Project Summary/Abstract
Post-traumatic stress disorder (PTSD) affects nearly 8% of the population and is more prevalent in women
than men. The neurobiological factors that contribute to this sex bias are largely unknown. This project
addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how transcriptional
regulation of memory-related genes differs in males and females in support of fear memory. Recently our
group demonstrated that male and female rats differentially engage signaling mechanisms within the prefrontal
cortex (PFC) during the formation of a fear memory, and our preliminary findings point to sex differences in
epigenetic modification of prefrontal genes. Currently, the transcriptional regulation of episodic memory within
cognitive systems is poorly understood, and the impact of sex on this dynamic process is all but unknown. This
represents a significant challenge to developing effective treatment for disorders such as PTSD. The objective
of this proposal is to determine how DNA hydroxymethylase TET enzymes functionally regulate fear memory
formation and to identify sex-specific methylomic signatures of aversive experience. DNA 5-
hydroxymethylation (5-hmC), a major regulator of active (increased) transcription in cells, has been recently
implicated in fear memory formation in the brain. However, whether sex differences exist in the engagement of
DNA 5-hmC mechanisms during fear memory formation remain equivocal. In our preliminary studies we found
that in the rat prefrontal cortex the DNA hydroxymethylase Tet2 increased in females while Tet1 decreased in
males following learning in a trace fear conditioning paradigm. The central hypothesis is that TET-mediated
DNA 5-hydroxymethylation of sex-specific gene targets in the prefrontal cortex facilitates the formation of fear
memory. The approach uses unbiased genome-wide analysis (NIMH Strategy 1.2, Research Priority B) in
combination with viral-mediated transcriptional control to test the functional link between TET activity and
memory (NIMH Strategy 1.1, Research Priority D; NIMH Strategy 1.2, Research Priority B). Aim 1 will identify
the sex-specific gene targets of TET1 and TET2-mediated DNA 5-hmC in the prefrontal cortex following fear
learning, using next generation whole genome chromatin immunoprecipitation (ChIP) and hydroxymethylated
DNA immunoprecipitation (hmeDIP) sequencing methods. Aim 2 will use cutting-edge CRISPR-dCas9
technology to manipulate the expression of Tet1 and Tet2 in the prefrontal cortex of males and females during
trace fear conditioning, which will determine the sex-dependent functional role of cortical TET1 and TET2 in
fear memory formation. The proposed research is significant because it is expected to provide a (epi)genomic
map of memory formation in a sexually dimorphic brain region, the medial prefrontal cortex, that is needed for
understanding and even identifying new genetic biomarkers of sex-biased emotional and cognitive deficits in
psychiatric illness.