In my lab we take the unconventional position that we are analog creatures in a world increasingly viewed as digital. We find evidence for this in proteins, which perform the bulk of the chemistry needed for life to persist. Proteins typically lie at the nexus of multiple signaling pathways that alter both their structure and function. Previously these signals have been modeled to act as simple switches turning proteins on or off. However, there is increasing evidence that multiple signals converge simultaneously on a given proteins. This creates a conundrum: how does a single protein molecule simultaneously integrate the input from multiple signals and adopt a specific function? Are proteins just a series of dipswitches or integrated energy continua? Our work has shown that the structure and dynamics of proteins make them very poor switches, instead predisposing them to fine-tuning. We are testing the hypothesis that fine-tuning is an essential property of proteins that is required for life and must be understood to interpret signaling networks and derive specific therapies.

In my lab we study the glucocorticoid receptor (GR), a steroid activated transcription factor that is the target of drugs used to treat a variety of conditions from asthma to cancer. GR is an ideal protein to understand the biology and mechanism of fine-tuning, which we approach with three general projects:

Acute Lymphoblastic Leukemia (ALL)

ALL arises when white blood cells do not undergo apoptosis despite an arrest in development. Apoptosis is itself a fine-tuned response to positive and negative signals encountered during the development in these immune cells. Glucocorticoids help to induce apoptosis in both T and B lineage lymphoblasts that have not developed properly. Having evaded apoptosis, these cells are treated with high-dose glucocorticoids to further push the balance toward cell-death in patients with this disease.

Somewhat surprisingly, glucocorticoids, such as dexamethasone, are very effective particularly in treatment of childhood B cell precursor ALLs (BCP-ALL). However they do not work in all cases, and when they do not, the prognosis for these patients is poor. In my lab we are studying how glucocorticoids work when they are effective and why they don’t when they fail. We use genomic methods such as RNAseq and ChIPseq to understand how GR is functioning in \ two patient populations, those who are sensitive and resistant, and whether treatments can be developed to resensitize resistant patients.

In addition to this project, we are also trying to answer the fundamental question of why some steroids, in this case glucocorticoids, are more effective than others. We are trying to make sense of the finding that dexamethasone is a more potent anti-leukemic drug than prednisone, even when the latter is administered at substantially higher doses. Our hypothesis is that rather than simply being more “potent”, dexamethasone and prednisone induced expression of different panels of genes, and that some of these result in more efficient leukemic blast apoptosis or sensitization to subsequent treatment.

Primary, causative, GR gene regulatory mechanisms

A primary goal of both of these studies is to find GR regulated genes that are required for induction of apoptosis and are misregulated in patients for whom glucocorticoids are not effective. We have recently begun to study these genes in mechanistic detail. In addition to using classical techniques, we are also employing genome–wide RNAi screening to identify factors that are required for properly regulation of these critical gene.

Structural and biophysical bases of fine-tuning

The glucocorticoid receptor (GR) is inactive until it encounters cortisol, it’s physiological ligand. Once bound, GR translocates to the nucleus where it associates with DNA where it both activates and represses gene expression. The degree to which it activates and represses is modulated by signals, such as ligand chemistry, phosphorylation, acetylation, partner protein binding, combinatorial control at genomic response elements, and importantly, the sequence to which it is bound. It is our goal to understand how signals modulate the structure and dynamics of GR to specifically direct its activity.

Our initial focus is on DNA sequence. DNA is a sequence-specific allosteric regulator that not only changes the structure of the DNA binding domain, but send signals to deploy distant domains. We are trying to define the atomic wires that transmit this signal, and to see how the wires emanating from other signals are integrated to fine-tune protein conformation. We employ primarily structural methods, including X-ray crystallography and nuclear magnetic resonance, as well as biochemical, biophysical, and computational methods.