Monday, March 28, 2022

Genetic Link to Fear Memories Found Hiding Within Mice's "Junk DNA"

ADRAM is an experience-dependent long noncoding RNA that drives fear extinction through a direct interaction with the chaperone protein 14-3-3. Wei Wei et al. Cell Reports, Vol 38, Iss 12, Mar 22 2022.


• Targeted RNA sequencing reveals learning-induced lncRNAs in the adult brain

• ADRAM is critical for the formation of fear extinction memory

• ADRAM coordinates the epigenomic regulation of Nr4a

Summary: Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.


Here, we report the discovery of widespread experience-dependent lncRNA activity in the adult ILPFC, and further reveal a significant number of inducible eRNAs that respond selectively to fear extinction learning. This class of lncRNA was first discovered at scale more than a decade ago by the Greenberg group who identified thousands of sites outside known promoter regions in primary cortical neurons stimulated with KCl in vitro, which exhibited features of enhancer elements, including binding of CBP and the deposition of the histone modification H3K4me1 (). Transcriptional activity at these sites showed a positive correlation with downstream mRNA expression, suggesting a context-specific permissive relationship between eRNAs and their proximal mRNA partners.  went on to functionally characterize neuronal enhancers and identify another histone modification, H3K27ac, as a key marker of their active state. An overlay of our lncRNA capture-seq data with learning-induced enhancer signatures in the adult brain (), as well as our cell-type-specific ATAC-seq signatures in learning-activated Arc+ neurons, revealed that there are many experience-dependent lncRNAs in the ILPFC that are endowed with features of activity-inducible eRNAs. Notably, all six of the validated eRNA-associated protein-coding gene candidates have been shown to be involved in plasticity, suggesting that this class of lncRNA is, in general, permissively involved in the regulation of experience-dependent gene expression.
One of the most interesting findings of our study beyond the necessary role of ADRAM in fear extinction is that it binds directly to the Nr4a2 promoter; however, in doing so it does not form an R-loop or promote chromosome looping. trans-Acting lncRNAs are known to form triplex structures on double-stranded DNA using a Hoogsteen base-pairing rule in the DNA target (). These structures are distinct from R-loops and could represent a mechanism by which lncRNAs act in a combinatorial manner to simultaneously serve as both guides and scaffolds. Indeed, examination of the 1 kb upstream promoter sequence of NR4A2 revealed two sites proximal to the TSS, with 25 nucleotide long complementary sequences found within exon III of ADRAM. Notably, these sites overlap with G-quadruplex motifs that are predicted to enable triplex formation. These findings suggest that ADRAM functions as a guide via a direct interaction with the Nr4a2 promoter and may do so via the formation of an RNA:DNA triplex at sites of structural reactivity. Future studies will investigate whether dynamic DNA structure states are the key to how lncRNAs find their genomic targets to regulate gene expression in an experience-dependent manner.
The 14-3-3 family of evolutionarily conserved chaperone proteins is ubiquitously expressed in the brain and highly enriched at the synapse () being involved in a variety of neuronal processes, including synaptic plasticity (). Our discovery of a direct interaction between ADRAM and 14-3-3 extends the capabilities of this class of chaperones to include functional activity as both an RNA-binding protein and a molecule that exerts its influence through protein-protein interactions. This is not without precedent as many proteins are able to interact with RNA, DNA, and other proteins. For example, YY1 interacts with both RNA and DNA, as well as other proteins, to promote its role as a regulator at the Xist locus (). Together with the observation that 14-3-3 is involved in learning and memory (), and our demonstration of how 14-3-3 interacts with eRNA to facilitate gene expression in fear extinction, these findings advance our understanding of the functional importance of this class of chaperones in the brain.
Histone modifications at neuronal enhancers also appear to be a requirement for the induction of activity-dependent genes and are particularly important in the case of rapidly induced immediate-early genes (). We found a broad overlap with H3K27ac, an open chromatin ATAC signature in activated neurons, and the expression of lncRNAs. Previous work has shown that eRNA activity often precedes, and then drives, the expression of immediate-early genes, such as c-Fos, which occurs via a direct interaction with the histone acetyltransferase domain of CBP (). In addition, a large number of eRNAs have been shown to bind to CBP, correlating with the expression of downstream genes that require CBP for their induction (). Our data on the functional relationship between ADRAM, HDAC3, HDAC4, CBP, and Nr4a2 agree with these observations and, importantly, extend the findings to include the ILPFC where they are critically involved in fear extinction. Our conclusion is that ADRAM functions as both a guide and a scaffold to epigenomically regulate extinction learning-induced Nr4a2 expression. There are now many examples of multifunctional lncRNAs. For example, in dopaminergic neurons, antisense Uchl1 regulates the expression of Uchl1 in the nucleus and then shuttles to the cytoplasm where it promotes Uchl1 translation (). Furthermore, owing to its modular domain structure, Neat1 functions in cis to coordinate the deposition of learning-related repressive chromatin modifiers along the genome () and in trans to govern paraspeckle assembly by influencing phase separation ().
In summary, the discovery of an lncRNA that is required for fear extinction deepens our understanding of learning-induced epigenomic mechanisms by integrating the modular function of enhancer-derived lncRNAs with key epigenomic processes involved in memory, and answers the long-standing question of how certain HDACs and CBP coordinate to confer their influence on localized gene regulation with a high degree of state-dependent selectivity. LncRNAs therefore provide a bridge to link dynamic environmental signals with epigenomic mechanisms of gene regulation. Together, these findings broaden the scope of experience-dependent lncRNA activity, and underscore the importance of considering eRNAs in the adult cortex as potential therapeutic targets for fear-related neuropsychiatric disorders.

 Limitations of the study

In this work, we identify an enhancer-derived lncRNA that is necessary for the formation of fear extinction memory in male mice. However, an examination of ADRAM and Nr4a2 mRNA expression in the ILPFC after extinction learning in randomly cycling females revealed no increase in ADRAM expression in the female ILPFC. In contrast, Nr4a2 was induced by training in both males and females (Figure S9). These results indicate that, although Nr4a2 may be generally induced by experience in both sexes, it may not be regulated by ADRAM under conditions where successful extinction does not occur. With respect to sex differences in fear extinction, we have also previously found that male and female mice respond differently to the standard extinction protocol used in our laboratory (

) and that the electrophysiological signature in the prelimbic PFC during fear extinction is very different between males and females (


). Therefore, it is highly plausible that there are female-specific molecular mechanisms that are involved in fear-related learning. Future studies on the role of lncRNAs in fear extinction in females will require the use of a sex-specific learning protocol to reveal brain region-specific molecular mechanisms underlying memory in these mice. In addition, although we have demonstrated that 14-3-3 is a key regulatory protein that interacts with ADRAM, there were other candidates identified by mass spectrometry that have not been validated. At this stage, these data should therefore be considered preliminary until further experiments are carried out. Finally, it is not yet known whether ADRAM is necessary for other forms of learning.