Project description:Biochemical fractionation of HEK293 nuclei and RNA-seq of chromatin-associated and soluble-nuclear RNA. Nuclei from three biological replicates were isolated by detergent lysis, fractionated, then three chromatin and three soluble RNA samples were converted to cDNA using Illumina TruSeq stranded protocol, and sequenced on Illumina HiSeq2000
Project description:Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using a novel subcellular fractionation and fluorescent activated cell sorting method to enrich for diseased neuronal nuclei without TDP-43 from post-mortem FTD-ALS human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing and DNA damage. Loss of nuclear TDP-43 was also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs.
Project description:Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using a novel subcellular fractionation and fluorescent activated cell sorting method to enrich for diseased neuronal nuclei without TDP-43 from post-mortem FTD-ALS human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing and DNA damage. Loss of nuclear TDP-43 was also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs.
Project description:The composition and dynamics of tightly bound chromatin associated RNAs during mitosis remains elusive. Here we report identification of chromatin enriched (cheRNAs) by biochemical nuclear fractionation coupled with RNA-seq in both interphase and mitotic phase of A549 and HeLaS3 cell lines. We show that highly abundant cheRNAs, mostly small noncoding RNAs, are largely maintained in mitotic chromatin, and constitute a substantial part of chromatin throughout cell cycle. We also show that the mitotic retained cheRNAs tend to be cell type nonspecific. We speculate that these cell type nonspecific cheRNAs might play roles in chromatin state and gene expression control. We anticipate the panoramic landscape of RNA composition of chromatin both in interphase and M-phase would shed light on understanding the structure and function of chromatin in M phase and would also help the understanding of some fundamental biological functions.
Project description:Nuclear chromosomes transcribe far more RNA than required to code for protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin and most nuclear RNA from the nucleus, but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome “scaffold.” RNA-seq analysis reveals most RNA in the nuclear scaffold is repeat-rich, non-coding, and predominantly derived from introns of nascent transcripts. This repeat-rich (C0T-1) RNA inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei, demonstrating RNA physically antagonizes a propensity for chromatin to condense. C0T-1 hnRNA co-distributes on euchromatin with several known scaffold proteins including scaffold attachment factor A (SAF-A). We further show that RNA is required for SAF-A to interact with chromatin and to form structurally embedded scaffold-attachment regions (SARs) in the nuclear genome. Collectively, results indicate nascent transcripts serve a dynamic structural role in the open architecture of active chromosome territories
Project description:We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 80mM or 150mM NaCl after digestion contain predominantly mononucleosomes and represent calssical 'active' chromatin. Profiles of these low-salt-soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.3 and correspond closely to profiles of RNA polymerase II. Nearly quantitative recovery of chromatin is obtained with 600mM NaCl, however, the remaining insoluble chromatin is enriched in actively transcribed regions. Salt-insoluble chromatin likely represents oligonucleosomes that are attached to large protein complexes. Both low-salt extracted and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture bound and unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics. Keywords: Chromatin affinity-purification on microarray For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin, or oligo(dT)-primed cDNA to randomly fragmented genomic DNA from S2 cell nuclei.
Project description:Spatial transcriptomics aims to understand how the ensemble of RNA molecules in tissues and cells is organized in 3D space. Here we introduce Proximity RNA-seq, which identifies co-localization preferences for pairs or groups of chromatin-associated, nuclear-retained and nascent RNAs in cell nuclei. Proximity RNA-seq is based on massive-throughput RNA barcoding of sub-nuclear particles in water-in-oil emulsion droplets, followed by sequencing.