Project description:We found that TLE3 in colonic macrophages mainly occupied distal intergenic regions (48%) and gene body regions (47%) with limited occupancy at the gene proximal regions (5%).
Project description:Transcriptional effectors of white adipocyte-selective gene expression have not been described. TLE3 is a white-selective cofactor that acts reciprocally with the brown-selective cofactor Prdm16 to specify lipid storage and thermogenic gene programs. When expressed at elevated levels in brown fat, TLE3 counters Prdm16, suppressing brown-selective genes and inducing white-selective genes, resulting in impaired fatty acid oxidation and thermogenesis. To further test the functional consequences of the changes in BAT phenotype in aP2-TLE3 Tg mice, we challenged them with cold exposure at 4C. aP2-TLE3 Tg mice had an impaired ability to respond to cold exposure compared to littermate control mice, as evidenced by a marked drop in core body temperature. Transgenic mice expressing TLE3 from the aP2 enhancer and wild type mice were housed individually without food and bedding immediately before the start of experiments. Mice were allowed free access to water and placed at 4C for 5 hours while core body temperature was monitored every hour using a rectal probe. For microarray experiments of BAT, RNA was extracted and pooled from 4 mice in each group.
Project description:H3K27Ac CUT&Tag-Seq to characterize the epigenetic profiles in colonic macrophages from Tle4KO (Tle4fl/fl, Lyz2-Cre) and WT (Lyz2-Cre) mice
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.
Project description:To reveal the role of MCM8 in suppressing R-loop accumulation, we performed the CUT&TAG assay using the S9.6 antibody to map genome-wide R-loops in Mcm8 wildtype MEFs and Mcm8 knockout MEFs. We also conducted the CUT&TAG assay to detect genome-wide R-loops in Ddx5 downregulated MEFs by adenovirus infection and in control MEFs. To investigate the underlying molecular mechanism of MCM8 suppressing R-loops, we conducted the DNA sequencing of libraries from CUT&TAG assay using the antibody against FLAG in HEK293 cells transfected with FLAG-MCM8 plasmid and using the S9.6 antibody in HEK293 cells. Besides, an IgG control and control of RNH1 overexpression were included.
Project description:We developed scNanoSeq-CUT&Tag, a streamlined method by adapting a modified CUT&Tag protocol to Oxford Nanopore sequencing platform for efficient chromatin modification profiling at single-cell resolution. We firstly tested the performance of scNanoSeq-CUT&Tag on six human cell lines: K562, 293T, GM12878, HG002, H9, HFF1 and adult mouse blood cells, it showed that scNanoSeq-CUT&Tag can accurately distinguish different cell types in vitro and in vivo. Moreover, scNanoSeq-CUT&Tag enables to effectively map the allele-specific epigenomic modifications in the human genome andallows to analyze co-occupancy of histone modifications. Taking advantage of long-read sequencing,scNanoSeq-CUT&Tag can sensitively detect epigenomic state of repetitive elements. In addition, by applying scNanoSeq-CUT&Tag to testicular cells of adult mouse B6D2F1, we demonstrated that scNanoSeq-CUT&Tag maps dynamic epigenetic state changes during mouse spermatogenesis. Finally, we exploited the epigenetic changes of human leukemia cell line K562 during DNA demethylation, it showed that NanoSeq-CUT&Tag can capture H3K27ac signals changes along DNA demethylation. Overall, we prove that scNanoSeq-CUT&Tag is a valuable tool for efficiently probing chromatin state changes within individual cells.
Project description:This study aimed to adapt CUT&Tag to Plasmodium falciparum samples as an efficient and sensitive alternative to classical ChIP-sequencing. We compare H3K9me3 and HP1 CUT&Tag with ChIP-seq datasets, showing successful establishment of CUT&Tag in P. falciparum. Next we aimed to scale down required input material for our CUT&Tag reactions and generated high-quality HP1 tracks with as little as 10.000 nuclei. To minimise potential sample loss we tested feasibility of utilising (frozen) saponin parasite isolates as input material instead of nuclei, which proved to be viable. Lastly, we deployed our new technique Dimerisation-induced Biotinylation-CUT&Tag (DiBioCUT&Tag) to catch transient interactions by biotinylation of strongly associated proteins such as histones. We tested this technique on HP1 and compared standart CUT&Tag with DiBioCUT&Tag. Furthermore, we explored interactions of the transcription factor BDP5, which we were previously unable to succesfully ChIP.