Unknown,Transcriptomics,Genomics,Proteomics

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ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS

ABSTRACT: Many DNA-hypermethylated cancer genes are occupied by the polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that approximately 75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESC and adult stem/progenitor cells. A large number of these genes are key developmental regulators and a subset, which we call the "DNA hypermethylation module", comprise a portion of the PcG target genes that are downregulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA-hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions. The goal was to analyze the chromatin in MSCs, osteoblasts and U2OS cells of genes that are methylated in the osteosarcoma cell line U2OS. Cell culture: MSCs derived from adult bone marrow were cultured and differentiated to osteoblasts as described by Jaiswal et al., 1997 (J Cell Biochem 64, 295-312). U2OS cells were cultured in McCoy's 5A medium supplemented with 10% FBS. ChIP-seq: Cells were crosslinked in 3.7% formaldehyde, lysed and sonicated to obtain chromatin fragments in the range of 200 to 600 base pairs. Sonicated chromatin was incubated with primary antibody, H3K4Me3 (Millipore, 07-473) or H3K27Me3 (kind gift from Thomas Jenuwein/ Nicholas Shukeir), overnight at 4°C, followed by capturing primary antibodies by adding ProteinA/G magnetic beads (DynaBeads) and incubating at room temperature for 3-4 hrs. Captured chromatin was washed in low- and high-salt buffers, as well as TE. Chromatin fragments were subjected to simultaneous elution, de-crosslinking and Proteinase-K treatment at 65°C in elution buffer followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Sequencing and alignment: Purified DNA was used to prepare the sequencing library and sequenced on Applied Biosystems SOLiD (V3). Sequencing reads were aligned to hg18 (NCBI 36) using Bioscope 1.2.1. The software guarantees finding all alignments between the first 25 base pairs of the read (seed) and the reference sequence with up to 2 mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and -2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported. Peak identification: Aligned reads were analyzed using Model-based Analysis of ChIP-Seq (MACS) to detect regions enriched for the histone marks (called peaks) with default settings. Input was used as background for peak identification.

ORGANISM(S): Homo sapiens

SUBMITTER: hari easwaran

PROVIDER: E-GEOD-35573 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This track depicts high throughput sequencing of long RNAs (>200 nt) from RNA samples from tissues or subcellular compartments from ENCODE cell lines. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome. 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. Sample preparation and sequencing: K562 and GM12878 total cell, total RNA: Standard Illumina Pair-end kit with the sole exception that a "tagged" random hexamer was used to prime the 1st strand synthesis: 5'-ACTGTAGGN6-3'. The addition of this tag is what permits us to make strand assignments for the reads. The sequence of the tag is reported in the 5' end of the read. Asymmetric PCR can place the tag on either the 1st or 2nd read depending on which strand it used as a template. Strand assignments are made by looking for the tag at the 5' end of either read 1 or read 2. Read 1 is physically linked to read 2. Therefore, if a tag is present on one end strand assignments are made for both ends. We noted during analysis that the tags are generally 5' truncated. We only "strand" reads that contain ACTGTAGG, CTGTAGG, TGTAGG, GTAGG. Between 63-68% of reads could be stranded in these libraries. It is possible to cull additional stranded reads that contain non-templated TAGG, AGG, GG, or G sequences at their 5' end. The peak in insert size distribution is between 200-250 nucleotides. K562 cytosol, polyA+ RNA: Oligo-dT selected poly-A+ RNA was RiboMinus-treated according to the manufacturer's protocol (Invitrogen). The RNA was treated with tobacco alkaline pyrophosphatase to eliminate any 5' cap structures and hydrolyzed to ~200 bases via alkaline hydrolysis. The 3' end was repaired using calf intestinal alkaline phosphatase, and poly-A polymerase was used to catalyze the addition of Cs to the 3' end. The 5' end was phosphorylated using T4 PNK, and an RNA linker was ligated onto the 5' end. Reverse transcription was carried out using a poly-G oligo with a defined 5' extension. The inserts were then amplified using oligos targeting the 5' linker and poly-G extension. This cloning protocol generated stranded reads that were read from the 5' ends of the inserts. The library was sequenced on a Solexa platform for a total of 36 cycles; however, the reads underwent post-processing, resulting in trimming of their 3' ends. Consequently, the mapped read lengths are variable. Analysis: K562 and GM12878 total cell, total RNA: Tags were removed from the 5' ends of the reads in accordance to their lengths and strand assignments made. Subsequently, the reads were trimmed from their 3' ends to a final length of 50 nucleotides and were mapped using NexAlign, a program developed by Timo Lassman, RIKEN. We allowed up to 2 mismatches across the entire length and only report reads that mapped to a single/unique locus in the assembled hg18 genome. K562 cytosol, polyA+ RNA: Reads were mapped to the human (hg18, March 2006) assembly using Nexalign, with only uniquely mapping (one loci), exactly matching (no mis-matches) aligned reads reported in the processed files, as follows: 1) Collect the read sequences from Illumina non-filtered output files. 2) Filter out all reads that contain undefined nucleotides ('N'). 3) Perform iterative alignment/C-tail chopping algorithm (below). On each alignment step, the reads are aligned to the genome with 100% identity. All reads that align to a single locus are withdrawn from the alignment pool and only the reads that could not be aligned continue to the next step. a) Align to the hg18 genome using Nexalign 1.3.3 (© Timo Lassmann) without chopping off any nucleotides. b) Chop off any C-blocks (until the first non-C) at the ends of the reads. c) Align to the genome -> remove and save those that align. d) Chop off any non-Cs until the next C. e) Chop off C-block until the next non-C. f) Align to the genome -> remove and save those that align. g) Repeat steps d, e, and f until the reads align to the genome, or chopping results in the reduction of the reads' lengths to below 16 (default), or there are no non-Cs left.

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Jonathan Preall jpreall@cshl.edu (Generation 0 Data from Hannon Lab), Carrie Davis davisc@cshl.edu (experimental), Alex Dobin dobin@cshl.edu (computational), Wei Lin wlin@cshl.edu (computational), Tom Gingeras gingeras@cshl.edu (primary investigator)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). hg18: This data was produced by Hannon lab part of Cold Spring Harbor as part of the ENCODE Project. The series depicts NextGen sequencing information for RNAs between the sizes of 20-200 nt isolated from RNA samples from tissues or sub cellular compartments of cell lines. hg19: This track depicts NextGen sequencing information for RNAs between the sizes of 20-200 nt isolated from RNA samples from tissues or sub cellular compartments from ENCODE cell lines. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome. hg19: This cloning protocol generates directional libraries that are read from the 5' ends of the inserts, which should largely correspond to the 5' ends of the mature RNAs. The libraries were sequenced on a Solexa platform for a total of 36, 50 or 76 cycles however the reads undergo post-processing resulting in trimming of their 3' ends. Consequently, the mapped read lengths are variable. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf hg18: Small RNAs between 20-200 nt were ribominus treated according to the manufacturer's protocol (Invitrogen) using custom LNA probes targeting ribosomal RNAs (some datasets are also depleted of U snRNAs and high abundant microRNAs). The RNA was treated with Tobacco Alkaline Pyrophosphatase to eliminate any 5' cap structure. Poly-A Polymerase was used to catalyze the addition of C's to the 3' end. The 5' ends were phosphorylated using T4 PNK and an RNA linker was ligated onto the 5' end. Reverse transcription was carried out using a poly-G oligo with a defined 5' extension. The inserts were then amplified using oligos targeting the 5' linker and poly-G extension and containing sequencing adapters. The library was sequenced on an Illumina GA machine for a total of 36, 50 or 76 cycles. Initially 1 lane is run. If an appreciable number of mappable reads are obtained, additional lanes are run. Sequence reads underwent quality filtration using Illumina standard pipeline (Gerlad). The read lengths may exceed the insert sizes and consequently introduce 3' adaptor sequence into the 3' end of the reads. The 3' sequencing adaptor was removed from the reads using a custom clipper program, which aligned the adaptor sequence to the short-reads, allowing up to 2 mismatches and no indels. Regions that aligned were "clipped" off from the read. The trimmed portions were collapsed into identical reads, their count noted and aligned to the human genome (NCBI build 36, hg18 unmasked) using Nexalign (Lassmann et al., not published). The alignment parameters are tuned to tolerate up to 2 mismatches with no indels and will allow for trimmed portions as small as 5 nucleotides to be mapped. We report reads that mapped 10 or fewer times. Data obtained from each lane is processed and mapped independently. The processed/mapped data from each lane is then complied as a single track without additional processing and submitted to UCSC. Consequently, identical reads within a lane were collapsed and their value is reported as the "transfrag" signal value. However, the redundancy between lanes has not been eliminated so the same transfrag may appear multiple times within a signal. hg19: Small RNAs between 20-200 nt were ribominus treated according to the manufacturer's protocol (Invitrogen) using custom LNA probes targeting ribosomal RNAs (some datasets are also depleted of U snRNAs and high abundant microRNAs). The RNA was treated with Tobacco Alkaline Pyrophosphatase to eliminate any 5' cap structures. Poly-A Polymerase was used to catalyze the addition of C's to the 3' end. The 5' ends were phosphorylated using T4 PNK and an RNA linker was ligated onto the 5' end. Reverse transcription was carried out using a poly-G oligo with a defined 5' extension. The inserts were then amplified using oligos targeting the 5' linker and poly-G extension and containing sequencing adapters. The library was sequenced on an Illumina GA machine for a total of 36, 50 or 76 cycles. Initially, one lane was run. If an appreciable number of mappable reads were obtained, additional lanes were run. Sequence reads underwent quality filtration using Illumina standard pipeline (GERALD). The Illumina reads were initially trimmed to discard any bases following a quality score less than or equal to 20 and converted into FASTA format, thereby discarding quality information for the rest of the pipeline. As a result, the sequence quality scores in the BAM output are all displayed as "40" to indicate no quality information. The read lengths may exceed the insert sizes and consequently introduce 3' adapter sequence into the 3' end of the reads. The 3' sequencing adapter was removed from the reads using a custom clipper program (available at http://hannonlab.cshl.edu/fastx_toolkit/), which aligned the adapter sequence to the short-reads using up to 2 mismatches and no indels. Regions that aligned were "clipped" off from the read. Terminal C nucleotides introduced at the 3' end of the RNA via the cloning procedure are also trimmed. The trimmed portions were collapsed into identical reads, their count noted and aligned to the human genome (version hg19, using the gender build appropriate to the sample in question - female/male) using Bowtie (Langmead B. et al). The alignment parameter allowed 0, 1, or 2 mismatches iteratively. We report reads that mapped 20 or fewer times. Discrepancies between hg18 and hg19 versions of CSHL small RNA data: The alignment pipeline for the CSHL small RNA data was updated upon the release of the human genome version hg19, resulting in a few noteworthy discrepancies with the hg18 dataset. First, mapping was conducted with the open-source Bowtie algorithm (http://bowtie-bio.sourceforge.net/index.shtml) rather than the custom NexAlign software. As each algorithm uses different strategies to perform alignments, the mapping results may vary even in genomic regions that do not differ between builds. The read processing pipeline also varies slightly, in that we no longer retain information regarding whether a read was 'clipped' off adapter sequence.

Dataset Information

ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS

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