CUT&RUN of histone modifications, chromatin remodelers, and transcription factors in forelimbs, hindlimbs, liver and branchial arches of E10.5 and E11.5 JAX/Swiss mouse embryos
Ontology highlight
ABSTRACT: Using CUT&RUN, we systematically measured the genome-wide binding profiles of key transcription factors and cofactors that track the activity of ontogenetically relevant signaling pathways in developing mouse tissues. This produced numerous genome-wide biding tracks for different tissues at two developmental stages in biological duplicate. Submitted files include raw fastq files, bigwig files from merged biological replicates, and peak sets.
Project description:The Wnt/β-catenin signaling pathway plays crucial roles in nearly all parts of embryonic development and adult stem cell homeostasis. Its aberrant activation has been linked to many diseases such as developmental irregularities and various severe forms of cancer, with colorectal cancer (CRC) as a prime example. While much work has been dedicated to uncovering effective therapeutics to block oncogenic Wnt signaling, such interventions have not proven trivial because of the broad activity of Wnt throughout the adult body and the difficulty in finding suitable molecular targets. We have previously identified the developmental transcription factor TBX3 as a participant of the Wnt-mediated transcriptional regulation. Here we examine the genome-wide binding pattern of TBX3 in the human CRC cells lines HCT116 (25 replicates), DLD1 (2 replicates) and SW620 (2 replicates), by employing CUT&RUN (C&R) with Low-Volume and Urea (LoV-U; Zambanini et al., 2022).
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: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: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.
Project description:We performed 8 C&R tests in HEK293T human cells and 8 in mouse embryonic tissues from JAX Swiss mice by using either IgG or anti-HA antibodies. To increase diversity in this test, we used both the original C&R protocol and our recently developed C&R-LoV-U version for non-DNA-binding transcriptional co-factors. The aim was to experimentally validate our generated CUT&RUN blacklists containing problematic high signal regions.
Project description:The HASTER promoter region is a cis-regulatory element that stabilizes the transcription of HNF1A, preventing silencing or overexpression. We have generated a mouse model where the promoter of Haster has been specifically deleted in liver (Haster loxP/loxP; AlbCre). In liver the prevailing consequence is upregulation of HNF1A. We performed HNF1A, H3K4me3 and H3K27ac ChIP-seq to assess the impact of HNF1A upregulation on the chromatin landscape of Haster KO liver.
Project description:Using ChIP-Seq analysis we define genes induced in macrophages through transcriptional activation by STAT1 and search for the gain of function in mutant Stat1Y701F compared to the Stat1-/- mice.
Project description:CUT&RUN was performed for Sox2 on ex-vivo dissected visual thalamic nuclei from P0 mice, revealing context specific activity of Sox2 binding in differentiated neurons.
Project description:RNA:DNA hybrids accumulate at the vicinity of DNA double-strand breaks (DSBs) and were shown to regulate homologous recombination repair. The mechanism responsible for the formation of these non-canonical RNA:DNA structures remains unclear although they were proposed to arise consequently to RNA Polymerase II or III loading followed by DSB-induced de novo transcription at the break site. Here, we found no evidence of RNA polymerases recruitment at DSBs. Rather, strand-specific R-loop mapping revealed that RNA:DNA hybrids are mainly generated at DSBs occurring in transcribing loci, from the hybridization of pre-existing RNA to the 3’ overhang left by DNA end resection. We further identified the H3K4me3 reader Spindlin 1 and the transcriptional regulator PAF1 as promoting RNA:DNA hybrid accumulation at DSBs, through their role in mediating transcriptional repression in cis to DSBs. Altogether, we provide evidence that RNA:DNA hybrids accumulate at DSBs occurring in transcribing loci as a result of DSB-induced transcriptional shut-down.