Upon WNT/beta-catenin pathway activation, stabilized beta-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 beta-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 beta-catenin binding profiles, uncovering direct WNT/beta-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 believe that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/beta-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

BackgroundCleavage 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, indicating that these are false positives. 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.ResultsWe compile suspect lists based on CUT & RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT & RUN data from our and other labs, we show that the CUT & RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT & RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified.ConclusionsWe propose that removing these problematic regions can substantially improve peak calling in CUT & RUN experiments, resulting in more reliable datasets.

Wnt signaling orchestrates gene expression via its effector, (3-catenin. However, it is unknown whether (3-cat-enin binds its target genomic regions simultaneously and how this impacts chromatin dynamics to modulate cell behavior. Using a combination of time-resolved CUT & RUN against (3-catenin, ATAC-seq, and perturba-tion assays in different cell types, we show that Wnt/(3-catenin physical targets are tissue-specific, (3-catenin "moves"on different loci over time, and its association to DNA accompanies changing chromatin accessi-bility landscapes that determine cell behavior. In particular, Wnt/(3-catenin progressively shapes the chro-matin of human embryonic stem cells (hESCs) as they undergo mesodermal differentiation, a behavior that we define as "plastic."In HEK293T cells, on the other hand, Wnt/(3-catenin drives a transient chromatin open-ing, followed by re-establishment of the pre-stimulation state, a response that we define as "elastic."Future experiments shall assess whether other cell communication mechanisms, in addition to Wnt signaling, are ruled by time, cellular idiosyncrasies, and chromatin constraints. A record of this papers transparent peer review process is included in the supplemental information.

Genome-wide binding assays aspire to map the complete binding pattern of gene regulators. Common practice relies on replication-duplicates or triplicates-and high stringency statistics to favor false negatives over false positives. Here we show that duplicates and triplicates of CUT&RUN are not sufficient to discover the entire activity of transcriptional regulators. We introduce ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), a pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regions, the targets of the co-factor beta-catenin, and those of the transcription factor TBX3, in human colorectal cancer cells. The ICEBERG datasets allow benchmarking of individual replicates, comparing the performance of peak calling and replication approaches, and expose the arbitrary nature of strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG establishes a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that underlie novel mechanisms of colorectal cancer progression. Graphical Abstract

Gene regulators physically associate with the genome, in a combinatorial fashion, to drive tissue-specific gene expression. Uncovering the genome-wide activity of all gene regulators across tissues is therefore needed to understand gene regulation during development. Here, we take a first step towards this goal. Using CUT&RUN, we systematically mapped genome-wide binding profiles of key transcription factors and co-factors that mediate ontogenetically relevant signaling pathways in select mouse tissues at two developmental stages. Computation of the datasets unveiled tissue- and time-specific activity for each gene regulator. We identified 'popular' regulatory regions that are bound by a multitude of regulators, which tend to be more evolutionarily conserved. Consistently, they lie near the transcription start site of genes for which dysregulation results in early embryonic lethality. Moreover, the human homologs of these regions are similarly bound by many gene regulators and are highly conserved, indicating a retained relevance for human development. This work constitutes a decisive step towards understanding how the genome is simultaneously read and used by gene regulators in a holistic fashion to drive embryonic development.

Wnt signaling plays a pivotal role during development and homeostasis. Upon pathway activation, CTNNB1 (also known as beta-catenin) drives the expression of target genes from regulatory regions bound by TCF/LEF transcription factors. Gene regulation, however, entails the interplay between sequence information and 3D genome structure, yet the impact of Wnt signaling on genome structure has been poorly explored. Here, we investigate how Wnt signaling influences CTCF and cohesin, key regulators of 3D genome organization. We identify a series of novel CTCF binding sites that emerge upon Wnt stimulation: CTCF Redistributions Under Wnt (RUW). RUW sites are characterized by CTCF, cohesin, and TCF/LEF occupancy, and are dependent on beta-catenin. Beta-catenin and CTCF colocalize upon pathway activation, and disruption of selected binding sites perturbs target gene regulation. Moreover, Wnt signaling reorganizes the 3D genome as evidenced by genome-wide alterations in CTCF-bound loops. This work reveals a previously unexplored role for CTCF in the regulation of Wnt signaling.