A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets

Abstract

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.

Time-resolved analysis of Wnt-signaling reveals β-catenin temporal genomic repositioning and cell type-specific plastic or elastic chromatin responses

“…Third, both cell types display statistical enrichment for motifs of all four individual TCF/LEF proteins as primary transcription factor footprinting in the sequences underlying β-catenin peaks (Figure 1E, F). Finally, these datasets recapitulate the previously observed genomic distribution of β-catenin in Wnt-ON conditions across functionally diverse regions and with respect to average distance to annotated genes (Figure 1G, H) (Cantù et al, 2018;Zambanini et al, 2022). Notably, our time-resolved CUT&RUN-LoV-U dataset robustly reproduced the b-catenin binding patterns observed via single-time-point ChIP-seq (performed after 24 hours of CHIR stimulation) in a previous report (Doumpas et al, 2019).…”

“…Here, Wnt/β-catenin signaling can be reliably activated by administration of the GSK3 inhibitor CHIR99021 (hereafter CHIR), which induces β-catenin stabilization, nuclear translocation, and activation of downstream targets (Figure 1A) (Doumpas et al, 2021; Wray et al, 2011). We applied CHIR for up to 3 consecutive days and performed CUT&RUN-LoV-U (Zambanini et al, 2022) targeting β-catenin at selected time-points after Wnt pathway stimulation (Figure 1B). This approach generated a time-resolved, cell-specific compendium of the genome-wide β-catenin chromatin occupancy profiles (Table S1).…”

“…Only a few datasets have been previously produced with CUT&RUN-related technologies (i.e., mostly ChIP-seq) to identify the physical targets of b-catenin, due to the difficulty in detecting the behavior of this protein (Zambanini et al, 2022). By their own experimental design, these must be interpreted as snapshots of targets occurring at an arbitrarily chosen moment after stimulation.…”

“…All experiments were performed in triplicate (n = 3 for each cell type at each time-point). Cells were filtered through a 40 μm cell strainer (KKE3.1, Carl Roth) and processed for CUT&RUN (Zambanini et al, 2022). 500,000 cells/sample were harvested using either Trypsin EDTA 0.25% (HEK293Ts) or EDTA 0.5 mM in PBS (hESCs) and washed twice in DPBS (Cat.…”

“…To address the first question, here we leveraged on a novel CUT&RUN-LoV-U (Cleavage Under Targets & Release Using Nuclease - Low Volume - Urea) (Zambanini et al, 2022) protocol to establish the full sets of β-catenin target genes detectable over time in two paradigmatic human cell types. We consider CUT&RUN and related technologies particularly powerful for this, as they allow for distinguishing high-confidence direct targets from indirect ones – that is, several genes could be transcriptionally upregulated as secondary or tertiary effects, and WREs could be found in silico but might not have functional relevance in vivo .…”