Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Sequencing-based ssDNA detection

ABSTRACT: Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method, single-stranded DNA (ssDNA) sequencing (SSDS), that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS consists of a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired. Development and validation of the method, SSDS, for the specific detection of ssDNA-derived and dsDNA-derived fragments in sequencing libraries and enrichment of ssDNA-derived fragments. SSDS was used to detect meiotic DSBs in 9R/13R mice.

ORGANISM(S): Mus musculus

SUBMITTER: Pavel Khil

PROVIDER: E-GEOD-34592 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA.

Khil Pavel P PP Smagulova Fatima F Brick Kevin M KM Camerini-Otero R Daniel RD Petukhova Galina V GV

Genome research 20120224 5

Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we ha ...[more]

PMID: 22367190

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Project description:Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain yet poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known on how these are selected and controlled the ultimate cellular effectors of terminal differentiation. We addressed this question during late Drosophila embryogenesis when the finely tuned expression of a TF, Ovo/Shavenbaby (Svb), triggers the morphological differentiation of epidermal trichomes. We used chromatin immunoprecipitation and microarray profiling to identify Svb downstream target genes and show that Svb directly regulates a large set of terminal effectors of trichome formation. Functional assays delineated 18 Svb bound sequences driving specific expression of trichome effectors, with highly similar pattern and dynamics. Coupling computational modeling to functional dissection, we further investigated the regulatory logic of these enhancers. We find that these “terminal” enhancers harbor remarkable features with respect to their functional architectures. Trichome enhancers display weak if any clustering of Svb binding sites. Moreover, the in vivo function of each site relies on its intimate context, with a critical importance of the nucleotides flanking Svb binding sites. Finally, we identify additional cis-regulatory motifs, showing a broad diversity of positioning among trichome enhancers, and that critically contribute to their activity. Taken together, these results show that trichome formation is underpinned by unexpectedly diverse modes of regulation, and shed light on the functional architecture of enhancers governing a terminal differentiation program. Chromatin from 12-14 hour old embryos expressing svb tagged with GFP (Kondo T, Plaza S, Zanet J, Benrabah E, Valenti P, Hashimoto Y, Kobayashi S, Payre F, Kageyama Y: Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science 2010, 329:336-339) was collected in duplicate. Chromatin was immunoprecipitated using an antiGFP antibody and input chromatin was used as a control.

Dataset Information

Sequencing-based ssDNA detection

Publications

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA.

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