Results of DNA sequencing for ChIP of Lhx8
To complete ChIP efficiently, the overexpressed-Lhx8 PC12 line was constructed successfully by infection with LV-Lhx8 when MOI was 50. The infection efficiency reached 100%, and Western blot showed that the protein expression of Lhx8 in LV-Lhx8 PC12 cells was higher than that in the negative control group (P < 0.01; Fig. 1A–D). The enriched Lhx8 target DNA fragments were collected by ChIP. Gel electrophoresis revealed that the length of ultrasonic fragmentation of DNA was about 150–250 bp (Fig. 1E), and ChIP sequencing results clarified 1.00% out of the whole gene profile, which Lhx8 targets upstream of the promoter within 1 k bp (Fig. 1F,G). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the significant enrichment pathways of target genes regulated by Lhx8 involve neuron development and differentiation, neuron projection, nervous system development, axon conduction, glutamatergic synapses, and other pathways related to nervous system development (Fig. 1H,I).
Identification of the Lhx8 downstream target gene
From genes that Lhx8 targets upstream of the promoter within 1 k bp, we used KEGG and Gene Ontology (GO) analysis to screen out 9 genes related to NSC development: Stk36, Ppp3cc, Acr, Ppp1r13b, Kifc3, Capn3, Hspa1b, Moy1d, and Kcnip3. RT PCR results showed that the RNA expression of Stk36, Ppp3cc, Accr, Ppp1r13b, Kifc3, and Capn3 in the LV-Lhx8 PC12 line had greatly changed compared to that in the LV-NC PC12 cell line (Fig. 2A–I). Based on ChIP gene data that we previously collected, the expression of Ppp3cc, Ppp1r13b, Capn3, and Acr changed in the deafferented hippocampus niche (Fig. 2J). RT PCR showed the mRNA expression levels of Capn3 and Acrl were significantly down-regulated in the FFT niche (Fig. 2K–N). Capn3 is a candidate gene for the Lhx8 target in the following study.
Capn3 interacts with Lhx8 in the LV-Lhx8 PC12 cell line
The relationship of Capn3 and Lhx8 was explored using the gain- and loss-function assay of Capn3 in the LV-Lhx8 PC12 cell line. The LV-Lhx8 PC12 transfected with Capn3 plasmid showed nearly 100% green fluorescent protein (GFP) positivity at a transfection concentration of 0.75 µl (Fig. 3A). The mRNA and protein levels of Capn3 in the LV-Lhx8 PC12 transfected with Capn3 plasmid were higher than those in the NC group (Fig. 3B,C). The protein level of the cholinergic neuron marker ChAT was also lower in LV-Lhx8 PC12 transfected with Capn3 plasmid (Fig. 3D,E). In the loss-function assay, siRNA-Capn3 was applied to down-regulate the expression level of Capn3 in the LV-Lhx8 PC12. The transfection efficiency reached nearly 100% at the transfection concentration of 20 nM si-Capn3. RT PCR and Western blot results showed si-Capn33# could greatly down-regulate the expression level of Capn3 in LV-Lhx8 PC12 (Fig. 3F–I). Contrary to the gain-function, the expression level of ChAT in LV-Lhx8 PC12 transfected with si-Capn33# was higher than that in the NC group (Fig. 3J).
The results of the luciferase reporter gene assay revealed that Lhx8 could interact with the promoter region of Capn3, which may trigger the negative regulation of the cholinergic neuron marker ChAT (Fig. 3K).
Capn3 negative regulates the differentiation of hippocampus NSCs into cholinergic neurons
Our previous studies indicated transcription factor Lhx8 is closely related to the development of cholinergic neurons, which promotes continuous expression of ChAT in the differentiation of hippocampus NSCs. To explore the potential role of Capn3 in the differentiation of hippocampus NSCs into cholinergic neurons, the hippocampus NSCs were infected with lentivirus LV-Capn3 at an MOI of 5, 10, and 20. Fluorescence analysis showed the infection efficiency reached 83.45 ± 5.28% at an MOI of 10, while the infection efficiency reached 84.22 ± 4.68% at an MOI of 5 (P > 0.05; Fig. 4A,B). In differentiation assays, NSCs differentiated in vitro for 7 days were subjected to MAP2/ChAT immunofluorescence double labeling to detect their differentiation into cholinergic neurons. Compared with the control group, NSCs that overexpressed Capn3 had less differentiation into MAP2/ChAT-positive cholinergic neurons (Fig. 4C,D; P < 0.001), and Western blot showed the expression of ChAT protein was also significantly lower than in the control group (Fig. 4F,G; P < 0.001). In the loss-function assay, 3 siRNA-Capn3 were designed and synthesized to transfect NSCs. The transfection efficiency reached nearly 100% at the transfection concentration of 20 nM si-Capn3 (Fig. 4H,I). RT PCR and Western blot results showed si-Capn33# could greatly down-regulate the expression level of Capn3 in NSCs (Fig. 4J,K). In a differentiation assay, modified NSCs differentiated in vitro for 7 days were subjected to MAP2/ChAT immunofluorescence double labeling to detect their differentiation into cholinergic neurons. Compared with the control group, NSC transfected with si-Capn33# showed greater differentiation into MAP2/ChAT-positive cholinergic neurons, and Western blot showed the expression of ChAT protein was also significantly higher than in the control group (Fig. 4L,M). Altogether, this implies that Capn3, acting as a downstream target of Lhx8, could negatively regulate the differentiation of hippocampus NSCs into cholinergic neurons.