Upon WNT/b-catenin pathway activation, stabilized b-catenin travels to the nucleus where it associates with the TCF/LEF transcription factors, constitutively bound to genomic Wnt Responsive Elements (WREs), to activate target gene transcription. Discovering the binding profile of b-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as prime technique for mapping the binding profile of DNA-interacting proteins. Here we present a modified version of CUT&RUN, named LoV-U (Low Volume and Urea), that enables the robust and reproducible generation of b-catenin binding profiles, uncovering direct WNT/β-catenin target genes in human cells, as well as in cells isolated from developing mouse tissues. CUT&RUN-LoV-U outperforms original CUT&RUN when targeting co-factors that do not bind the DNA, can profile all classes of chromatin regulators, 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.

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 pioneer-ing 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.

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.

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