Project description:induced motor neurons (iMNs) differentiated from hiPSC line were separated on axonal and somatodendritic compartments. Isolated subcellular compartments were subjected to RNA-seq.
Project description:induced motor neurons (iMNs) differentiated from hiPSC line were separated on axonal and somatodendritic compartments. Isolated subcellualr compartments were subjected to RNA-seq. FUSR244RR-ALS model was compared with isogenic control in which R244RR mutation was corrected to WT.
Project description:Using a transcriptional network derived from 2000 breast cancer gene expression profiles we identify the master regulators (MRs) of FGFR2 signalling. To validate the identified regulons, we examined whether there was enrichment of TF binding near the transcription start sites (TSS) of genes found in the regulons of a particular MR. For ESR1 and SPDEF, ChIP-seq experiments were performed in MCF-7 cells, while existing data was analysed for FOXA1 (Hurtado et al. Nature Genetics, 43:27–33, 2010) and GATA3 (Theodorou, et al., Genome Res 23: 12-22, 2013). ChIP-seq experiments were performed on three biological replicates per each transcription factor. For each sample, 36bp single-end reads were obtained. Peak regions were identified in all ChIP-seq TF data sets using the peak caller algorithm MACS (Zhang et al., Genome Biology, 9(9):R137, 2008) with default parameters.
Project description:In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3M-NM-^T cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements at the borders of subtelomeres and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and components of the nuclear envelope by maintaining chromatin structure required for anchoring DNA insulators to nuclear pores. For MNase samples, duplicate mutant mononucleosome fractions were compared with duplicate WT mononucleosomes.
Project description:Polycomb repression of gene expression is critical for development, with a pivotal role for trimethylation of lysine 27 of histone H3 (H3K27me3) deposited by Polycomb Repressive Complex 2 (PRC2). While the function and regulation of PRC2 have been extensively studied, the mechanism(s) by which it is recruited to specific genomic targets has remained largely elusive, in particular in vertebrates. Here we identify the PRC2-associated protein Mtf2 as a novel DNA methylation-sensitive PRC2 recruiter in mouse embryonic stem cells (mESCs). Mtf2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit Ezh2 to genomic targets is drastically impaired in Mtf2 knock-out mESCs, resulting in largely reduced H3K27me3 deposition. Mtf2 selectively binds regions with high density of closely spaced unmethylated CpG-containing motifs with a locally unwound helical structure. This binding is dependent on one of the Mtf2 PHD domains, a protein domain shared among Pcl homologs, and an Mtf2-specific domain. The sequences bound by Mtf2 are enriched in PRC2-repressed CpG island-containing targets in zebrafish, Xenopus, mouse and human, suggesting that Mtf2-mediated PRC2 recruitment to unmethylated genomic regions is conserved among vertebrates.
Project description:We explored the RNA binding properties of LINE-1 ORF1p, both free and in the L1 RNP, using a recently developed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation technique (PAR-CLIP) to comprehensively identify ORF1p binding sites in the transcriptome of human cells (HEK293T). Our results show that ORF1p binds to a wide range of cellular mRNAs, with an enrichment for binding at the 3’ UTR. Our data also show that ORF1p binds very strongly with retrotransposable RNA, i.e., L1, Alu and SVA. PAR-CLIP analysis of L1 RNPs and free ORF1p RNA binding profiles, comparison to HuR RNA binding profile
Project description:This project aims to study human repetitive elements on human chromosome 21 (HsChr21) in a heterologous mouse environment (Tc1 mouse). The in vivo, HsChr21-wide enrichment of active histone modified regions (H3K4me3) and transcription factor binding sites was profiled by ChIP-seq in Human and Tc1 mouse tissue in order to determine the unbiased regulation of the human genome.
Project description:By 4C-seq protocol we investigated DNA contacts across the genome by the FLC gene in the model plant Arabidopsis thaliana in order to explore a potential role of long-distance chromosomal interactions in the regulation of flowering.
Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).