Project description:We performed high numbers of replicates of CUT&RUN LoV-U against H3K4me3, β-catenin, and the negative control IgG in human colorectal cancer HCT116 cells over two independent rounds of experiments to discover the complete set of binding events.
Project description:In our attempts to profile different regulators of the WNT/b-catenin transcriptional complex, CUT&RUN failed to produce consistent binding patterns of the non-DNA-binding b-catenin. We developed a modified CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible b-catenin binding profiles. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested, as shown by datasets targeting the TCF/LEF transcription factors and various histone modifications. 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.
Project description:Serine-threonine kinase receptor-associated protein (STRAP) is upregulated in breast, colorectal and lung cancers, promoting their growth. We identify the upregulation of STRAP in hepatocellular carcinomas. Elevated STRAP endows tumor cells with growth advantage by reprograming a variety of metabolic processes and signaling pathways critical for hepatocellular carcinoma progression. Especially, enhanced Wnt/β-catenin signaling is likely to be a major effector of its tumor-promoting role.
Project description:SOX6 CUT&RUN on HUDEP1 over expressing SOX6-Flag. The experiment is done using and anti Flag Ab to assist the genome wide binding profile of SOX6 in HUDEP1 (Human Umbilical cord blood-Derived Erythroid Progenitor-1).
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin-driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. A first question concerns cell-specificity: how this response is integrated into lineage-specific choices is still unknown. A second question concerns time: it is not known whether β-catenin associates with its targets simultaneously or in a time-dependent fashion. For instance, while TCF/LEF and other components of the Wnt transcriptional complex are constitutively associated with the chromatin, it is β-catenin arrival, upon Wnt induction, that launches target genes transcription. Therefore, discovering the dynamics of the genome-wide β-catenin binding pattern is required to unambiguously define the direct targets of Wnt signaling To address these questions, we realized a time-resolved atlas of β-catenin genome-wide occupancy in two human cell types, human embryonic kidney cells 293T (HEK293T) and human embryonic stem cells (hESCs). To this end, we treated HEK293T and hESCs with the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days, and assessed β-catenin binding via CUT&RUN-LoV-U (Zambanini et al., 2022) 90 minutes, 4 hours, 24 hours and 3 days after the onset of the stimulation. This approach allowed us to establish that β-catenin repositions to different genomic loci along stimulation time, showing that a definition of Wnt target genes must take into account the time-dimension. Moreover, β-catenin physical targets are largely cell-type specific, as only a subset of them is present across the examined contexts.