Characterization of nucleosome occupancy in mammalian cells

• Main Author: Cook, April D
• Other Authors: Kingston, Robert Edward
• Language: en_US
• Published: Harvard University 2014
• Subjects: Genetics; Chromatin; MNase; nucleosome
• Online Access: http://dissertations.umi.com/gsas.harvard.inactive:11748; http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070019

Chromatin 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.