Abstract
The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure.
Highlights
Cholinergic neurons are critical for communicating information to a wide range of target cells using the neurotransmitter acetylcholine (ACh)[1,2]
We explored the novel finding that 82-kDa choline acetyltransferase (ChAT) can facilitate and participate in an epigenetic response in Aβ -exposed human neural cells
We found that 1) nuclear localized 82-kDa ChAT forms aggregates in Aβ 1–42 -treated cells; 2) 82-kDa ChAT is associated with chromatin after exposure of cells to Aβ 1–42; and 3) 82-kDa ChAT increases its association with gene introns and promoters after Aβ -exposure of cells. 82-kDa ChAT protein aggregates in neural cell nuclei are co-localized with special AT-rich binding protein 1 (SATB1), a protein involved in anchoring DNA to the nuclear matrix for either chromatin activation or repression
Summary
82-kDa ChAT sub-nuclear localization is altered after acute exposure of cells to Aβ. To evaluate the subcellular localization of 82-kDa ChAT and changes that may occur after acute exposure of cells to oligomeric Aβ 1–42, we imaged retinoic acid-differentiated SH-SY5Y cells stably expressing heterologous 82-kDa ChAT at 4 h after exposure to either vehicle or Aβ 1–42 by confocal microscopy. After resveratrol treatment we observed no significant differences for SATB1 siRNA transfected cells (50 ± 8%) compared to control siRNA (57 ± 4%) or non-transfected (55% ± 3%) cells These data indicate that SATB1 is required for the Aβ 1–42 -induced aggregates. We annotated peaks contained in intergenic components for both proteins, and found that, in agreement with the genomic features, there was a higher number of associated genes after exposure of cells to Aβ 1–42 for both 82-kDa ChAT (4507) and SATB1 (4672) compared to vehicle treatment (3700 and 2615, respectively) (Fig. 6A). After exposure for 4 h with 100 nM Aβ 1–42, we observed no significant changes in total APP steady-state mRNA levels for SH-SY5Y cells stably expressing 82-kDa ChAT after a mock transfection, control siRNA or siRNA targeted to SATB1 compared to cells that were not transfected. We measured the size of the individual clusters and found no significant difference in any of the conditions (box on Fig. 8B[h,i])
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