Search

Wnt signaling drives nuclear translocation of β-catenin and its subsequent association with the DNA-bound TCF/LEF transcription factors, which dictate target gene specificity by recognizing Wnt responsive elements across the genome. β-catenin target genes are therefore thought to be collectively activated upon Wnt pathway stimulation. However, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. Here we followed Wnt target gene expression in human embryonic stem cells after Wnt pathway stimulation at a single-cell resolution. Cells changed gene expression program over time consistent with three key developmental events: i) loss of pluripotency, ii) induction of Wnt target genes, and iii) mesoderm specification. Contrary to our expectation, not all cells displayed equal amplitude of Wnt target gene activation; rather, they distributed in a continuum from strong to weak responders when ranked based on the expression of the targetAXIN2. Moreover, highAXIN2did not always correspond to elevated expression of other Wnt targets, which were activated in different proportions in individual cells. This uncoupling of Wnt target gene expression, which was also identified in single colorectal cancer cells with hyper-activated Wnt signaling, underlines the necessity to identify additional mechanisms that explain the heterogeneity of the Wnt/β-catenin-mediated transcriptional outputs in single cells.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions. For this reason, we have compiled blacklists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls by the CUT&RUN peak caller SEACR. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN blacklist regions can persist even when peak calling is performed with SEACR against a negative control, and after ENCODE blacklist removal. Moreover, we experimentally validated the CUT&RUN Blacklists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture >80% of the peaks identified. We propose that removing these problematic regions prior to peak calling can substantially improve the performance of SEACR-based peak calling in CUT&RUN experiments, resulting in more reliable peak datasets.

Wnt signaling orchestrates gene expression via its effector β-catenin. Whether β-catenin targets genomic regions simultaneously or in a temporal fashion, and how this impacts the chromatin dynamics to modulate cell behavior, is currently unknown. Here we find that β-catenin binds different loci at each time-point after stimulation, implying that the definition of Wnt-targets is fundamentally temporal. This process is intrinsically cell-type specific. In fact, Wnt/β-catenin progressively shapes the chromatin of human embryonic stem cells consistent with their mesodermal differentiation: we call this genomic response plastic. In embryonic kidney cells, on the other hand, Wnt/β-catenin drives a transient chromatin opening, followed by a re-establishment of the pre-stimulation state: a response that we define elastic. Finally, the Wnt-induced transient chromatin opening requires β-catenin, suggesting a previously unappreciated pioneering role for this molecule. We submit that the plastic-vs-elastic behavior constitutes part of the mechanism explaining how Wnt/β-catenin drives divergent cell-fate decisions during development and homeostasis.

Upon WNT/β-catenin pathway activation, stabilized β-catenin travels to the nucleus where it associates with the TCF/LEF family of transcription factors, which constitutively bind to genomic Wnt Responsive Elements (WREs), to activate transcription of target genes. Discovering the binding profile of β-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage Under Target and Release Using Nuclease (CUT&RUN) has recently emerged as a prime technique for mapping the binding profile of chromatin interacting proteins. In our attempts to profile different regulators of the WNT/β-catenin transcriptional complex, CUT&RUN performed reliably when targeting transcription factors such as TCF/LEF, but it failed to produce consistent binding patterns of the non-DNA-binding β-catenin. Here, we present a biochemical modification of the CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible β-catenin binding profiles. CUT&RUN-LoV-U uncovers direct WNT/β-catenin target genes in human cells, as well as in ex vivo cells isolated from developing mouse tissue. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested and is well suited for simultaneous processing of several samples. We submit that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/β-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

The forkhead box transcription factor FOXQ1 is aberrantly induced in various cancers, and contributes to tumour growth and metastasis. It has been suggested that FOXQ1 exacerbates cancer by activating the oncogenic Wnt/β-catenin signalling pathway. However, the mode of action of FOXQ1 in the Wnt pathway remains to be resolved. Here, we report that FOXQ1 is a bimodal transcriptional activator of Wnt target gene expression in normal and cancer cells. Using co-immunoprecipitation, proximity proteomics, and reporter assays, we show that FOXQ1 engages the Wnt transcriptional complex to promote gene expression via TCF/LEF transcription factors. In parallel, FOXQ1 differentially regulates the expression of Wnt target genes independently of β-catenin and TCF/LEFs, which is facilitated by spatially separated activator and repressor domains. Our results suggest that FOXQ1 is a novel component of the Wnt transcriptional complex that reinforces and specifies Wnt signalling in a context-dependent manner.

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

Genetic alterations in humanBCL9genes have repeatedly been found in congenital heart disease (CHD) with as-of-yet unclear causality. BCL9 proteins and their Pygopus (Pygo) co-factors can participate in canonical Wnt signaling via binding to β-catenin. Nonetheless, their contributions to vertebrate heart development remain uncharted. Here, combining zebrafish and mouse genetics, we document tissue-specific functions in canonical Wnt signaling for BCL9 and Pygo proteins during heart development. In a CRISPR-Cas9-based genotype-phenotype association screen, we uncovered that zebrafish mutants forbcl9andpygogenes largely retain β-catenin activity, yet develop cardiac malformations. In mouse, both systemic and lineage-specific loss of the Pygo-BCL9-β-catenin complex caused heart defects with outflow tract malformations, aberrant cardiac septation and valve formation, and compact myocardium hypoplasia. Mechanistically, these phenotypes coincide with transcriptional deregulation during heart patterning, and Pygo2 associates with β-catenin atcis-regulatory regions of cardiac genes. Taken together, our results establish BCL9 and Pygo as tissue-specific β-catenin co-factors during vertebrate heart development. Our results further implicate alterations inBCL9andBCL9Lin human CHDs as possibly causative.