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Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences): a novel strategy for high-resolution mapping of DNA binding sites

ABSTRACT: Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences): a novel strategy for high-resolution mapping of DNA binding sites

PROVIDER: PRJNA996661 | ENA |

REPOSITORIES: ENA

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Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences): a novel strategy for high-resolution mapping of DNA binding sites

Project description:DNA and protein interactions are essential for cellular processes and have major implications in cancer biology. DNA binding proteins, particularly transcription factors (TF), are crucial in regulating gene expression and downstream protein functions. Studying the intricate DNA-protein interactions helps unravel specific functions of TFs that are involved in cancer pathogenesis, identify regulatory pathways, and ultimately uncover therapeutic targets. Currently, the lack of high-throughput screening techniques makes research in cancer associated-TFs challenging. Therefore, we have developed an innovative Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences). Here, K562 tumor cells were coencapsulated with NK92 and antibody-conjugated (NFkB/RelA) magnetic beads in a microfluidic droplet-based system. After NK92 immune cells killed the K562 target cells, we collected RelA-bound DNA from the dead cells and analyzed DNA-binding sites using ChIP-seq. The results reveal the RelA genome-wide association study (GWAS) of the target K562 tumor cells in response to immune cell killing. The genome-wide mapping matched the expected profile of NFkB, validating the ability of our Iso-CUT&MAP method to accurately identify NFkB binding in response to NK92 killing of K562. This novel technique holds promise for precision medicine in understanding apoptosis biology and the influence of immune cell or chemotherapy-induced tumor cell killings.

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Mapping out genome wide binding sites of Prox1 by CUT&RUN approach in mouse cochlea

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.

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C-maf binding to dna in C-maf_ab vs Iso in Mus musculus

Project description:We report the application of Chip-seq to study the binding properties of C-maf. We determine the genetic and non genetic regions with the largest Chip-seq peaks in C-maf_ab compared to Isotype control antibody (Iso) samples.

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Cut&Tag of GLI3 binding during early organoid development

Project description:To investigate GLI3 TF targets during organoid fate decisions, we performed Cut&Tag of GLI3 binding

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Genome-wide mapping of MEF2A binding sites by CUT&Tag-seq in chicken primary myoblasts

Project description:In this study, CUT&Tag-seq technology was employed to investigate MEF2A binding sites across the entire genome of chicken primary myoblasts. CUT&Tag was performed using CUT&Tag Assay Kit for Illumina Pro (TD904-1) from Vazyme. Antibody targeting MEF2A as well as IgG were used.The final DNA library on a HiSeq PE150 platform was subjected for the analyses. This study provides a wide landscape of MEF2A target genes from chicken primary myoblasts, which supports the active role of MEF2A in avian muscle development.

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Genome-wide mapping of MEF2A binding sites by CUT&Tag-seq in chicken primary myoblasts

Project description:Genome-wide mapping of MEF2A binding sites by CUT&Tag-seq in chicken primary myoblasts

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Mapping out genome wide binding sites of Prox1 by CUT&RUN approach in mouse cochlea

Project description:Mapping out genome wide binding sites of Prox1 by CUT&RUN approach in mouse cochlea

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Transcriptional and translational effect of the lack of translation initiation factor eIF(iso)4E

Project description:One of the most regulated steps of translation initiation is the recruitment of an mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5M-BM-4end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [AteIF(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), the ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. For transcriptional analysis used total RNA of AteIF(iso)4E-1 seedlings of 15 days old to known the changes on transcripts leves by the eIF(iso)4E absence, using as control Wt seedlings. The experiments were performed in duplicate, and swap analysis were done. For translational analysis, used non-polysomal and polysomal RNA of AteIF(iso)4E-1 seedlings of 15 days old in order to known the transcripts that are modified in their translational levels by the eIF(iso)4E absence, using as control non polysomal and polysomal RNA of Wt seddlings.

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Cut-and-Run in situ factor profiling maps DNA binding and 3D contact sites at high resolution

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CUT&RUN of Sox2 binding in the mouse P0 visual thalamus (dLGN)

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2023-06-03 | E-MTAB-13004 | biostudies-arrayexpress

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