Figure 1.
Acetylated SINEs are a distinctive feature of NEE-induced genes.
(A and C) Average H3K9K14ac ChIPseq tag density of constitutively silent (CS, gray dashed line), expressed (CE, black dashed line), NEE-induced (NI, black solid line) and repressed (NR, gray solid line) genes in somatosensory cortex of control mice (A) and mice exposed to NEE for 45 minutes (C). For each gene group, the average density of ChIPseq reads per 100 bp window is plotted relative to the TSS. (B and D) Box and whiskers plots summarizing the distribution of ChIPseq tag density at +0/+0.5 kbp from TSS, highlighted in yellow in (A) and (C), for each of the four gene groups, in control (B) and NEE-stimulated (D) mouse cortex. Lower and upper whiskers indicate the minimum and maximum value of the distribution, respectively. The lower and upper limits of the box indicate the 25th and 75th percentile, respectively. The solid line denotes the median. The red lines below each box plot indicate whether the difference between the medians of two data sets is statistically significant (P<0.0001, two-tailed Mann-Whitney test). The tag density values for control (A, B) and NEE-stimulated (C, D) cortex are not directly comparable, as they have not been normalized across samples. (E) Ac1 and Ac2 motifs were found by applying motif inference on +Δac regions. (F) Average density of de novo acetylated SINEs distribution for constitutively silent (CS, gray dashed line), constitutively expressed (CE, black dashed line), NEE-induced (NI, black solid line) or NEE-repressed (NE, gray solid line) genes. For each gene group, the average density of de novo acetylated SINEs per 50 kbp window (at 5 kbp intervals) is plotted. Horizontal red bars represent regions where the SINE density of NI genes is significantly higher than that of CE genes (P<0.05, two-tailed Mann-Whitney test). Red arrowheads under the graph indicate the de novo acetylated SINEs detected within 100 kbp of the c-Fos and Gadd45b TSSs.
Table 1.
Acetylated SINEs proximal to TSSs of NEE activated genes.
Figure 2.
TFIIIC binds to de novo acetylated SINEs near inducible genes.
(A and B) Changes in H3K9K14ac at c-Fos (A) and Gadd45b (B) loci. From the top, gene locus structure (blue), SINEs (shades of gray) with c-FosRSINE1 (A) and Gadd45bB1F (B) highlighted, H3K9K14ac tag density per 100 bp in control conditions (CTR) and in response to NEE (NEE), and −log(p-value) of regions of statistical difference in H3K9K14ac levels are shown. Tag density profiles are not directly comparable, as they have not been normalized across samples. (C) Exposure to NEE increased histone H3 acetylation at c-FosRSINE1 and Gadd45bB1F and not at TSSs. Adult mice were either exposed to NEE for 45 min or left untreated, somatosensory cortex was dissected and subjected to ChIP using either H3K9K14ac or histone H3 antibodies, followed by qPCR. Histograms show the ratio of immunoprecipitation efficiency between H3K9K14ac and H3 antibodies relative to total chromatin input (average and s.e.m. of at least 6 mice per condition are shown; *, P<0.05, Student's t-test). (D and E) Mouse somatosensory cortex (D) and primary cortical neurons (E) were subjected to Gtf3c1 ChIP followed by qPCR targeting c-FosRSINE1 and Gadd45bB1F. Histograms show the efficiency of immunoprecipitation relative to total chromatin input, expressed as percentage of total input. 5S rRNA gene locus was used as positive control. Jdp2B1, GapdhB4 and a genomic region devoid of SINEs and H3K9K14ac ChIPseq tags (ctrl) were used as negative control (average and s.e.m. of at least 3 experiments are shown; *, P<0.05, Student's t-test). (F) Gtf3c1 binding at c-FosRSINE1 and Gadd45bB1F increased in response to depolarization. Mouse primary cortical neurons were either exposed to 50 mM KCl for 45 min or left untreated, and subjected to ChIP using Gtf3c1 antibodies, followed by qPCR. Histograms show the immunoprecipitation efficiency of Gtf3c1 and control IgG antibodies relative to total chromatin input. Five previously identified c-Fos enhancers [16] showed no significant Gtf3c1 binding (average and s.e.m. of 3 experiments are shown; *, P<0.05, Student's t-test). (G) p300 was recruited to c-FosRSINE1 in response to depolarization. Mouse primary cortical neurons were either exposed to 50 mM KCl for 45 min or left untreated, and subjected to ChIP using p300 antibodies, followed by qPCR. Histograms show the immunoprecipitation efficiency of Gtf3c1 antibodies relative to control IgG (average and s.e.m. of 3 experiments are shown; *, P<0.05, Student's t-test).
Figure 3.
TFIIIC controls activity-dependent genes transcription and SINE acetylation.
(A) Representative images of cortical neurons transfected with a GFP expression vector and either control or Gtf3c5 siRNA. Neurons were transfected at DIV2, after three days exposed to 50 mM KCl for 45 minutes or left untreated, and subjected to quantitative RNA-FISH analysis of c-Fos mRNA. Maximal z-projections of confocal stacks of transfected cells are shown. In GFP-expressing cells, c-Fos mRNA ribonucleoparticles (red) were detected by FISH (reconstructed cell edges are shown in green). Nuclei were stained with DAPI (blue). Ribonucleoparticles showing low levels of c-Fos mRNA are indicated by arrowheads. (B) Quantitative analysis of RNA-FISH experiments. For each neuron, total fluorescence intensity of all c-Fos mRNA particles was calculated. Average and s.e.m. of at least 25 cells per condition are shown (*, P<0.05; **, P<0.01; ***, P<0.001, two-way ANOVA). (C, D, E and F) Mouse primary cortical neurons were infected with lentiviral particles driving the expression of a short hairpin RNA targeting either firefly luciferase (shLUC, as a negative control) or Gtf3c5 (shGtf3c5). 4 days later cells were stimulated with either 50 mM KCl for 45 minutes or left untreated, then subjected to RNA extraction and cDNA synthesis, followed by qRT-PCR analysis of c-Fos and Gadd45b pre-mRNA (C, E) and mRNA (D, F). Silencing of Gtf3c5 was sufficient to drive a significant enhancement of activity-dependent transcription, as assessed both at the level of pre-mRNA (C, E) and fully processed RNA (D, F). (G) Primary cortical neurons were infected with lentiviral particles driving the expression of a short hairpin RNA targeting either firefly luciferase (shLUC, as a negative control) or Gtf3c5 (shGtf3c5). 4 days later cells were stimulated with either 50 mM KCl for 45 minutes or left untreated, then subjected to ChIP using either H3K9K14ac or histone H3 antibodies, followed by qPCR. Lentiviral-mediated silencing of Gtf3c5 enhanced histone H3 acetylation at c-FosRSINE1, Gadd45bB1F and not at c-Fos TSS and GapdhB4. Histograms show the ratio of immunoprecipitation efficiency between H3K9K14ac and H3 antibodies relative to total chromatin input (average and s.e.m. of 5 experiments are shown; *, P<0.05, Student's t-test).
Figure 4.
Inducible genes relocate to TFs in response to depolarization.
(A) Representative images of single confocal sections of immuno-DNA FISH experiments showing nuclear localization of c-Fos, Gadd45b, Csn2, Gapdh, Bdnf and Fcf1 loci (green) relative to TFs. TFs were detected by RNAPII-ser5P immunostaining (red), in cortical neurons either stimulated with 50 mM KCl for 45 minutes or left untreated. Nuclei were stained with DAPI (blue). For each image series, the distance between the centre of the FISH signal and the nearest TF is indicated (top right inset). Scale bars, 2 µm (images on the left) and 0.5 µm (magnified images). (B) Box and whiskers plot of the distribution of the distance between c-Fos, Gadd45b, Csn2, Gapdh, Bdnf and Fcf1 loci and the nearest TF. Neurons were maintained either in basal conditions or exposed to 50 mM KCl for 45 minutes. Whiskers denote the 90th and 10th percentiles, box edges denote the 75th and 25th percentiles, solid lines denote medians, dashed lines denote averages. (C) Percentage of co-localization with TFs of c-Fos, Gadd45b, Csn2, Gapdh, Bdnf and Fcf1 gene loci both in basal conditions an in response to KCl (*, P<0.05, Fisher's exact test). For each condition shown in (B) and (C), 31 to 64 FISH signals were analysed. (D) DIV10 cortical primary neurons were stimulated with 50 µM bicuculline for 45 minutes, either in the presence of 1 µM tetrodotoxin (TTX) or alone, and analysed by immuno-DNA FISH. Histograms show the percentage of co-localization with TFs of c-Fos gene locus (*, P<0.05, Fisher's exact test; n = 36 to 41 FISH signals per condition). (E) Cortical primary neurons were stimulated with 50 mM KCl for 45 minutes, either in the presence of 5 µM nifedipine, 50 µg/ml DRB or alone, and analysed by immuno-DNA FISH. Histograms show the percentage of co-localization with TFs of c-Fos gene locus (*, P<0.05, Fisher's exact test; n = 30 to 41 FISH signals per condition).
Figure 5.
TFIIIC controls activity-dependent relocation of neuronal genes.
Two days after plating, mouse primary cortical neurons were transfected with control or Gtf3c5 siRNA in combination with an eGFP-actin expression vector. After three days, neurons were stimulated with 50 mM KCl for 45 minutes or left untreated and analysed by immuno-DNA FISH targeting c-Fos, Gadd45b and Csn2 loci. (A) Representative images of single confocal sections of immuno-DNA FISH experiments showing the nuclear localization of c-Fos and Gadd45b loci (green) relative to TFs, detected by RNAPII-ser5P immunostaining (red). GFP staining was confined outside the nuclear space (stained with DAPI, blue) and did not interfere with the FISH signal. For each image series, the distance between the centre of the FISH signal and the nearest TF is indicated (top right inset). Scale bars, 2 µm (images on the left) and 0.5 µm (magnified images). (B) Percentage of co-localization with TFs of c-Fos, Gadd45b and Csn2 gene loci both in basal conditions an in response to KCl in neurons transfected with either control or Gtf3c5 siRNA (*, P<0.05, Fisher's exact test; n = 32 to 44 FISH signals per condition).
Figure 6.
TFIIIC regulates dendritic growth.
(A) Representative images of cortical neurons transfected with a GFP-expressing vector alone or in combination with either control or Gtf3c5 siRNA. Neurons were cultured for 2 days in basal conditions or in presence of 50 mM KCl, followed by GFP immunostaining. Shown are the enhanced profiles of neurons after reconstruction with a trainable segmentation tool. (B) Sholl profiles of neurons maintained in basal conditions (above) or exposed to KCl (below). For each distance point, the average number of intersections and s.e.m. are shown. At least 25 cells per condition were analysed (*, P<0.05; **, P<0.01; ***, P<0.001, two-way ANOVA). (C) Total length of the dendritic processes of the cells analysed in (B). Average and s.e.m. are shown (*, P<0.05; **, P<0.01, two-way ANOVA).
Figure 7.
A model for TFIIIC-dependent regulation of activity-regulated transcription.
(A and B) In resting conditions (left), TFIIIC is bound to SINEs located in the proximity of poised, activity dependent genes. TFIIIC keeps gene loci away from transcription factories, thereby inhibiting transcription. (A) In response to depolarization, p300 is recruited to SINEs and co-regulated genes come in close proximity within shared TFs, where they become transcriptionally activated. (B) Alternatively, upon depolarization TFIIIC undergoes a functional switch, p300 is recruited to SINEs and genes relocate to TFs where they become transcriptionally activated.