In the meantime, work by Polakis, Clevers, Vogelstein and others had revealed that dysregulated Wnt\/beta-catenin signalling can be a major cancer driver, especially for colorectal cancer, and that hyperactive beta-catenin is a potent oncogene in many tissues. However, beta-catenin was thought to be undruggable, as there are no known enzymes to inhibit below beta-catenin. Indeed, the only known effectors downstream of beta-catenin were the TCF\/LEF factors, which bind to an extensive surface of beta-catenin that is shared with beta-catenin antagonists such as APC and Axin.<\/p>\n\n\n\n
We therefore designed a suppressor screen based on activated Armadillo expressed in the fly eye, to identify new factors required for its activity, expecting some of the suppressor hits to be co-activators of TCF that are conserved in humans. We thus discovered Pygopus (Pygo1 and Pygo2 in humans), a nuclear factor essential for the activity of activated Armadillo throughout fly development. Molecularly, Pygo proteins use their PHD fingers to recognise histone H3 tail tri-methylated at lysine 4 (an epigenetic hallmark for active transcription) while also associating with Armadillo\/beta-catenin through the Legless\/BCL9 adaptor discovered by the Basler group in 2002. <\/p>\n\n\n\n
Unexpectedly, Drosophila<\/em> Pygo turned out to be associated constitutively with Wingless-responsive loci through its chromatin-binding PHD finger. Remarkably, the requirement for pygo<\/em> function can be overcome by high nuclear concentrations of Armadillo or by genetic deletion of the transcriptional co-repressor Groucho (known to bind TCF). We therefore proposed the \u2018Armadillo-loading model\u2019, according to which Pygo acts as an anti-repressor, by associating with Wingless-responsive genes in the absence of Wingless signalling to poise Wingless-responsive enhancers for signal activation. Pygo could thus capture Armadillo through the Legless\/BCL9 adaptor and load it onto TCF by displacing the Groucho co-repressor (which, like its mammalian TLE orthologs, was known to keep Wnt-responsive genes inactive in the absence of Wingless\/Wnt signalling). <\/p>\n\n\n\n While our Armadillo-loading model incorporated everything we knew at the time about Pygo and other nuclear factors mediating Wnt-dependent transcription, it contained one glaring unknown \u2013 namely the physical link (\u2018factor X\u2019) that guides Pygo to TCF target genes in the absence of Wnt signalling. Factor X was likely to bind to the N-terminal NPF signature motif of Pygo, essential for its function in flies and required for its binding to TCF target genes. However, identifying this NPF-binding factor proved to be a formidable challenge, which took several years to solve: unexpectedly, factor X turned out to be a complex between two proteins, which we called ChiLS. We discovered ChiLS in a proteomics screen as a complex that binds directly and specifically to the NPF motif of Pygo. ChiLS comprises a homo-dimer of a large protein called Chip\/LIM-homeodomain binding protein (Chip\/LDB) and a homo-tetramer of Single-strand DNA-binding protein (SSDP, also known as SSBP). The ChiLS complex was already known from mammalian haematopoietic cells in which it associates with the Locus Control Region of the beta-globin gene cluster and other remote enhancers, exerting key functions in haematopoiesis. ChiLS is tethered to its cognate enhancers by sequence-specific enhancer-binding proteins, either LIM homeodomain proteins to which it binds directly, or bHLH or GATA factors to which it binds via LIM-only (LMO) adaptors. In the murine intestine, Ldb1<\/em> was also known to function in the Wnt-dependent crypt stem cell compartment, whereas in fly imaginal discs, Chip<\/em> and ssdp<\/em> had been implicated in Notch and Wingless responses of remote transcriptional enhancers. Importantly, LIM-only 2 (LMO2) is an oncogene that, upon inappropriate expression in T cells, drives acute leukaemia by blocking their normal T cell differentiation programme.<\/p>\n\n\n\n We discovered that ChiLS is the core component of a multi-protein complex termed the Wnt enhanceosome, which associates with the Ultrabithorax<\/em> and labial<\/em> midgut enhancers via Groucho-TCF to drive a Wnt-dependent transcriptional programme. ChiLS thus primes these HOX genes for responses to Wingless and its cooperating signals, and for ON-OFF switches that integrate their inputs with those from lineage-specific factors, including LIM and LMO proteins, GATA and bHLH factors. Its scaffold is BCL9\/Legless, a large intrinsically disordered protein that links beta-catenin with Pygo through its direct binding to ChiLS and Groucho\/TLE. Our work in flies and human cells thus revealed that the Wnt enhanceosome imparts context-specificity on Wnt-responsive selector genes, providing a molecular explanation for the context-dependence of TCF\/LEF factors. Notably, Notch is one of the signals feeding into the Wnt enhanceosome, priming it for the OFF state and reinstalling Groucho\/TLE-dependent repression on Wnt target genes upon cessation of Wnt signalling.<\/p>\n\n\n\n We also discovered Hyd\/UBR5 as a HECT E3 ubiquitin ligase essential for Wnt-dependent derepression of Wnt target genes. In Drosophila<\/em> imaginal discs, the hyd<\/em> phenotype mimics that of pygo<\/em>, and its function for Wingless responses is partially bypassed in the absence of groucho<\/em>. In human cells, UBR5 ubiquitylates TLE during Wnt signalling, thereby relieving TLE-dependent repression of Wnt target genes, possibly via remodelling compacted chromatin.<\/p>\n\n\n\n Our genetic analysis of the mouse intestine revealed that pygo<\/em> and bcl9<\/em> synergise to drive stem cell-like transcriptional programmes in Apc<\/em> mutant tumours while antagonising their cell differentiation. Remarkably, loss of Bcl9 essentially cured Apc<\/em> mutant mice (developed by the Tomlinson group as the prime model for early-stage colorectal cancer) of their neoplastic disease, by suppressing tumourigenesis and restoring a near-healthy life span in these otherwise moribund mice. This indicated the potential of BCL9 as a therapeutic target at the early stages of colorectal cancer.<\/p>\n\n\n\n We have recently determined the architecture of the ChiLS core complex and the position of its Pygo-binding pocket and proposed a structural model, now validated by X-ray crystallography in collaboration with Wenqing Xu (ShanghaiTech University). This work will pave the way towards determining the structure of the complete Wnt enhanceosome.<\/p>\n\n\n