Widespread rearrangement of 3D chromatin organization underlies Polycomb-mediated stress-induced silencing [ChIP]
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ABSTRACT: Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences. Analysis of the distribution of architectural proteins, chromatin proteins and histone modifications in Drosophila Kc167 cells. Cells were grown at 25 C (NT) or heat shocked for 20 min at 36.5 C (HS). Both control and reference samples are included. For some of the samples, replicates are also included.
Project description:Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences. Analysis of 3D chromatin organization using Hi-C in Drosophila Kc167 cells. Cells were grown at 25 C and heat shocked for 20 min at 36.5 C. Cells were also treated with flavopiridol or triptolide to inhibit transcription elongation or initiation, respectively. Cells were depeleted of Cap-H2 or Rad21 using RNAi. Finally, cells depleted of RAd21 were subjected to heat shock at 36.5 C for 20 min.
Project description:Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences.
Project description:Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences.
Project description:The DNA in humans and many animals is compartmentalised in topologically associating domains (TADs). In Drosophila, several architectural proteins are enriched at TAD borders, but we are still missing evidence that these proteins have a functional role in TAD maintenance. Here, we show that depletion of BEAF-32, Cp190 and Chro leads to changes in TAD organisation and chromatin loops. Their depletion affects mainly TAD borders in heterochromatin, while euchromatin TAD borders are resilient to these mutants. Furthermore, transcriptomic data identified thousands of genes displaying differential expression in these mutants and that majority of differentially expressed genes are in TADs that are reorganised. In contrast, we observed a lower effect on gene expression by the loss of chromatin loops. Our work identified for the first time a functional role for architectural proteins at TAD borders in Drosophila and a strong link between TAD reorganisation and changes in gene expression.
Project description:Topologically associating domains (TADs) are widely recognized as fundamental elements of the 3D structure of the eukaryotic genome. However, while the structural importance of the insulator protein CTCF together with cohesin at TAD borders in mammalian cells is well established, the absence of such co-localization at most TAD borders in recent Hi-C studies of D. melanogaster is enigmatic, raising the possibility that these TAD border elements are not generally conserved among metazoans. Using in situ Hi-C with sub-kb resolution, we show that the genome of D. melanogaster is almost completely partitioned into more than 4,000 TADs (median size, 13 kb), nearly 7-fold more than previously identified. The overwhelming majority of these TADs are demarcated by pairs of Drosophila-specific insulator proteins, BEAF-32/CP190 or BEAF-32/Chromator, indicating that these proteins may play an analogous role in Drosophila as that of the CTCF/cohesin pair in mammals. Moreover, we find that previously identified TADs enriched for inactive chromatin are predominantly assembled from the higher-level interactions between smaller TADs. In contrast, the closely-spaced small TADs in regions previously thought to be unstructured “inter-TADs” are organized in an open configuration with far fewer TAD-TAD interactions. Such structures can also be identified in some “inter-TAD” regions of the mammalian genome, suggesting that larger assemblages of small self-interacting TADs separated by a “burst” of adjacent small, weakly interacting TADs may be a conserved, basic characteristic of the higher order folding of the metazoan genome.
Project description:Compartmentalisation of the genome as topologically associating domains (TADs) may have a regulatory role in development and cellular functioning, but the mechanism involved in TAD establishment is still unclear. Here, we present the first high-resolution contact map of Drosophila melanogaster neuronal cells (BG3) and identify different classes of TADs by comparing this to genome organisation in embryonic cells (Kc167). We find new interactions during differentiation in neuronal cells, which are reflected as enhanced long-range interactions. This is supported by pronounced enrichment of CTCF at TAD borders. Furthermore, we observed strong divergent transcription, together with RNA Polymerase II occupancy, and an increase in DNA accessibility at the TAD borders. Interestingly, TAD borders that are specific to neuronal cells are enriched in enhancers controlled by neuronal specific transcription factors. Our results suggest that TADs are dynamic across developmental stages and reflect the interplay between insulators, transcriptional states and enhancer activities.
Project description:Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.
Project description:Transcriptional coactivators of YAP/TAZ play key roles in cancers through transcriptional outputs. However, the mechanisms of their transactivation remain unclear, and effective and safe targeting solutions are lacking. Here, we discovered that YAP/ TAZ possess a hydrophobic transactivation domain (TAD). Knockout of TADs prevents tumor establishment due to growth defects and enhanced immune attack. TADs facilitate preinitiation complex (PIC) assembly by promoting TFIID recruitment in a TAF4-dependent manner and enhance RNA polymerase II (Pol II) elongation through MED15-dependent mediator complex recruitment. The bindings of TAD to the surface hydrophobic groove of MED15 induce its folding of the helix and hydrophobic interactions of TAD-MED15 boosts YAP-MED15 co-condensations thereby enhancing transcriptional hub formation and the expression of an oncogenic and immune suppressive transcriptional program. Synthesized small peptide TJ-M11 selectively disrupts the interactions of TADs with MED15 and TAF4, thereby suppressing tumor growth and sensitizing immunotherapy. This study indicates TADs of YAP/TAZ exhibit dual functions in PIC assembly and Pol II elongation, relying on hydrophobic interactions.
Project description:Maintenance of gene expression programs requires cell-type specific barcoding of genomes through euchromatin interspaced with facultative heterochromatin domains where PRC1/2 hinder chromatin accessibility thereby repressing genes inside such domains. At heterochromatin-euchromatin borders, regulation of heterochromatin spreading may depend on transcriptional activity, histone modifiers, and chromatin barrier insulators or the borders of topological associated domain (TADs). Here we show that depletion of H3K36 di- or tri-methyl histone methyl transferases dMes-4/NSD or Hypb/dSet2 induces H3K27me3 spreading at H3K27me3 borders, accounting for the repression of hundreds of genes upon depletion of these enzymes. dMes-4/NSD influences genes within active chromatin hubs and flanked by H3K27me3 borders demarcated by a TAD border, unlike dHypb/dSet2 that protects genes near H3K27me3 borders in absence of a TAD border. Insulator mutants that disrupt 3D chromatin hubs recapitulate only the H3K27me3 spreading observed upon depletion of dMes-4/NSD, and not of Hypb/dSet2. Hi-C data demonstrate how dMes-4/NSD directly block the propagation of the long-range interactions marking the inactive TADs, onto neighbor active regions, unlike Hypb/dSet2. Our data thus show a division of labor between dMes-4/NSD and Hypb/dSet2 to protect genes against H3K27me3 silencing, highlighting a direct influence of H3K36me marks on the demarcation of H3K27me3 domains within repressive TADs.
Project description:Maintenance of gene expression programs requires cell-type specific barcoding of genomes through euchromatin interspaced with facultative heterochromatin domains where PRC1/2 hinder chromatin accessibility thereby repressing genes inside such domains. At heterochromatin-euchromatin borders, regulation of heterochromatin spreading may depend on transcriptional activity, histone modifiers, and chromatin barrier insulators or the borders of topological associated domain (TADs). Here we show that depletion of H3K36 di- or tri-methyl histone methyl transferases dMes-4/NSD or Hypb/dSet2 induces H3K27me3 spreading at H3K27me3 borders, accounting for the repression of hundreds of genes upon depletion of these enzymes. dMes-4/NSD influences genes within active chromatin hubs and flanked by H3K27me3 borders demarcated by a TAD border, unlike dHypb/dSet2 that protects genes near H3K27me3 borders in absence of a TAD border. Insulator mutants that disrupt 3D chromatin hubs recapitulate only the H3K27me3 spreading observed upon depletion of dMes-4/NSD, and not of Hypb/dSet2. Hi-C data demonstrate how dMes-4/NSD directly block the propagation of the long-range interactions marking the inactive TADs, onto neighbor active regions, unlike Hypb/dSet2. Our data thus show a division of labor between dMes-4/NSD and Hypb/dSet2 to protect genes against H3K27me3 silencing, highlighting a direct influence of H3K36me marks on the demarcation of H3K27me3 domains within repressive TADs.