Project description:Amartya Sanyal mailto:amartya.sanyal@umassmed.edu (Wet Lab), Bryan R. Lajoie mailto:bryan.lajoie@umassmed.edu, Gaurav Jain mailto:gaurav.jain@umassmed.edu (Dry Lab), Job Dekker mailto:job.dekker@umassmed.edu (Principal Investigator) This track contains chromatin interaction data generated using the 5C (Chromatin Conformation Capture Carbon Copy) method by the ENCODE group (Dekker Lab) located at the University of Massachusetts, Worcester, MA. This track shows the significant looping interactions between transcriptional start sites (TSS) and distal regulatory elements in the context of the 44 ENCODE pilot regions spanning 1% of the human genome. Although the DNA is a linear sequence, the chromatin, which is packed and organized inside the nucleus, does not function linearly. This is most clearly illustrated by the fact that genes are often regulated by elements that are located hundreds of kilobases away in the linear genome. Imaging techniques have shown that regulatory elements can act over large genomic distances by engaging in direct physical interactions with target genes, resulting in the formation of chromatin loops. Based on these observations, we have envisaged that the spatial organization of the genome resembles a three-dimensional network that is driven by physical associations between genes and regulatory elements, both in cis (within the same chromosome) and in trans (between different chromosomes) (Dekker, 2006). Apart from imaging technology which is labor intensive and low-throughput, long-range chromatin looping interactions can be detected using the Chromosome Conformation Capture (3C) technology (Dekker et al., 2002). The 3C method employs formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. Cells are subsequently lysed and chromatin is digested with a restriction enzyme of choice. The digested fragments are then ligated under dilute conditions to facilitate intramolecular ligation. The result is a genome-wide interaction library of ligation products corresponding to all possible chromatin interactions. Specific ligation products can then be detected by PCR using specific primer pairs. The 5C method was developed to dramatically increase 3C throughput (Dostie et al., 2006; Dostie and Dekker, 2007). The 5C method greatly increases the scale of chromatin interaction detection by replacing the PCR detection step of 3C with ligation-mediated amplification (LMA). LMA is advantageous due to a much higher level of multiplexing by using thousands of primers in a single reaction to detect millions of chromatin interactions (ligation junctions) in parallel. The LMA step effectively "copies" 3C ligation products into much smaller 5C ligation products that precisely correspond to ligation junctions formed during the 3C procedure. The products of the multiplexed LMA reaction constitute the 5C library. The composition of the 5C library is determined using high-throughput DNA sequencing. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf The aim of the pilot study was to generate a "connectivity map" between transcription start sites (TSS) and distal regulatory elements within the 44 ENCODE PILOT regions. In the current scheme, 5C primers were designed for all HindIII restriction fragments. Reverse primers were designed on fragments containing the TSS of annotated genes. Forward primers were designed on all other fragments. This design allowed for the interrogation of all TSS with all other restriction fragments, thus generating an interaction map between TSS and regulatory elements. For gene desert ENCODE pilot regions (for example ENr313), an altered scheme of forward and reverse primers was designed. Primers were selected for relative uniqueness using a custom 15-mer frequency table and BLAST. A custom hexamer barcode was added to each primer to ensure the sequence was unique relative to the primer pool being used. Primers were also selected for the appropriate melting temperature and GC-content and a universal tail sequence for amplification. The 44 ENCODE regions were analyzed in two groups using two separate 5C primer pools. The first group (ENm) contained the manually-picked ENCODE regions, ENm001-014 and ENr313. The second group (ENr) contained the 30 randomly-picked ENCODE regions. The two 5C primer pools were made by pooling 5C primers for interrogating long-range interactions in the two groups of ENCODE regions. The primer pool for the ENm group contained a total of 3,150 primers (476 reverse 5C primers and 2674 forward 5C primers). This primer pool allowed interrogation of a total of 1,272,824 interactions. Of these, 83,427 interactions were between fragments that were both located in the same ENCODE region. This primer pool for the ENr group contained a total of 3,152 primers (505 reverse 5C primers and 2647 forward 5C primers). This primer pool allowed interrogation of a total of 1,336,735 interactions. Of these, 34,859 interactions were between fragments that were both located in the same ENCODE region. In total, 981 reverse primers and 5,321 forward primers were designed (corresponding to ~77.1% (6,302/8,174) of all HindIII fragments in the 44 ENCODE regions). Currently, data for two biological replicates have been generated for ENCODE Tier I (GM12878 and K562), Tier II (HeLa-S3), and H1 human embryonic stem cells (H1-hESC), spanning 14 ENCODE manual regions along with one random region (ENr313) as well as 30 random regions separately using high-throughput paired-end sequencing in the Illumina GA2 platform. The looping interactions, which are detected in both the biological replicates, are considered significant.

Project description:Amartya Sanyal mailto:amartya.sanyal@umassmed.edu (Wet Lab), Bryan R. Lajoie mailto:bryan.lajoie@umassmed.edu, Gaurav Jain mailto:gaurav.jain@umassmed.edu (Dry Lab), Job Dekker mailto:job.dekker@umassmed.edu (Principal Investigator) This track contains chromatin interaction data generated using the 5C (Chromatin Conformation Capture Carbon Copy) method by the ENCODE group (Dekker Lab) located at the University of Massachusetts, Worcester, MA. This track shows the significant looping interactions between transcriptional start sites (TSS) and distal regulatory elements in the context of the 44 ENCODE pilot regions spanning 1% of the human genome. Although the DNA is a linear sequence, the chromatin, which is packed and organized inside the nucleus, does not function linearly. This is most clearly illustrated by the fact that genes are often regulated by elements that are located hundreds of kilobases away in the linear genome. Imaging techniques have shown that regulatory elements can act over large genomic distances by engaging in direct physical interactions with target genes, resulting in the formation of chromatin loops. Based on these observations, we have envisaged that the spatial organization of the genome resembles a three-dimensional network that is driven by physical associations between genes and regulatory elements, both in cis (within the same chromosome) and in trans (between different chromosomes) (Dekker, 2006). Apart from imaging technology which is labor intensive and low-throughput, long-range chromatin looping interactions can be detected using the Chromosome Conformation Capture (3C) technology (Dekker et al., 2002). The 3C method employs formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. Cells are subsequently lysed and chromatin is digested with a restriction enzyme of choice. The digested fragments are then ligated under dilute conditions to facilitate intramolecular ligation. The result is a genome-wide interaction library of ligation products corresponding to all possible chromatin interactions. Specific ligation products can then be detected by PCR using specific primer pairs. The 5C method was developed to dramatically increase 3C throughput (Dostie et al., 2006; Dostie and Dekker, 2007). The 5C method greatly increases the scale of chromatin interaction detection by replacing the PCR detection step of 3C with ligation-mediated amplification (LMA). LMA is advantageous due to a much higher level of multiplexing by using thousands of primers in a single reaction to detect millions of chromatin interactions (ligation junctions) in parallel. The LMA step effectively "copies" 3C ligation products into much smaller 5C ligation products that precisely correspond to ligation junctions formed during the 3C procedure. The products of the multiplexed LMA reaction constitute the 5C library. The composition of the 5C library is determined using high-throughput DNA sequencing. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Yijun Ruan mailto:ruanyj@gis.a-star.edu.sg). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE Project. It shows the locations of protein factor mediated chromatin interactions determined by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) data (Fullwood et al., 2010) extracted from five different human cancer cell lines (K562 (chronic myeloid leukemia), HCT116 (colorectal cancer), HeLa-S3 (cervical cancer), MCF-7 (breast cancer), and NB4 (promyelocytic)). A chromatin interaction is defined as the association of two regions of the genome that are far apart in terms of genomic distance, but are spatially proximate to each other in the 3-dimensional cellular nucleus. Additionally, ChIA-PET experiments generate transcription factor binding sites. A binding site is defined as a region of the genome that is highly enriched by specific Chromatin ImmunoPrecipitation (ChIP) against a transcription factor, which indicates that the transcription factor binds specifically to this region. The protein factors displayed in the track include estrogen receptor alpha (ERa), RNA polymerase II (RNAPII), and CCCTC binding factor (CTCF). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a global de novo high-throughput method for characterizing the 3-dimensional structure of chromatin in the nucleus. In the ChIA-PET protocol, samples were cross-linked and fragmented, then subjected to chromatin immunoprecipitation. The DNA fragments that were brought together by the chromatin interactions were then proximity-ligated. During this proximity-ligation step, the half-linkers (created by the fragmentation) containing flanking MmeI sites (type IIS restriction enzymes) were first ligated to the DNA fragments and then ligated to each other to form full linkers. Full linkers bridge either two ends of a self-circularized fragment, or two ends of two different chromatin fragments. The material was then reverse cross-linked, purified and digested with MmeI. MmeI cuts 20 base pairs away from its recognition site. Tag-linker-tag (paired-end tag, PET) constructs were sequenced by ultra-high-throughput methods (Illumina or SOLiD paired-end sequencing). ChIA-PET reads were processed with the ChIA-PET Tool (Li et al., 2010) by the following steps: linker filtering, short reads mapping, PET classification, binding site identification, and interaction cluster identification. The high-confidence binding sites and chromatin interaction clusters were reported. Chromatin interactions identified by ChIA-PET have been validated by 3C, ChIP-3C, 4C and DNA-FISH (Fullwood et al., 2009).

Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE Project. This track displays genome-wide maps of histone modifications in different cell lines (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType) using ChIP-seq high-throughput sequencing. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Cells were cross-linked with 1% formaldehyde, and the reaction was quenched by the addition of glycine. Fixed cells were rinsed with PBS, lysed in nuclei lysis buffer, and the chromatin was sheared to 200-500 bp fragments using a Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 °C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross-linking in the immunoprecipitated DNA was reversed and treated with RNase-A. Following proteinase K treatment, the DNA fragments were purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. A quantity of 20-50 ng of ChIP DNA was end-repaired, followed by the addition of adenine, ligation to Illumina adapters, and creation of a Solexa library for sequencing. ChIP-seq affinity was directly measured through the raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). One percent false discovery rate thresholds (FDR 1.0%) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36-mers. ChIP-Seq affinities (Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm. All tracks have a False Discovery Rate of 1% (FDR 1.0%). Data were verified by sequencing biological replicates displaying a correlation coefficient > 0.9.

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE Project. This track shows DNaseI sensitivity measured genome-wide in different cell lines using the Digital DNaseI methodology (see below), and DNaseI hypersensitive sites. DNaseI has long been used to map general chromatin accessibility and DNaseI hypersensitivity is a universal feature of active cis-regulatory sequences. The use of this method has led to the discovery of functional regulatory elements that include enhancers, insulators, promotors, locus control regions and novel elements. For each experiment (cell type) this track shows DNaseI sensitivity as a continuous function using sequencing tag density (Raw Signal), and discrete loci of DNaseI sensitive zones (HotSpots) and hypersensitive sites (Peaks)." For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Digital DNaseI was performed by DNaseI digestion of intact nuclei, isolating DNaseI 'double-hit' fragments as described in Sabo et al. (2006), and direct sequencing of fragment ends (which correspond to in vivo DNaseI cleavage sites) using the Solexa platform (36 bp reads). Uniquely mapping high-quality reads were mapped to the genome. DNaseI sensitivity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). DNaseI sensitive zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. DNaseI hypersensitive sites (DHSs or Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.

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Chromatin Interactions by 5C from ENCODE/University of Washington

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