11 research outputs found
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High-resolution mapping of transcription factor binding sites on native chromatin
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions
Characterization and analysis of repetitive centromeres
Thesis (Ph.D.)--University of Washington, 2017Centromeres are specialized regions of eukaryotic chromosomes that ensure faithful transmission of genetic information at each cell division. The molecular architecture of centromeres is defined by evolutionarily dynamic protein and DNA components, which have been proposed to contribute to the origin of new species, while defects in centromeres have been linked to human disease. Centromeres are embedded in regions composed of large arrays of head-to-tail 'satellite' DNA elements, which are not amenable to many conventional genomic analyses. Here, I describe the development of methods for the analysis of repetitive genomic regions and apply these tools to study primate centromeres, which are composed of ~170-bp alpha-satellite units. Although centromeric DNA is known to be polymorphic in humans, comprehensive cataloguing of variants at centromeres has not been possible. To gain insight into centromeric genetic variation, I developed a method that uses single-molecule sequencing for analyzing characteristic sequence periodicities called higher-order repeats that arise in human centromeres. The application of this approach to catalogue inter-individual, population-scale, and disease-associated structural variation identified extensive polymorphism in centromeres associated with binding sites for CENP-B, a sequence-specific DNA binding protein. This work also defined a set of functionally important alpha-satellite dimeric units that are underrepresented in current centromere models and demonstrated aberrations in centromeric sequence in breast cancer. I suggest a role for CENP-B in the evolution and maintenance of higher-order periodicities in centromeric arrays. Although alpha-satellite is present at the centromeres of most primates, the precise mechanisms of evolution of centromeric DNA and the contribution of genetic sequence to the specification of centromere identity remain unresolved. I examined centromere evolution in primates using a combination of data from different whole-genome sequencing methods. This approach demonstrated the presence of higher-order periodicities in all primates and identified an important role for CENP-B in shaping centromeric repeat organization. Further analysis of alpha-satellite uncovered interspecific variation in the presence of short inverted repeats, which may form hairpin and stem-loop structures. Based on these data, I propose a genetic mechanism for centromere specification that depends on the formation of cruciform or other non-B-form nucleic acid structures. Taken together, this work enables the cataloguing of variation in satellite DNA, defines important evolutionary transitions in primate centromeres, and advances a model for primate centromere evolution and a theory for centromere specification
Influence of Exogenous PlGF on Apoptosis in the H9c2 Cardiomyoblast Cell Line
Advisor: Ronald TorryPlacenta growth factor (PlGF) is known to inhibit apoptosis in cells such as trophoblast or endothelial cells. Although PlGF is expressed in heart tissue in vivo and in vitro, little is known about its antiapoptotic
effects in cardiomyocytes. H9c2 cells (a rat cardiomyoblast cell line) were used to determine
if PlGF can protect these cells from oxidative-induced apoptosis. Cells were cultured and then
treated with hydrogen peroxide (H2O2) to induce apoptosis. An H2O2 dose response curve was performed and showed increased concentrations of H2O2 were associated with increased percent apoptosis: 25uM H2O2 = 36.77±8.9%; 50uM H2O2 = 47.53±4.6%; and 100uM H2O2 = 61.36±5.1%
apoptosis. Some cultures were then pre-treated with either 10ng/mL or 50ng/mL of exogenous recombinant mouse P1GF for 24 hours and then incubated with 50uM H2O2 for 2 hours. Cells were stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent stain that binds strongly to DNA and then fixed with ice cold acetone. Apoptotic cells were recognized by the condensed, fragmented, and degraded nuclei with fluorescence microscopy. Random fields of cells from each treatment group were chosen to count the apoptotic cells and percent of apoptosis was calculated for each field. Control cultures
showed 12.41±0.5% apoptosis, H2O2+0ng/mL PlGF = 46.49±1.5% apoptosis, H2O2+10ng/mL PlGF = 30.48±5.9% apoptosis, and H2O2+50ng/mL PlGF = 33.70±5.8% apoptosis. In conclusion, we
have developed a consistent method to induce apoptosis in H9c2 cells and used this to show that although exogenous PlGF tended to protect H9c2 cells from oxidative-induced apoptosis, this difference did not attain statistical significance.Drake University, Pharmaceutical, Biomedical, and Admininstrative Science Departmen
Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling
An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types
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Massively multiplex single-molecule oligonucleosome footprinting.
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure
Recommended from our members
Massively multiplex single-molecule oligonucleosome footprinting.
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure