Project description:Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development.
Project description:Characterizing the relationship between genome form and function requires methods both for zooming out, to globally survey the genomic architectural landscape, and for zooming in, to investigate regions of interest at high resolution. High throughput methods based on chromosome conformation capture (3C) have greatly advanced our understanding of the three-dimensional (3D) organization of genomes, but are limited in resolution by their reliance on restriction enzymes. Here we describe a method for comprehensively mapping global chromatin contacts that uses DNaseI for chromatin fragmentation, leading to greatly improved efficiency and resolution. Coupling this method with DNA capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 1,030 lincRNA promoters in two human cell lines, and identified thousands of high-confidence lincRNA promoter-associated chromatin contacts at 1 kilobase pair (kbp) resolution. Detailed analysis of these contacts reveals that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the polycomb repressive complex. Our results provide the first glimpse of the cell-type-specific 3D organization of lincRNA genes. DNase Hi-C assay (x1 replicate) in H1 and K562 cells, respectively. Targeted DNase Hi-C assays of the 220kb promoter-enhancer library (x1 replicate) and the 5Mb lincRNA promoter library (x2 replicate), in H1 and K562 cells, respectively.
Project description:Drosophila Haspin kinase phosphorylates Histone H3 at threonine 3 at centromeric heterochromatin and either lamin- or polycomb-enriched euchromatic regions, being required for nuclear organization of interphase cells and polycomb-dependent gene silencing.
Project description:Chromatin is organized into a 3D interwoven tapestry of multi-layer architectural features important for controlling gene expression. How distinct layers influence each other and quickly they quickly they respond to cellular environment is unclear. Using Hi-C in Drosophila melanogaster, we measure how 3D chromatin organization responds to cellular hyperosmotic stress. In combination with the hd-pairing method, we demonstrate that chromosome unpairing represents a fast an reversible response to hyperosmotic stress. We identify a novel function of the Z4 protein as an anti-pairer, which we demonstrate is necessary for changes to chromatin organization during hyperosmotic stress. Finally, we demonstrate how changes to pairing impacts the other interwoven layers of 3D chromatin organization.
Project description:Chromatin is organized into a 3D interwoven tapestry of multi-layer architectural features important for controlling gene expression. How distinct layers influence each other and quickly they quickly they respond to cellular environment is unclear. Using Hi-C in Drosophila melanogaster, we measure how 3D chromatin organization responds to cellular hyperosmotic stress. In combination with the hd-pairing method, we demonstrate that chromosome unpairing represents a fast an reversible response to hyperosmotic stress. We identify a novel function of the Z4 protein as an anti-pairer, which we demonstrate is necessary for changes to chromatin organization during hyperosmotic stress. Finally, we demonstrate how changes to pairing impacts the other interwoven layers of 3D chromatin organization.
Project description:Chromatin is organized into a 3D interwoven tapestry of multi-layer architectural features important for controlling gene expression. How distinct layers influence each other and quickly they quickly they respond to cellular environment is unclear. Using Hi-C in Drosophila melanogaster, we measure how 3D chromatin organization responds to cellular hyperosmotic stress. In combination with the hd-pairing method, we demonstrate that chromosome unpairing represents a fast an reversible response to hyperosmotic stress. We identify a novel function of the Z4 protein as an anti-pairer, which we demonstrate is necessary for changes to chromatin organization during hyperosmotic stress. Finally, we demonstrate how changes to pairing impacts the other interwoven layers of 3D chromatin organization.
Project description:Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. We investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of five H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. H3K9me2 was regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments were regulated by different MTases. H3K9me2 in A compartments was primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments was regulated by all five MTases. Furthermore, decreased H3K9me2 correlated with changes to the more active compartmental state that accompanied transcriptional activation.