The use of high-throughput sequencing to investigate histone modifications in the non-coding portions of the human genome.

摘要

The higher order structure of eukaryotic chromosomes is complex, due to the fact that nearly six feet of DNA needs to be packaged into the nucleus of a cell. This packaging requires multiple levels of organization from the raw double helix up to a completely folded chromosome. The fundamental level of this organization is the wrapping of DNA around nucleosomes consisting of histone proteins. These histones can be post-transcriptionally modified through the addition of acetyl or methyl groups to individual amino acid residues. I have investigated the enrichment of specific histone lysine methylation states throughout the human genome in hematopoietic cells.;In Chapter 2, I examine the genome-wide locations of 18 different histone acetylations using chromatin immunoprecipitation coupled with high-throughput sequencing. The locations of these acetylations were analyzed in concert with the previously determined locations of 19 histone methylations. I clustered the modifications into groups which were found to correspond to gene expression or repression.;In Chapter 3, I analyze the enriched histone modifications within different portions of the genome. Notably, there were many cases where different methylation states of the same histone residue were found to have differential enrichment. For example, the trimethyaltion of H3K9 (H3K9me3) was found to be strongest in repressed heterochromatin whereas the monomethylated form of H3K9 (H3K9me1) was found to be strongest in actively transcribed genes. I then performed a deeper analysis of the histone modification variation within the non-coding portions of the genome.;Finally, in Chapter 4, I look specifically at the profile of H3K9me3 enrichment throughout the human genome. This modification is generally considered to be repressive and corresponds to constitutive heterochromatin, but recent studies have found it to be enriched at actively expressed genes. For this analysis, I developed a novel repetitive matching technique to interpret the alignment of high-throughput sequencing reads. I found that there are significant levels of H3K9me3 both within genes and in non-genic regions of the genome. This finding indicates that H3K9me3 has a function exclusive of gene expression and it is hypothesized to be important for the higher level organization of chromatin into chromosomes.