Aided by next-generation sequencing and epigenomics, the identification of regulatory elements such as promoters and enhancers is progressing at a rapid pace. Two distinct histone modifications can be used to identify and distinguish these elements. This involves the methylation states of histone H3 at lysine 4. Trimethylation (H3K4me3) and monomethylation (H3K4me1) are uniquely localized to promoters and enhancers respectively. Employing ChIP- seq, chromatin immunoprecipitation coupled with next-generation sequencing, with antibodies against these modifications allows one to construct genome-wide maps in a variety of species. However, these modifications often mark such elements in a cell-specific manner, especially in the case of enhancers with H3K4me1. Therefore, if we are to have a comprehensive list of these regulatory elements, maps will need to be constructed from most cell types of the body. Here in lies a major hurdle. For the purpose of ChIP-seq, ChIP is most commonly performed on a large number of cells, ranging from 10^8 to 10^6. This scale is unattainable for most cell types, especially if the goal is to mov away from cell lines and focus on primary cell types. Without reducing the scale of ChIP-seq, it will prove challenging to continue the pace at which we are currently identifying such key regulatory elements. Recently, a number of attempts have been made to scale down input material for the library amplification, with fewer focusing on small-scale ChIP, and even less on small-scale chromatin fragmentation. We are proposing a method, with viable alternatives, that we believe will reduce the scale of ChIP-seq by 2-4 orders of magnitude, possibly as low as 1-100 cells. This method takes into account the entire process from chromatin fragmentation to library amplification. Our approach is greatly aided by the use of in vitro transposition to add sequencing adaptors to chromatin prior to ChIP. Ultimately, this method may help us achieve single cell analysis.

Public Health Relevance

We are developing a method to aid the identification of functional regulatory elements, such as enhancers and promoters. Our approach is to reduce the scale of input material required for ChIP-seq by several orders of magnitude. This method alleviates several steps currently required during library preparation, which may provide an added benefit of reducing cost.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HG006767-01
Application #
8309590
Study Section
Special Emphasis Panel (ZHG1-HGR-M (J2))
Program Officer
Pazin, Michael J
Project Start
2012-04-24
Project End
2014-03-31
Budget Start
2012-04-24
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$231,563
Indirect Cost
$81,563
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195