Files

Abstract

In the eukaryotic genome, DNA is organized into nucleosomes, which are comprised by 147bp of DNA wrapped around a core of eight histone proteins. These histones can then be post-translationally modified to regulate the function and status of the associated genomic region. The primary method to determine the genomic distribution of these modifications is chromatin immunoprecipitation (ChIP). However, this method, as traditionally practiced, has many problems hampering its interpretability insofar as it is non-quantitative and has indeterminate specificity. Internally calibrated ChIP (ICeChIP) can address some of these issues by employing nucleosomal internal standards, but many open questions remain as to the specificity of commercially available antibodies and many paradigms which are less easily resolved by traditional native ICeChIP. Here, I present my work on methods to extend the use cases of ICeChIP and applications therein. First, I show that many commercially available antibodies against H3K4 methylation states are of low quality and that common methods of antibody validation fail to reflect performance in ChIP, ultimately showing that this low specificity contributed to incorrect biological conclusions in several high-profile studies. Second, I describe our work on the study of bivalency, in which we developed a sequential form of ICeChIP to study nucleosomes bearing both H3K4me3 and H3K27me3, ultimately showing that many paradigms concerning such a combination are incorrect. Third, I describe our development of denaturative ICeChIP and use it to study the role of H3K79me2 in MLL-rearranged leukemias. Finally, I discuss our development of SmartMap, a tool to allocate next-generation sequencing reads that align ambiguously to the genome, demonstrate its ability to improve read depth at regions with low-mappability, and use it to study the role of histone modifications at repetitive elements. Overall, this work shows the power of using specific and quantitative methods in studying histone modifications.

Details

Actions

PDF

from
to
Export
Download Full History