Article Figures & Data
Figures
- Figure 1.
Nab2 ribosome-associated mRNA expression after repeated cocaine exposure. A, Male mice received intraperitoneal injection of cocaine (20 mg/kg) for 7 consecutive days. NAc punches were harvested 24 h after last cocaine injection. B, qRT-PCR analysis shows a trend toward an increase in Nab2 mRNA expression in the NAc after repeated cocaine exposure in male mice (saline n = 6, cocaine n = 7). C, Transcription repression associated H3K9me2 and H3K27me3 marks show bidirectional enrichment on Nab2 promoter after repeated exposure to cocaine. A transcription activation associated H3K4me3 mark showed an increased enrichment on Nab2 promoter after repeated exposure to cocaine (H3K4me3 saline n = 6, cocaine n = 6; H3K9me2 saline n = 6, cocaine n = 5; H3K27me3 saline n = 6, cocaine n = 5). D, Histone demethylase KDM1A showed an increased enrichment on both Nab2 and Egr3 promoters after repeated exposure to cocaine (Nab2 saline n = 6, cocaine n = 6; Egr3 saline n = 6, cocaine n = 5). E, D1-Cre-RT and D2-Cre-RT mice allow cell subtype specific isolation of ribosome associated mRNA. F, Repeated cocaine exposure reduced Nab2 mRNA expression in D1-MSNs while inducing Nab2 mRNA expression in D2-MSNs (D1-MSNs saline n = 11, cocaine n = 8; D2-MSNs saline n = 9, cocaine n = 8). Each bar represents mean ± SEM. *p < 0.05, **p < 0.01.
- Figure 2.
Cell subtype-specific CRISPRa transcriptional activation and CRISPRi transcriptional interference. A, Illustration of a Cre recombinase-dependent expression of a catalytically dead saCas9 fused with VP64 or KRAB in Neuro2a cell line. Transfected cells are harvested 48 h after transfection. B, C, Immunocytochemistry of Neuro2a cells transfected with CRISPRi and CRISPRa systems displays a Cre-inducible expression as observed by GFP (green) and HA-Tag (red) expression. Blue represents DAPI signal. Scale bar, 50 μm.
- Figure 3.
CRISPRi and CRISPRa manipulation of Nab2 and Egr3 transcription. A, qRT-PCR shows Nab2-targeted CRISPRi repressed Nab2 mRNA while inducing Egr3 mRNA expression, and Egr3-targeted CRISPRi repressed Egr3 mRNA while inducing Nab2 mRNA expression in Neuro2a cells (n = 9 per condition). B, Conversely, Nab2-targeted CRISPRa activates Nab2 mRNA expression, and Egr3-targeted CRISPRa activates Egr3 mRNA expression in Neuro2a cells (n = 9 per condition). C, Western blots using Neuro2a cells transfected with Nab2-targeted CRISPRi show downregulation of NAB2 and upregulation of EGR3, while cells transfected with Nab2-targeted CRISPRa show upregulation of NAB2 and downregulation of EGR3. Consistently, Neuro2a cells transfected with Egr3-targeted CRISPRi samples show downregulation of EGR3 and upregulation of NAB2, while cells transfected with Egr3-targeted CRISPRa show upregulation of EGR3 but did not change NAB2 levels (n = 3 per condition). D, Cut&Run of Neuro2a cells transfected with CRISPRi show HA-tag enrichment on the promoters of respective genes targeted by sgRNAs. Transcriptional activation marks, H3K4me3 and H3K27ac, display reduced enrichment on the promoters of respective genes targeted by sgRNAs compared with lacZ controls (n = 7-9 per condition). E, Cut&Run of Neuro2a cells transfected with CRISPRa show HA-tag, H3K4me3, and H3K27ac enrichment on the promoters of respective genes targeted by sgRNAs compared with lacZ controls (n = 9 per condition). Error bar indicates mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.
- Figure 4.
Kdms display bidirectional expression patterns in D1-MSNs and D2-MSNs after repeated cocaine exposure. A, Kdm1a, Kdm6a, and Kdm5c have GC-rich EGR3 putative binding sites in the promoter and near the transcription start site. B, Male D1-Cre-RT and D2-Cre-RT mice received seven daily injections of cocaine (20 mg/kg). Tissue punches were collected 24 h after last injection. C, qRT-PCR shows increased Kdm1a mRNA in D1-MSNs, while it decreased in D2-MSNs (D1-MSNs saline n = 11, cocaine n = 11; D2-MSNs saline n = 11, cocaine n = 11). D, Consistently, qRT-PCR shows increased Kdm6a mRNA levels in D1-MSNs, while it decreased in D2-MSNs (D1-MSNs saline n = 11, cocaine n = 11; D2-MSNs saline n = 11, cocaine n = 10). E, qRT-PCR shows decreased Kdm5c mRNA levels in D2-MSNs, while it did not change in D1-MSNs (D1-MSNs saline n = 11, cocaine n = 12; D2-MSNs saline n = 12, cocaine n = 11). F, No changes were observed in Kdm4a mRNA levels in either D1- or D2-MSNs (D1-MSNs saline n = 6, cocaine n = 6; D2-MSNs saline n = 5, cocaine n = 5). Error bar indicates mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001.
- Figure 5.
Development of a Cre- and light-inducible Opto-CRISPR-KDM1A system. A, Illustration of light-inducible dCas9 and KDM1A fusion complex using CIBN and CRY2 heterodimers. Neuro2a cells received 2 h of 1 mW blue light stimulation. B, Immunocytochemistry of Neuro2a cells transfected with Opto-CRISPR-KDM1A display a Cre-inducible expression as observed by GFP (green), HA-Tag (red), and Flag-Tag (magenta) expression. Blue represents DAPI signal. Scale bar, 50 μm. C, qRT-PCR demonstrates cells transfected only with DIO-CRY2-FLAG-KDM1A, and Cre vectors do not show significant changes in Nab2 or Egr3 mRNA levels compared with cells transfected with complete Opto-CRISPR-KDM1A targeted by lacZ sgRNA after 2 h of blue light stimulation (n = 3 per condition). D, qRT-PCR demonstrates that Neuro2a cells transfected with Opto-CRISPR-KDM1A with lacZ sgRNA in no light condition and 2 h of 1 mW blue light stimulation do not display significant differences in Nab2 and Egr3 mRNA levels (n = 3 per condition). Error bar indicates mean ± SEM.
- Figure 6.
Opto-CRISPR-KDM1A mediated inhibition of Nab2 and Egr3 transcription. A, qRT-PCR demonstrates that 2 h of blue light stimulation downregulated Nab2 mRNA while inducing Egr3 mRNA expression in cells transfected with Nab2-targeted Opto-CRISPR-KDM1A. Conversely, 2 h of blue light stimulation induced Nab2 mRNA while downregulating Egr3 mRNA expression in cells transfected with Opto-CRISPR-KDM1A and Egr3 sgRNA (n = 9 per condition). B, Western blots represent 2 h of blue light stimulation downregulated NAB2 levels, while upregulating EGR3 levels in cells transfected Opto-CRISPR-KDM1A and Nab2 sgRNA. EGR3 levels were downregulated, while NAB2 levels were unchanged in cells transfected with Opto-CRISPR-KDM1A and Egr3 sgRNA (n = 3 per condition). C, Cut&Run on blue light stimulated Neuro2a cells transfected with Opto-CRISPR-KDM1A shows enrichment of Opto-CRISPR-KDM1A complex on the promoters of respective genes targeted by sgRNAs compared with lacZ sgRNA controls. Transcriptional activation marks, H3K4me3 and H3K27ac, have reduced enrichment on the promoters of respective genes targeted by sgRNAs compared with lacZ sgRNA controls (n = 9 per condition). Error bar indicates mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.
- Figure 7.
Development of a Cre- and light-inducible Opto-CRISPR-p300 system. A, Illustration of light-inducible dCas9 and p300 fusion complex using CIBN and CRY2 heterodimers. Neuro2a cells received 2 h of 1 mW blue light stimulation. B, Immunocytochemistry of Neuro2a cells transfected with Opto-CRISPR-p300 system demonstrates a Cre-inducible expression as observed by GFP (green), HA-Tag (red), and Flag-Tag (magenta) expression. Blue represents DAPI signal. Scale bar, 50 μm. C, qRT-PCR demonstrates that the expression of DIO-CRY2-FLAG-p300core alone does not induce changes in Nab2 or Egr3 mRNA levels compared with cells transfected with Opto-CRISPR-p300 targeted by lacZ sgRNA after 2 h of blue light stimulation (n = 3 per condition). D, qRT-PCR demonstrates Neuro2a cells transfected with Opto-CRISPR-p300 targeted by lacZ sgRNA in no light condition and 2 h of 1 mW blue light stimulation do not display significant differences in Nab2 and Egr3 mRNA levels (n = 3 per condition). Error bar indicates mean ± SEM.
- Figure 8.
Opto-CRISPR-p300-mediated activation of Nab2 and Egr3 transcription. A, qRT-PCR demonstrates that 2 h of blue light stimulation induced Nab2 mRNA and Egr3 mRNA expression in cells transfected with Nab2 or Egr3 sgRNAs, respectively, and Opto-CRISPR-p300 (n = 9 per condition). B, Western blots demonstrate that 2 h of blue light stimulation induced NAB2 levels while downregulating EGR3 levels in cells transfected with Opto-CRISPR-p300 and Nab2 sgRNA. EGR3 levels were upregulated, while NAB2 levels were unchanged in cells transfected with Opto-CRISPR-p300 and Egr3 sgRNA (n = 3 per condition). C, Cut&Run of blue light-stimulated Neuro2a cells transfected with Opto-CRISPR-p300 display enrichment of Opto-CRISPR-p300 complex on the promoters of respective genes targeted by sgRNAs compared with lacZ sgRNA controls. Transcriptional activation marks, H3K4me3 and H3K27ac, have increased enrichment on the promoters of respective genes targeted by sgRNAs compared with lacZ sgRNA controls (n = 9 per condition). Error bar indicates mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.
- Figure 9.
Proposed model of Nab2 and Egr3 regulation after repeated exposure to cocaine. Nab2 expression is decreased and Egr3 expression is increased in NAc D1-MSNs after repeated exposure to cocaine. Oppositely, Nab2 expression is increased and Egr3 expression is decreased in NAc D2-MSNs after repeated exposure to cocaine.
