Major areas of interest for research projects in the lab include the following:
Transcriptional regulation of Brain-Derived Neurotrophic Factor (BDNF)
BDNF plays numerous important roles in brain development and plasticity by affecting the development and function of synapses. The expression of BDNF is exquisitely sensitive to neuronal activity, and transcriptional regulation of Bdnf synthesis is the major mode of activity-dependent BDNF induction. Thus the study of Bdnf transcription can offer important insights into synapse to nucleus to synapse signaling. We have used behavioral and biochemical analyses of knockout mice to study the role of the unique transcription factor Calcium-Response Factor (CaRF) in this process, revealing that the regulation of Bdnf by CaRF is selective for specific brain regions and stimulus conditions. In related studies in the lab, which are still ongoing, we have identified additional transcriptional targets of CaRF that suggest a role for this transcription factor in the regulation of neuronal signaling cascades that underlie homeostatic synaptic plasticity. We are also continuing to identify new transcriptional regulators of the Bdnf gene, with the goal of understanding mechanisms of stimulus-selective regulation of immediate-early genes in neurons.
Methyl-DNA complex protein 2 (MeCP2) in monoamine-regulated neuronal plasticity
Although psychostimulants and antidepressants rapidly bind their pharmacological targets in the brain, chronic exposure to these substances is required for their effects on addictive- or depressive-like behaviors. This observation indicates that slower processes downstream of monoamine receptor activation, which include the induction of new gene transcription, are essential for the action of these drugs on the neural circuits that underlie these behaviors. A growing body of evidence suggests that epigenetic mechanisms of chromatin regulation, such as histone modifications and DNA methylation, contribute to the long-lasting changes in gene expression induced by repeated exposure to these psychoactive compounds. In collaboration with Dr. William C. Wetsel, Director of the Duke University Mouse Behavioral and Neuroendocrine Analysis Core Facility, we have been investigating the role of the methyl-DNA binding protein MeCP2 in this process. We showed that genetic manipulation of MeCP2 expression in the nucleus accumbens of adult mice modulates behavioral adaptations to amphetamine, implicating MeCP2 for the first time as a modulator of psychostimulant-induced reward. We further discovered that activation of dopamine and serotonin receptors in the brain drives the phosphorylation of MeCP2 at a specific site (Ser421) in selected neural circuits relevant for reward- and stress-related behaviors, thus suggesting a mechanism to link regulation of MeCP2 function with the pharmacological action of psychostimulants and antidepressants. Currently we are studying the biological importance of monoamine-regulated MeCP2 Ser421 phosphorylation in mice bearing a germline mutation that changes Ser421 to Ala, rendering MeCP2 non-phosphorylatable at this site. This mouse model selectively disrupts only the stimulus-regulated functions of MeCP2, providing us with a unique opportunity to address the importance of DNA-methylation dependent chromatin plasticity in the behavioral plasticity induced by psychostimulants and antidepressants.
Epigenomics of neuronal development
Cellular differentiation requires the precise spatial and temporal orchestration of gene expression programs. Progressive changes in gene transcription during development are driven by epigenetic modifications of genomic DNA and its associated histone proteins, collectively called chromatin, that differentially alter the access of DNA regulatory sequences to the transcriptional machinery. A growing body of evidence shows that chromatin-regulatory proteins can be modulated in neurons by environmental stimuli, raising the intriguing possibility that early life experience may impact brain development by inducing plasticity of the neuronal epigenome. However it is largely unknown when during development neuronal chromatin is subject to epigenetic regulation, where in the genome the key gene regulatory elements are located that mediate neuronal differentiation, and to what extent chromatin structure and state can be modulated by extrinsic stimuli in postmitotic neurons. We are using the differentiation of mouse cerebellar granule neurons as a model system to address this question because the vast numbers of these neurons in the brain and their very well-characterized stages of differentiation make them ideal for comparing biochemical analyses of chromatin regulation with biological differentiation of neurons during brain development. We have identified histone-modifying enzymes that are required to turn on neuronal differentiation genes during granule neuron maturation, giving new insights into the molecular mechanisms that control this process. Now in collaboration with genome biologist Dr. Gregory E. Crawford at the Duke Institute for Genome Science and Policy, we are undertaking an effort using high-throughput sequencing to map the genome-wide distribution of open chromatin in differentiating cerebellar granule neurons with the goal of understanding how changes in chromatin structure guide neuronal differentiation. These studies have the potential to reshape the conceptual landscape for the development of models to explain environmental and transcriptional contributions to brain development.
New technologies for studying activity-regulated neuronal transcription
A subset of brain-specific genes shows very rapid induction of transcription in response to neural activity, and many of these gene products act to directly modulate synaptic function. Understanding the kinetic parameters of induced transcription is of particular importance in neurons, where tightly regulated patterns of synaptic activity are the key source of information that drives plasticity. Historically, biochemical methods have been applied to characterize the molecular mechanisms that transduce synaptic activity into new gene transcription. However this methodology yields only static snapshots of transcriptional activity averaged across a cell population. Thus these studies provide little insight into the temporal response properties of activity-regulated genes. In addition, biochemical studies of gene transcription lose all information about cell-to-cell variability in this response, which is crucial for understanding the dynamics of plasticity in real circuits. To fill this gap in knowledge, together with Dr. Sridar Raghavachari at Duke Neurobiology we are working to develop new molecular and computational methods that will permit an analysis of activity-regulated transcription that is both induced by physiologically relevant patterns of activity and measured in single neurons in real time. Our innovative technological platform combines multiple methodologies to achieve this goal, including site-specific modification of endogenous immediate-early genes through ES cell recombineering, live fluorescent imaging of gene transcription in single neurons, light-gated activation of neuronal firing patters, and noise analysis of gene transcription in individual neurons.
