Mechanisms of Altered Gene Regulation in Neurodegenerative Disease

Epigenetic features, such as DNA methylation, are regulated to control chromatin structure and gene expression. In neurons, altered DNA methylation has been linked to age-related neurodegenerative diseases including Alzheimer's disease and related dementias (ADRD), yet the relationship between DNA methylation and neurodegeneration remains unclear. Unlike dividing cells that can passively demethylate DNA during replication, neurons use active DNA demethylation, which involves excision of methylated cytosines and DNA repair. We recently developed a new method, Synthesis-Associated Repair sequencing (SAR-seq), to capture and sequence sites of recurrent DNA synthesis or repair. Using this method, we discovered ~30,000 sites of DNA synthesis across the neuronal genome at enhancers, likely playing key roles regulating active DNA demethylation. To investigate the relationship between methylation and neurodegenerative disease-associated proteins, we used human induced pluripotent stem cell-derived neurons (i3Neurons) and CRISPR interference (CRISPRi) to knock down TDP-43, a hallmark of ALS, FTD and AD. In TDP43-knockdown (KD) i3Neurons, we observed substantial reduction of SAR-seq intensity. TDP-43 is an RNA-binding protein and splicing repressor that prevents intronic sequences, called cryptic exons, from being included into mature RNA transcripts. We performed RNA-seq in TDP-43 KD i3Neurons and found 100s of destabilized transcripts with cryptic exons. We determined that two of the destabilized genes are also essential for neuron survival and belong to the TFIIH complex, which regulates transcription initiation. We found that TFIIH components localize to SAR-seq sites and TFIIH inhibition reduces SAR-seq intensity, suggesting that TFIIH acts upstream of active demethylation. Furthermore, we found that TDP-43 KD i3Neurons display reduced transcription, and that rescue of TFIIH components in these neurons restored transcriptional activity. These data are consistent with a model where TDP-43 splices TFIIH components, which are required for transcription, which then acts upstream of active DNA demethylation, and suggest a novel mechanism through which TDP-43 may regulate transcription and DNA demethylation. Contact Greg Crawford (greg.crawford at duke dot edu) and Debby Silver (debra.silver at duke dot edu) with any questions.