Grant: Discovery Grants Program - Individual

The human genome is made up of equal parts DNA and proteins called histones. This organization, known as chromatin, not only helps to compact the genome in size, but the histone proteins also control accessibility of the underlying DNA and play important regulatory functions in major biological processes such as transcription and replication. Histones are subjected to many types of post-translational modifications (PTMs). Some modifications, such as phosphorylation and acetylation, act as quick on-off switches to turn genes on or off, whereas other PTMs, such as methylation, provide long term instructions on how to package chromatin (loosely or tightly) at different parts of the genome over many cell divisions. My lab's has had a long standing interest in elucidating intricate mechanisms involving histone PTMs and how they regulate gene expression (e.g., transcriptional activation or repression). For example, we found that H3 phosphorylation occurs in a growth factor-inducible manner in interphase cells and this functions to activate transcription of growth-promoting genes. In contrast, the same H3 phosphorylation occurs during mitosis to condense chromosomes prior to cell division. How this same histone modification performs contrasting functions at different cell cycle stages is still unclear and led us to hypothesize that H3 phosphorylation works in combination with other histone PTMs to specify distinct downstream effects. Indeed, we previously developed a novel technique to purify phosphorylated H3-containing nucleosomes (the physiological form of chromatin comprising DNA wrapped around two copies each of histones H2A, H2B, H3 and H4) and found that H3 phosphorylation triggers downstream histone acetylation, and together they activate transcription in interphase cells. Using this same technique, we will now test and identify other histone PTMs that might synergize with H3 phosphorylation during mitosis to mediate chromosome condensation. In addition, we will adapt this technique to purify nucleosomes that either have two copies of phosphorylated H3 (symmetrically modified) or with only one H3 that is phosphorylated (asymmetrically modified) for biochemical analyses. Recent studies suggested symmetric vs. asymmetric histone PTMs may have distinct functions, and we will directly test whether this is true for H3 phosphorylation as well, particularly in the context of H3 phosphorylation-coupled histone acetylation and transcriptional activation. Finally, we will test whether asymmetric H3 phosphorylation also affects the mitotic H3 function as well. Histone PTMs play important roles in the regulation of many nuclear processes, and the functions of symmetric vs. asymmetric histone PTMs is a new and almost completely unknown aspect of chromatin regulation. Our proposed work addressing this new area of research will yield significant and important new knowledge that will enhance our understanding of chromatin and histone PTM mechanisms.