Research

Some representative research areas are listed below.

Characterization of Histone Post-translational Modification Patterns

The genetic code linking DNA, RNA and proteins was decoded by pioneering scientists in the mid-20th century, and has resulted in a revolution in the field of biology. Nevertheless, not all changes in gene expression can be attributed to changes in DNA sequence, implying that other regulatory mechanisms controlling gene expression exist. Heritable changes in gene expression occurring without alterations in gene sequence are referred to as “epigenetic”, and regulation of epigenetic signaling underlies fundamental biological processes such as cell cycle progression and apoptosis. At the center of epigenetic mechanisms are critical chromatin regulatory networks such as DNA methylation and post-translational modifications (PTMs) of histone proteins. Single histone PTMs have been separately associated with either transcriptional activation or repression depending on the location of the single modification site, but what type of effect distinct combinations of simultaneously occurring modifications (Histone Codes) have upon transcriptional events remains unclear. We are specifically addressing these deficiencies by developing novel mass spectrometry based methods for rapid comparison of histone modifications from multiple cellular states such as from cancer cells and also for quantitative tracking of hundreds of combinatorial Histone Codes in a single experiment. It is hoped that we will be able to use cutting edge proteomic technology, biochemistry and genomics to correlate histone modifications with gene expression.

Proteomic-Genomic Approach for Investigating Histone Code Reading Proteins

In attempts to explain how the large type of number of histone PTMs correlate with different nuclear events, researchers have put forward many theories on the subject. One such theory is the Histone Code hypothesis postulated by David Allis and Thomas Jenuwein in 2001. The Histone Code hypothesis essentially states that the PTMs on histones may act as a binding platform that recruits other proteins with specialized binding domains to bind the histones at specific genomic locations. In support, several proteins have been identified to recognize very specific modification sites on different histones (Histone Code reading proteins). For example, heterochromatin protein 1 binds histone H3 methylated at Lys9, and this interaction has been linked to gene silencing and heterochromatin formation. We are taking an active approach to characterize the interactions of histones and their Histone Code reading proteins, including characterization of novel histone binding proteins using both proteomics and genomics techniques.

Characterization of Histone Modifying Enzyme Activity

Gene transcription mediated through histone modification networks are balanced by the activity of both enzymes that add and remove these post-translational modifications. While a variety of methyltransferases, demethylases, acetyltransferases, and deacetylases that modify PTM patterns on histones are known, the human genome contains a large number of other potential enzymes with similar catalytic domains, the targeted substrates of which remain unclear. We are using both RNA inhibition knockdown of these enzymes and quantitative proteomics to: First, clarify the substrate requirements of the known histone modifying enzymes, and second, identify novel enzymes involved in regulation the Histone Code. The significance of work is that several histone modifying enzymes are differentially expressed in many disease phenotypes such as cancer. Additionally, we have observed that several histone modifying enzymes or Histone Code reading proteins are also modified with many distinct types of modifications, thereby expanding our studies into the enzymes responsible for these modifications and thus allowing us to probe the interplay of these complex chromatin signaling networks.

In vivo Histone Post-translational Modification Dynamics

Although histone methylation, acetylation, phosphorylation and other modifications are known to be dynamically reversible processes, most studies only present a static snapshot of histone modifications. To follow dynamic histone modification fluxes, we are creating novel approaches using quantitative proteomics in combination with modification specific in vivo labeling for temporal analysis of the Histone Code. Using these approaches, we can monitor the progression and dynamics of specific histone modifications during it's cellular lifespan or after it's induction following certain biological processes. Current work includes to measure the rates of histone modification fluxes under different physiological conditions, external stimuli or following knockdown of known and potential histone modifying enzymes.

Systems-wide Readout of the Histone Code

How do alterations in the Histone Code affect cellular function at the proteome-wide level? This is one of the questions that drives us to take a large-scale view of the consequences of resetting the Histone Code. For these experiments, we are inhibiting histone modifying enzyme activity either by small molecule or RNA inhibition to induce a change in the Histone modification patterns normally observed in cells. Changes in these Histone modification patterns most likely coincide with changes in gene expression patterns, chromatin structure and transduce a multitude of further downstream effects. Using large-scale proteomic methodology and technology, we can identify and quantitatively monitor the expression and post-translational modification state of thousands of distinct proteins in related sets of experiments. Combining this "birds eye" proteomic working view of the entire cell (including distinct signaling pathways) with gene expression (microarray) experiments will allow us to gain a holistic perspective of the systems biology level of epigenetic control through Histone modification.