Research

Our lab uses a wide variety of molecular genetic approaches to study the neurobiology of memory formation and the development of obesity, including Pavlovian fear conditioning, objection location paradigm, diet-induced obesity protocols, in vivo CRISPR-dCas9, CRISPR-dCas13 and siRNA technology, pharmacology, subcellular fractionation protocols, western blotting, proteasome activity assays, immunohistochemistry, ELISAs, qRT-PCR, chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (meDIP), direct bisulfite sequencing, mass spectrometry and FACS sorting

Sex Differences in the Role of the Ubiquitin-Proteasome System During Fear Memory Formation

The formation of fear memories, such as those that underlie post-traumatic stress disorder (PTSD), require dynamic transcriptional and translational changes in the amygdala. Our work has shown that in addition to new protein synthesis, fear memory formation also requires increased ubiquitin-proteasome system mediated protein degradation in the amygdala. However, this work has only been done in males, so it is unclear if females requires similar changes in amygdala protein degradation. Importantly, females are 3 times more likely to develop PTSD than males, suggesting that they may differ in the molecular mechanisms regulating the development of this disorder

Our current work suggests that females do not need increases in protein degradation in the amygdala to form fear memories, but do need the process in general. This may be because females have increased baseline epigenetic regulation of the ubiquitin coding gene Uba52. Current experiments are examining the significance of this sex-specific altered Uba52 epigenetic regulation in the amygdala during the development of fear memories.

The Role of Linear Ubiquitination in Memory Formation

In addition protein degradation, the ubiquitin-proteasome system can also target proteins for other biological functions/fates. For example, proteins can acquire a unique polyubiquitin tag called linear ubiquitination, in which the ubiquitin molecules line up in a head-to-tail fashion on the target substrate. This form of ubiquitination is independent of the proteasome and the canonical protein degradation process. Instead, linear ubiquitinated proteins are marked for other cellular fates such as transcriptional control or cellular localization. However, this modification has never been examined in the brain

Using in vivo siRNA technology in combination with TUBE-based mass spectrometry and next generation RNA-seq technology, our current work examines the role of nuclear linear ubiquitination in transcriptional regulation in the amygdala during fear memory formation.

The Role of Hypothalamic DNA 5-Hydroxymethylation (5-hmC) in the Development of Obesity

The hypothalamus regulates appetite control. During the development of obesity, dynamic changes in gene transcription occur in the hypothalamus, particularly at genes involved in satiation and appetite stimulation/suppression. However, little is known about the mechanisms that regulate these changes in hypothalamic transcriptional processes.

Recently, we found that there are dynamic decreases in DNA 5-hydroxymethylation (5-hmC) in the hypothalamus during the development of obesity. However, it is unknown 1) what genes are targeted by these changes in 5-hmC, and 2) whether changes in DNA 5-hmC contribute to obesity development. Using CRISPR-dCas9 technology to increase or decrease DNA 5-hmC levels in the hypothalamus, our lab is currently testing the role of hypothalamic DNA 5-hmC in the development of obesity.

Epigenetic Mechanisms of Memory Modification Following Retrieval

The retrieval of a previously stored memory results in a temporary time window in which the memory can be modified, a process called reconsolidation. Previous work from our group has shown a role for epigenetic mechanisms in the hippocampus during the reconsolidation of a contextual fear memory, suggesting that memory modification requires epigenetic-mediated transcriptional control. However, little is known about how different epigenetic mechanisms are coordinated during the reconsolidation process.

Work in the lab is interested in 1) identifying the histone and DNA modifications in the hippocampus that are critical for memory modification following retrieval, and 2) determining how these epigenetic modifications are coordinated to specific genes during the reconsolidation process. Using in vivo siRNA technology in combination with chromatin immunoprecipitation and other biochemical techniques, our current project focuses on how double-stranded DNA breaks in the hippocampus can act as the initial signal to recruit other epigenetic mechanisms (histone and DNA methylation) to specific DNA regions following memory retrieval, which is necessary to initiate active gene transcription criticalfor the reconsolidation process.