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Deaton, Aimee

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Deaton

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Aimee

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Deaton, Aimee

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2013 - 20144

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Author's Publications: search.filters.isAuthorOfPublication.c4f8b477-d494-4f4b-9b82-2da1859fac89

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Now showing 1 - 4 of 4
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    Multiplexed Illumina sequencing libraries from picogram quantities of DNA
    (BioMed Central, 2013) Bowman, Sarah K.; Simon, Matthew D; Deaton, Aimee; Tolstorukov, Michael; Borowsky, Mark L; Kingston, Robert
    Background: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. Results: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Conclusions: Application of this method allows whole genome studies from samples where material or yields are limiting.
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    H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex
    (eLife Sciences Publications, Ltd, 2014) Bowman, Sarah K.; Deaton, Aimee; Domingues, Heber; Wang, Peggy I; Sadreyev, Ruslan; Kingston, Robert; Bender, Welcome
    The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a ‘stairstep’ pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity. DOI: http://dx.doi.org/10.7554/eLife.02833.001
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    Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming
    (2014) West, Jason A.; Cook, April; Alver, Burak; Stadtfeld, Matthias; Deaton, Aimee; Hochedlinger, Konrad; Park, Peter; Tolstorukov, Michael Y.; Kingston, Robert
    Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2, and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.
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    The structure of Polycomb-repressed chromatin in the bithorax complex
    (BioMed Central, 2013) Bowman, Sarah K.; Deaton, Aimee; Domingues, Heber; Bender, Welcome; Kingston, Robert