The goal of this proposal is to link histone post-translational modifications (HPTMs) to transcription rates and bursting frequencies. In all eukaryotes studied, transcription at individual gene loci exhibits random oscillations between active and inactive states, a phenomenon known as transcription bursting. Cell fate specification in embryos is driven by genes whose bursting characteristics determine synchrony and robustness during development, but the molecular interactions which generate the bursting frequencies observed in development are unknown. To understand how transcription controls development, it is necessary to uncover the molecular determinants that control the duration of bursts and the rates at which bursts occur in vivo. Eukaryotic transcription is characterized by the enrichment of HPTMs at enhancers and promoters. Despite the strong correlation between HPTMs and gene activity, it is unclear how HPTMs determine transcription rates and set bursting frequencies. Moreover, HPTMs occur in many combinations at promoters and enhancers, generating in theory many possible promoter states. Here, I will determine whether HPTMs confer multiple transcriptional states during development and whether those states determine specific rates of transcription bursting. The most powerful methods currently available to measure transcription rates are single mRNA FISH and live nascent site imaging. To quantitatively describe state transitions using these assays, I will implement a mathematical model of transcription states. The ?two-state? model has been widely used to quantitatively describe burst duration and frequency in terms of the average rates of switching between the active and inactive states. However, it is untested whether the simple two-state approach can accurately describe the transcription of a gene undergoing complex regulation during development, such as the gap gene hunchback. Here I will determine whether the two-state model is sufficient to describe bursting frequencies of endogenous hunchback. I will insert RNA loops into the endogenous hunchback locus to create an endogenous reporter. I will use single mRNA FISH and live-imaging of the endogenous hunchback reporter to measure transcriptional bursting kinetics. I will then determine how HPTMs affect the kinetics of hunchback transcription. I will maternally knockdown an array of histone methyltransferases, acetyltransferases, demethylases, and deacetylates and measure their effects on the transcriptional bursting of hunchback. For each HPTM knockdown, I will determine which mathematical model best describes the single mRNA FISH and live-imaging data. Together, these experiments will determine how changes in specific histone marks change hunchback bursting kinetics and promoter state.
Epigenetic information plays a pivotal role in cell fate decisions in development and prevalent diseases such as cancer. Understanding how epigenetic information regulates the changes in gene expression that underlie these cell fate decisions is crucial to developing effective therapies. Here we test how the epigenetic marks on histones modify gene expression levels.
