Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited.
The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects.
We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation.
We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear. / Doctor of Philosophy / Changes in epigenetic mechanisms, processes that control the expression of genes without changing the original sequences, play a crucial role in the formation and maintenance of memory.
Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, helping to understand post-traumatic stress disorder (PTSD). However, these majorly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited.
In the present study, we investigated some of these epigenetic mechanisms called histone modifications. In the brain, histone 3 lysine 4 trimethylation (H3K4me3), a histone modification, is critical for memory formation in the hippocampus, a key area for memory storage. However, it is still not fully understood how H3K4me3 is coordinated during memory formation. Another histone modification called H2B monoubiquitination (H2Bubi) helps recruit H3K4me3 to DNA and so is also crucial for memory formation. Here, using rat models, we found that the expression of 86 genes is changed during memory formation in the hippocampus and that this result is almost entirely dependent on the presence of H2Bubi. We also discovered that H2Bubi is critical for longterm memory formation only in neurons of the hippocampus, and not astrocytes (another type of brain cells).
Additionally, there is a connection between H3K4me3 and the phosphorylation of histone H2A.X, another epigenetic mechanism that co-occurs with DNA breaks and may serve as a markerfor studying these breaks. DNA breaks play a vital role during gene expression and could be triggered by neuronal activity during memory formation. We observed an increase in H2A.X phosphorylation and H3K4me3 levels in a memory-permissive gene five minutes after memory retrieval. Inhibition of DSBs, prior to retrieval abolished these changes, and impaired long-term memory. This suggests a critical role for DSBs in memory maintenance and that H2A.X phosphorylation is necessary for the recruitment of H3K4me3 to DNA.
Lastly, our data demonstrated that both males and females could learn fear indirectly from either sex by observing them undergoing auditory-cued fear conditioning. Additionally, we found distinct molecular patterns in the amygdala, a brain region involved in fear processing, depending on whether fear was directly or indirectly acquired, and it varied between sexes. Collectively, data from this dissertation reveals novel roles for histone modifications in memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115854 |
| Date | 25 July 2023 |
| Creators | Valajannavabpour, Shaghayegh |
| Contributors | Graduate School, Jarome, Timothy J., Rosenkranz, Jeremy Amiel, Olsen, Michelle Lynne, Mabb, Angela, Fox, Michael A. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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