Progress towards the human chromatome: Global and targeted analysis of chromatin proteins and their post-translational modifications using mass spectrometry-based proteomics

Eukaryotic DNA is packaged into chromatin, a highly organized protein-DNA complex. Chromatin is involved in virtually all DNA processes; changes in its composition and structure are sufficient to cause heritable changes. The basic unit of chromatin is the nucleosome which consists of DNA wrapped around an octameric histone core. Histone proteins, particularly their N-terminal tails, undergo diverse and pervasive patterns of post-translational modifications (PTMs). Combinations of such modifications are thought to constitute a 'histone code' that influences gene expression. Additional chromatin proteins and their PTMs contribute to the formation of chromatin domains with different gene expression levels. After decades of research, the complete protein composition and structure of chromatin and its domains remain insufficiently characterized.;This thesis aims to comprehensively characterize human chromatin proteins and their modifications both at the global level and at the single protein level using mass spectrometry based proteomics. On the global scale, we have identified nearly 2,000 distinct proteins to be chromatin components. Approximately 11% of these proteins are post-translationally modified. This is the most complete characterization of the protein components in chromatin to date, representing at least a 5-fold improvement over previous investigations. We have also performed the first large-scale proteomic investigation into the protein composition of (actively transcribed) euchromatin and (silenced) heterochromatin.;On the single protein level, we have developed various proteomics methods to greatly streamline the identification of differentially expressed histone codes. We have applied these methods to study the histone codes in euchromatin and heterochromatin. We find novel and distinct histone modifications for both euchromatic and heterochromatic regions. These findings provide new insights into the histone codes of gene activation and silencing, and will hopefully allow for additional discoveries involving the regulation and maintenance of these processes. Through a similar protocol, we investigated differentially expressed histone codes occurring throughout a critical cellular process known as apoptosis. Despite the fact that apoptosis involves intense chromatin remodeling, histone modification changes during this event have not been studied in detail. We find distinct histone modification changes in cells undergoing apoptosis; our findings seem to indicate that chromatin increases in heterochromatic character as apoptosis progresses.