Project description:The nuclear organization of the genome is accepted as an important feature of gene expression regulation. However, it has remained unknown what the spatial organization of a single transcribed gene is. Here, we made use of several long highly expressed mammalian genes to describe their structure and spatial arrangement during transcription. We demonstrate that an expressed gene forms a transcription loop with RNA polymerases moving along the loop and carrying nascent RNAs that undergo co-transcriptional splicing. Transcription loops dynamically modify their harboring loci and extend into the nuclear interior due to their intrinsic stiffness. We hypothesize that the stiffness of the transcription loop arises due to a dense decoration of gene-axis with multiple voluminous nascent ribonucleoprotein particles, thus creating a stiff polymer bottlebrush. and provide supporting evidence to this hypothesis. Our work suggests that transcription loop formation is a universal principle of eukaryotic gene expression.
Project description:The nuclear organization of the genome is accepted as an important feature of gene expression regulation. However, it has remained unknown what the spatial organization of a single transcribed gene is. Here, we made use of several long highly expressed mammalian genes to describe their structure and spatial arrangement during transcription. We demonstrate that an expressed gene forms a transcription loop with RNA polymerases moving along the loop and carrying nascent RNAs that undergo co-transcriptional splicing. Transcription loops dynamically modify their harboring loci and extend into the nuclear interior due to their intrinsic stiffness. We hypothesize that the stiffness of the transcription loop arises due to a dense decoration of gene-axis with multiple voluminous nascent ribonucleoprotein particles and provide supporting evidence to this hypothesis. Our work suggests that transcription loop formation is a universal principle of eukaryotic gene expression.
Project description:The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We now have generated a high-resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and mapped translocations from target DNA double-strand breaks (DSBs) within it via high-throughput genome-wide translocation sequencing. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high frequency DSBs at these loci and their co-localization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing-radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and sub-chromosomal domains in a manner directly related to pre-existing spatial proximity. Our studies reveal the power of combining two high-throughput genomic methods to address long-standing questions in cancer biology. Hi-C interaction maps for WT and ATM -/- G1-arrested AMuLV-transformed pro-B cell lines.
Project description:Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the posttranscriptional Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the post-transcriptional dynamics of mRNAs.
Project description:Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions, and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of compound mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me2/3, was redistributed to regions targeted by H3K9me2/3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that the two major repressive epigenome pathways exclusively but also coordinately maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.
Project description:How heat shock induces the heat shock response (HSR) - a gene expression program encoding chaperones and other protein homeostasis (proteostasis) factors - remains an unresolved question in eukaryotic cell biology. Here we show that subcellular localization of the conserved J-protein Sis1 is a key regulator of the HSR in yeast. Under nonstress conditions, nucleoplasmic Sis1 promotes interaction between the chaperone Hsp70 and the transcription factor Hsf1 to repress the HSR. Heat shock triggers Sis1 to localize to the periphery of the nucleolus and to condense on the ER surface. Sis1 recruits the proteasome to this spatial network along with disaggregases and the ribosome quality control complex. Through localization dynamics, Sis1 relays the condition of the proteome to Hsf1. We conclude that the activation state of the HSR is determined by the spatial organization of the proteostasis network.
Project description:GCC was used to determine the structure of E. coli grown in LB or treated with SHX. The bacterial genome is highly condensed into a nucleoid structure. Here we present global analyses of the genome spatial organization for two γ-proteobacteria: Escherichia coli and Pseudomonas aeruginosa by Genome Conformation Capture. Long distance interactions occurred within the E. coli and P. aeruginosa nucleoids with frequencies that were affected by growth condition and gene dosage. Spatial clustering of genes that are either up or down-regulated depended on the environmental signals, indicating a non-random functional organization of the nucleoid. The largest changes in gene expression upon amino acid starvation occurred in genes that participate in long-range interactions. These genes remained highly spatially clustered when transcript levels decreased. Environment specific interactions were related to DNA motifs but did not correlate with binding sites for nucleoid associated proteins. Overall we identify spatial organization as a significant factor in bacterial gene regulation and suggest that the prokaryotic operon is not simply a linear entity.