| Much remains unknown about the establishment and maintenance of chromatin states. Of particular interest are the numerous post-translational modifications of the histone proteins that comprise the core of the nucleosome, the basic unit of chromatin. These histone modifications play an important role in the regulation of gene activity. Currently, the "histone code" hypothesis is the dominant model in describing the function of these modifications, suggesting that they carry complex epigenetic information. However, the question of whether a "histone code" truly exists is a subject under much debate, and it is even less clear whether the epigenetic information these modifications carry is inheritable. While studies have probed this question on a limited scale, no study has yet examined this in global detail within a synchronous cell population. In this dissertation, a number of technologies were developed to enable such an analysis. ChIP-chip, a method combining Chromatin IP (ChIP) with DNA microarrays, is a method commonly used to rapidly and globally map epigenetically-relevant epitopes (e.g. transcription factors) in the genome. T7-based Linear Amplification of DNA (TLAD) was developed as an improvement over exponential-based methods of amplifying a random, complex mixture of DNA, a critical step in the ChIP-chip method. With this method, global studies of nucleosome occupancy were performed in mid-log yeast, where nucleosome depletion was observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. However, to enable single nucleosome-resolution mapping of histone modifications, micrococcal nuclease digestion was combined with Chromatin IP (ChIP) and a high-resolution tiling microarray. With these tools, the in vivo occurrence of combinations of 12 histone modifications on thousands of nucleosomes was examined in mid-log yeast, revealing that histone modifications do not occur independently but fall into two groups of co-occurring modifications, with no evidence for a deterministic code of many discrete states, suggesting a simple, redundant histone code. Preliminary analyses, from dynamic mapping of four of these histone modifications in synchronized yeast, reveal trends that are largely consistent with those observed in midlog yeast and tentatively suggest that histone methylation could be epigenetically inheritable. |
