Project description:The homeobox transcription factor Prox1 is critical for organogenesis including brain, retina, liver and pancreas and lymphatic system. Prox1 is transiently expressed in mouse cochlear hair cells (HCs) and supporting cells (SCs), however, it remains elusive about its in vivo DNA binding site and roles during cochlear development. Here, by using fresh cochlear tissues and Prox1 antibody, we firstly mapped out the genome-wide binding sites of Prox1 via Cleavage Under Targets and Release Using Nuclease (CUT&RUN). Gene function enrichment analysis suggests several potential functions of Prox1, one of which is regulating cell cycle progression. Secondly, to validate our CUT&RUN data, we further performed in vivo prox1 gain-of-function studies through genetic approaches. Onset of ectopic Prox1 at early embryonic otocyst leads to shorter cochlea with density of HCs and SCs distribution, suggesting a precocious cell cycle exit of cochlear sensory progenitor cells. These findings highlight the power of CUT&RUN in mapping DNA binding sites even in tissues (i.e. cochlea) with rare cells, as well as providing the first genetic evidence to support roles of Prox1 in regulating progenitor pools of cochlear progenitors.
Project description:Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, Cut-and-Run is performed in situ, allowing for both high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, Cut-and-Run yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. Cut-and-Run is simple to perform and at low temperatures is inherently robust, with extremely low backgrounds that make it especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, Cut-and-Run mapped high-resolution 3D directional interactions. We conclude that Cut- and-Run is a suitable complement or replacement for ChIP-seq that can also provide 3D mapping information.
Project description:CUT&RUN sequencing to define binding sites for SOX11 and IgG in the neuroblastoma cell lines IMR-32, CLB-GA and NGP as well as neuroblastoma cell line SH-EP after SOX11 overexpression for 48h. A SOX11 inducible overexpression system was generated using a Tet-on system.
Project description:To identify primary and direct targets of CHD7 during the early stages of cardiac commitment and differentiation, we decided to map the genome-wide binding sites of CHD7. We used an in vitro approach of directed differentiation of mouse embryonic stem cells (ESCs) towards cardiomyocytes (CM). We isolated cells at the cardiac progenitor (CP) stage (Day 5) and used CUT&RUN (“Cleavage Under Targets & Release Using Nuclease”) technique, followed by NGS. The assay was performed in 3 biological replicates using anti-CHD7, and IgG as negative control.
Project description:Recent studies have demonstrated that hepatocytes can be reprogrammed into biliary epithelial cell-like cells. An earlier work in our laboratory nominated Sox4 as an initiation factor of this reprogramming event. To investigate the effect of Sox4 on the change of chromatin landscape of hepatocytes, we exogenosly expressed Sox4 in adult hepatocytes, and profiled its binding sites in a genome wide manner by CUT&RUN-seq. We also profiled histone post-translational modifications of H3K4me1, H3K4me3, H3K27ac and H3K27me3 to explore the influence of Sox4 on the epigenetic landscape of hepatocytes.
Project description:Recent studies have demonstrated that hepatocytes can be reprogrammed into biliary epithelial cell-like cells. An earlier work in our laboratory nominated Sox4 as an initiation factor of this reprogramming event. To investigate the effect of Sox4 on the change of chromatin landscape of hepatocytes, we exogenosly expressed Sox4 in adult hepatocytes, and profiled its binding sites in a genome wide manner by CUT&RUN-seq. We also profiled histone post-translational modifications of H3K4me1, H3K4me3, H3K27ac and H3K27me3 to explore the influence of Sox4 on the epigenetic landscape of hepatocytes.