Project description:Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing, we mapped long-range chromatin interactions associated with RNA polymerase II in three different mouse cell lines and uncovered widespread promoter-centered interactions. These interactions further aggregated into higher-order clusters, in which proximal and distant genes are engaged through enhancer-promoter interactions. Comparative analyses of different cell lines imply that cell specific enhancer interactions are dynamic among different cell specific transcription, and suggest significant enrichment of enhancer-promoter interactions for cell specific manner. Overall, our study provides novel insights into the three-dimensional basis of transcription activity in mouse cells. RNA polymerase II (RNAPII) guided chromatin interactions were discovered by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study genome-wise the enhancer-promoter interactions. Three cell lines, namely mouse embryonic stem cell E14, Neural stem cell NS5 and neuroshpere cells were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al, 2009. The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al, 2010)
Project description:Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells. RNA polymerase II (RNAPII) bound chromatin interactions were extracted with Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study the transcription regulations with RNAPII-associated long-range chromatin interactions. Five cell lines, namely MCF7 (ATCC# HTB-22), K562 (ATCC# CCL-243), HCT116 (ATCC# CCL-247), HeLa (ATCC# CCL-2.2), and NB4 (Roussel and Lanotte, 2001) (provided by Dr. Sherman Weissman, Yale University), were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al (Fullwood et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009, 462(7269):58-64). The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al: ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol 2010, 11(2):R22).
Project description:Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2,264 SMC1A interactions, of which 1,491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue- specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues, but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin- associated interactions in the genome and highlight their role in establishing the regulatory architecture of development. Smc1a ChIA-PET, RNA-seq, chromatin state maps (H3K27ac, H3K27me3, H3K4m2), and CTCF and Smc1a binding in mouse embryonic limb bud (E11.5)
Project description:We report the application of single-molecule-based DNA sequencing technology for high-throughput profiling of long-range chromatin interactions in Sox2-deleted and wild type NSC, by improved ChIA-PET procedure (in situ ChIA-PET) with antiRNApolII antibodies. By obtaining sequences from chromatin immunoprecipitated DNA, we generated genome-wide maps of inter-molecular ligated DNA representing long-range interactions in chromatin.
Project description:We generated a genome-wide interaction map of regulatory elements in human cells (K562, GM12878) using Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) experiments targeting six broadly distributed factors. For data usage terms and conditions, please refer to https://www.encodeproject.org/about/data-use-policy Chromatin interactions identified by ChIA-PET for 4 different histone modifications (H3K4me1, H3K4me2, H3K4me3, H3K27ac), RAD21 and RNAPII in the K562 cell line, two biological replicates each. Additionally, chromatin interactions were identified by ChIA-PET in the GM12878 cell line for RAD21.
Project description:We report Proximity Ligation Assisted ChIP-sequencing (PLAC-seq), a method for comprehensive detection of long-range interactions associated with proteins of interest. PLAC-seq requires up to 500-fold less starting material compared to ChIA-PET and using experimentally determined input as control precisely reveals protein associated interaction upto single-element resolution. Application of PLAC-seq to mouse embryonic stem cells revealed a comprehensive map of regulatory interactions.
Project description:We developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by estrogen receptor α (ERα) in the human genome. We performed 454 and Illumina sequencing analyses. Keywords: Epigenetics Using 454, we examined 3 libraries: IHM001 (Estrogen Receptor ChIA-PET), IHM043 (Estrogen Receptor ChIP-PET) and IHM062 (IgG ChIA-PET) Using Illumina, we examined 4 libraries: IHM001 (Estrogen Receptor ChIA-PET replicate 1, Paired End Sequencing), IHH015 (Estrogen Receptor ChIA-PET replicate 2, Paired End Sequencing), H3K4me3 ChIP-Seq and RNA polymerase II ChIP-Seq
Project description:Estrogen receptor α (ERα) is key player in the progression of breast cancer. ERα binds to DNA and mediates long-range chromatin interactions throughout the genome, but the underlying mechanism in this process is unclear. Here, we show that AP-2 motifs are highly enriched in the ERα binding sites (ERBS) identified from the recent ChIA-PET of ERα. More importantly, we demonstrate that AP-2γ (also known as TFAP2C), a member of the AP-2 family which has been implicated in breast cancer oncogenesis, is recruited to chromatin in a ligand-independent manner and co-localized with ERα binding events. Furthermore, pertubation of AP-2γ expression disrupts ERα DNA binding, long-range chromatin interactions, and gene transcription. Using ChIP-seq, we show that AP-2γ and ERα binding occurs in close proximity on a genome-wide scale. The majority of these shared genomic regions are also occupied by the pioneer factor, FoxA1. AP-2γ is required for efficient FoxA1 binding and vice versa. Finally, we show that most ERBS associated with long-range chromatin interactions are co-localized with both AP-2γ and FoxA1. Together, our results suggest AP-2γ is an essential factor in ERα-mediated transcription, primarily working together with FoxA1 to facilitate ERα binding and long-range chromatin interactions. Gene expression profiling of negative control (NC) and AP-2γ siRNA transfected MCF-7, with and without E2 (estradiol) stimulation using microarray.