Zambanini G, Nordin A, Jonasson M, Pagella P, Cantù C
Development - (-) - [2022-11-10; online 2022-11-10]
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.
Clinical Genomics Linköping [Service]