Nanopore Sequencing to Identify Transposable Element Insertions and Their Epigenetic Modifications

Smits, Nathan; Faulkner, Geoffrey J.

doi:10.1007/978-1-0716-2883-6_9

Cited by 1 publication

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“…Test whether activity-induced eccDNAs are incorporated into distant sites in the chromosomes of a neuron, especially at DSBs formed during subsequent activation events. This is the most difficult step to assess, but may be approached using the biotin-dUTP technique to label activity-induced eccDNAs during a short interval following activation, i.e., stimulating cultured neurons with NMDA while incorporating biotin-dU into eccDNAs selectively within the subsequent 2 h. After a further interval of 24 h, to allow recovery of the cells and washout of the biotin-dUTP, the cultures can be stimulated a second time to induce new DSB sites to form, and 2 h later, the neurons’ chromosomal DNA can be isolated, fragmented by sonication or using rare-cutting restriction enzymes to produce fragments of a more controlled average size suitable for sequencing, and the fragments exposed to biotin pulldown to selectively isolate chromosomal DNA fragments that contain biotin-dU, followed by sequencing ( Smits and Faulkner, 2023 ).…”

Section: How To Test the Hypothesismentioning

confidence: 99%

Mobile circular DNAs regulating memory and communication in CNS neurons

Smalheiser

2023

Front. Mol. Neurosci.

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Stimuli that stimulate neurons elicit transcription of immediate-early genes, a process which requires local sites of chromosomal DNA to form double-strand breaks (DSBs) generated by topoisomerase IIb within a few minutes, followed by repair within a few hours. Wakefulness, exploring a novel environment, and contextual fear conditioning also elicit turn-on of synaptic genes requiring DSBs and repair. It has been reported (in non-neuronal cells) that extrachromosomal circular DNA can form at DSBs as the sites are repaired. I propose that activated neurons may generate extrachromosomal circular DNAs during repair at DSB sites, thus creating long-lasting “markers” of that activity pattern which contain sequences from their sites of origin and which regulate long-term gene expression. Although the population of extrachromosomal DNAs is diverse and overall associated with pathology, a subclass of small circular DNAs (“microDNAs,” ∼100–400 bases long), largely derives from unique genomic sequences and has attractive features to act as stable, mobile circular DNAs to regulate gene expression in a sequence-specific manner. Circular DNAs can be templates for the transcription of RNAs, particularly small inhibitory siRNAs, circular RNAs and other non-coding RNAs that interact with microRNAs. These may regulate translation and transcription of other genes involved in synaptic plasticity, learning and memory. Another possible fate for mobile DNAs is to be inserted stably into chromosomes after new DSB sites are generated in response to subsequent activation events. Thus, the insertions of mobile DNAs into activity-induced genes may tend to inactivate them and aid in homeostatic regulation to avoid over-excitation, as well as providing a “counter” for a neuron’s activation history. Moreover, activated neurons release secretory exosomes that can be transferred to recipient cells to regulate their gene expression. Mobile DNAs may be packaged into exosomes, released in an activity-dependent manner, and transferred to recipient cells, where they may be templates for regulatory RNAs and possibly incorporated into chromosomes. Finally, aging and neurodegenerative diseases (including Alzheimer’s disease) are also associated with an increase in DSBs in neurons. It will become important in the future to assess how pathology-associated DSBs may relate to activity-induced mobile DNAs, and whether the latter may potentially contribute to pathogenesis.

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Section: How To Test the Hypothesismentioning

confidence: 99%

Mobile circular DNAs regulating memory and communication in CNS neurons

Smalheiser

2023

Front. Mol. Neurosci.

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show abstract

Nanopore Sequencing to Identify Transposable Element Insertions and Their Epigenetic Modifications

Cited by 1 publication

References 38 publications

Mobile circular DNAs regulating memory and communication in CNS neurons

Mobile circular DNAs regulating memory and communication in CNS neurons

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