bending function of the HMG domain that was of primary importance; DNA bending enabling a three-way, CRD-dependent interaction between TCF/LEFs and other enhancer factors [16]. The exact identities of the ATF/CREB proteins were unknown and were never fully explored. The Grumolato study brings the past back to the present by identifying specific ATF interactors for TCF1 and LEF1 for the first time, and by showing that immune system cancers possess elevated, functional interactions. The current study does not highlight the DNA binding of ATF2 because it appears to be recruited by TCF1/LEF1 to the Wnt reporters in a protein–protein interaction mode. An emerging feature of TCF/LEFs that connects with these findings is a growing recognition of a functional split in the vertebrate family. That is, an increasing number of reports show that TCF4 and a fourth family member, TCF3, function as repressors, or at best, weak activators. More and more frequently it seems that TCF1 and LEF1 operate by opposing TCF3/TCF4 repression and providing strong activation. Since TCF1 and LEF1 Citation: Sprowl S, Waterman ML (2013) Past Visits Present: TCF/LEFs Partner with ATFs for b-Catenin– Independent Activity. PLoS Genet 9(8): e1003745. doi:10.1371/journal.pgen.1003745 Editor: H. Leighton Grimes, Cincinnati Children’s Hospital Medical Center, United States of America Published August 15, 2013 Copyright: s 2013 Sprowl, Waterman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this article. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marian.waterman@uci.edu August 2013 | Volume 9 | Issue 8 | e1003745