Person: Cook, April
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Cook
First Name
April
Name
Cook, April
3 results
Search Results
Now showing 1 - 3 of 3
Publication Characterization of nucleosome occupancy in mammalian cells(2014-10-21) Cook, April; Kingston, Robert Edward; Blower, Michael; Mostoslavsky, Raul; Garber, ManuelChromatin is a complex of genomic DNA, RNA, and associated proteins. Many of the processes that occur on chromatin regulate the accessibility of the genetic material of a cell. The nucleosome is the basic subunit of chromatin, composed of a histone octamer wrapped with approximately 150bp of DNA. Alterations to chromatin structure, including to nucleosomes and their location, underlie global transcriptional diversity. A striking example of this is the so-called "open" chromatin state in pluripotent cells, characterized by loosely bound chromatin proteins and rapid nucleosome turnover, that allows transcriptional flexibility for subsequent differentiation. In contrast, differentiated cells contain compacted chromatin that can selectively block access to DNA and subsequent transcription. Thus, characterizing the physical state of chromatin is important to understanding its regulatory state. Digestion of chromatin with micrococcal nuclease (MNase) and subsequent sequencing of the protected DNA fragments produces a map of nucleosome occupancy. Traditional MNase mapping experiments capture a snapshot of nucleosome occupancy, providing information about nucleosomes that are accessible at the level of digestion used. We analyzed regions of difference in nucleosome occupancy between embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and differentiated cell types using traditional MNase-seq and found that differences in pluripotent and differentiated cells are punctate and correlate with regulatory regions important for pluripotency and development. Further, our analysis shows ESCs and iPSCs to be vastly more similar to each other in their chromatin structure than to the differentiated cells. We then developed a new way of collecting and analyzing MNase-seq data that allows us to determine both nucleosome occupancy as well as the accessibility of DNA to regulatory factors. Our methodology discerns distinct physical states of chromatin and provides novel insights into the accessibility of regulatory regions. Additionally, we present a quantitative metric useful for characterizing local and global regions of the genome that should be useful in future cell type comparisons.Publication 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, RobertChromatin 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.Publication MNase titration reveals differences between nucleosome occupancy and chromatin accessibility(Nature Publishing Group, 2016) Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter; Kingston, Robert; Tolstorukov, Michael Y.Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.