cis-regulatory elements (CREs) regulate the expression of genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there ...remain major gaps in the functional characterization of CREs and the identification of their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.
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•A focused CRISPR screen identifies essential enhancers for pluripotency maintenance•Proximal pluripotency genes are identified by 4C-seq•Lrrc31 is found to regulate pluripotency through the JAK-STAT signaling pathway•Lrrc31 regulates the binding of STAT3 on super-enhancers of mESCs
Wang et al. identify important genomic regulatory elements through a CRISPR knockout screen. Through 4C-seq, they correlate their candidate enhancers with their target genes and zoom in on Lrrc31 as a pluripotency regulator. Lrrc31 is found to regulate pluripotency by affecting the phosphorylation of STAT3 through the JAK-STAT3 signaling pathway.
DNA fragmentation is a well-recognized hallmark of apoptosis. However, the precise DNA sequences cleaved during apoptosis triggered by distinct mechanisms remain unclear. We used next-generation ...sequencing of DNA fragments generated in Actinomycin D-treated human HL-60 leukemic cells to generate a high-throughput, global map of apoptotic DNA breakpoints. These data highlighted that DNA breaks are non-random and show a significant association with active genes and open chromatin regions. We noted that transcription factor binding sites were also enriched within a fraction of the apoptotic breakpoints. Interestingly, extensive apoptotic cleavage was noted within genes that are frequently translocated in human cancers. We speculate that the non-random fragmentation of DNA during apoptosis may contribute to gene translocations and the development of human cancers.
Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet ...available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.
How human modification of native habitats changes the feeding patterns and nutritional ecology of tropical birds is critical to conserving avian biodiversity, but tropical bird diets are laborious to ...investigate using the traditional methods of diet analysis. Stable isotope analysis provides a cost-effective and efficient proxy to identify general foraging patterns, especially when dietary shifts spanning multiple trophic levels have occurred due to ecosystem disturbance or transformation. To characterize the diets of forest bird species that persist in tropical agricultural countryside, we compared feather carbon (δ
13
C) and nitrogen (δ
15
N) isotope values of four species caught and radio-tracked in a 270 hectare forest reserve, smaller forest remnants (including mature forest, secondary forest, and riparian strips), and coffee plantations in mid-elevation (
ca.
800–1,400 m) southern Costa Rica. Bird habitat choice had a significant effect on diet composition as revealed by δ
13
C and δ
15
N values. Three of the four species studied showed evidence of significantly reduced consumption of invertebrates in coffee plantations, with the isotope values of two species (
Tangara icterocephala
and
Mionectes oleaginous
) indicating, by comparison, nearly a doubling of invertebrate consumption in forest remnants. Our results suggest that coffee plantations are deficient in invertebrates preferred by forest generalist birds that forage in both native forest remnants and coffee plantations. In this region, typical of mountainous American tropics, small forest remnants and a larger forest reserve provide critical dietary resources for native forest birds that utilize the agricultural countryside.
The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it ...is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.
Gene expression is the most fundamental level at which the genotype leads to the phenotype of the organism. Enabled by ultra-high-throughput next-generation DNA sequencing, RNA-Seq involves shotgun ...sequencing of fragmented RNA transcripts by next-generation sequencing followed by in silico assembly, and is rapidly becoming the most popular method for gene expression analysis. PolyA+ RNA-Seq analyses of normal human adult tissue samples such as Illumina's Human BodyMap 2.0 Project and the RNA-Seq atlas have provided a useful global resource and framework for comparisons with diseased tissues such as cancer. However, these analyses have failed to provide information on polyA-RNA, which is abundant in our cells. The most recent advances in RNA-Seq analyses use ribosomal RNA-depletion to provide information on both polyA+ and polyA-RNA. In this paper, we describe the use of Illumina's HiSeq 2000 to generate high quality rRNA-depleted RNA-Seq datasets from human fetal and adult tissues. The datasets reported here will be useful in understanding the different expression profiles in different tissues.
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces (7, 2, 12). Such architectures are not random and involve ...interactions between gene promoters and regulatory elements (13). The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination (1, 14). Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures (5,6). Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions (8). We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cells are treated with ...formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA-PET for genome-wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies.
Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation. To reduce biases, large numbers of colonies are plated and scraped from ...solid-surface agar. This process is time consuming, tedious and limits scaling up. At the same time, multiple displacement amplification (MDA) has been recently developed as a method for in vitro amplification of DNA. However, MDA has no selection function for the removal of ligation multimers. We developed a novel method of briefly introducing ligation reactions into bacteria to select single insert DNA clones followed by MDA to amplify. We applied these methods to a Gene Identification Signatures with Paired-End diTags (GIS-PET) library, which is a complex transcriptome library created by pairing short tags from the 5′ and 3′ ends of cDNA fragments together, and demonstrated that this selection and amplification strategy is unbiased and efficient.
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