Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning

Abstract

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning.

Keywords: CREB; ChIP assays; Tet1; classical conditioning; histones.

Figure 1.

In vitro model of eye blink classical conditioning. (A) Isolated preparation of the pons in which suction electrodes are used for paired stimulation of the auditory nerve CS (the “tone”) and the trigeminal nerve US (the “air puff”) while recording neural activity from the abducens nerve that generates the neural correlate of the blink response. (B) Representative physiological records show neural discharge in the abducens nerve characteristic of the unconditioned blink response (upper panel) and after acquisition of conditioning in which a burst is recorded during the auditory nerve CS that represents a conditioned response (CR, arrow; lower panel). (C) Acquisition curve of learned responses generated by paired conditioning stimulation. CRs are typically recorded by the second pairing session and asymptote with continued training. Unpaired pseudoconditioning stimuli generate no CRs.

Figure 2.

Rapid conditioning-dependent methylation and demethylation of tBDNF promoter regions and differential effect of DNMT inhibitors. (A) tBDNF partial gene structure showing 3 5' non-coding exons (I-III) and the 3' protein coding sequence (IV) with its 3' UTR. Transcription start sites are indicated by the arrows. Promoter and exon regions that contain the CpG sites shown in B are indicated by the gray bars. Diagonal lines represent gaps in sequence data. (B) Bisulfite sequencing PCR (BSP) analysis of the methylation status of CpG sites in promoter and exonic regions I-III from naïve preparations (N) and those pseudoconditioned (Ps1) or conditioned for one training session (C1; 25 min in duration). 3 × 7 clones/group. Transcription start sites are indicated by the arrows. Promoter and exon I show relatively low levels of methylation that do not change with conditioning. Promoter II undergoes significant methylation of specific CpG sites after conditioning, particularly those localized to the BHLHB2 and C/EBP transcription factor binding sites (one-way ANOVA followed by Fisher's post-hoc test, P < 0.05 and P = 0.005, respectively). Promoter III undergoes significant demethylation notably at the CREB binding site (one-way ANOVA followed by Fisher's post-hoc test, P = 0.02). Values in this and all subsequent figures represent means ± SEM. (C) Timing of promoter II methylation and promoter III demethylation after the onset of conditioning training. Total methylation levels for promoter II (CpG sites 1–13) and III (sites 3–13) are shown. Levels of methylation for promoter II are significantly elevated after just 5 min of conditioning and are further increased after 15 and 25 min (F_(3,48) = 3.72, P = 0.01). Data for the BHLHB2 (CpG site 8; one-way ANOVA followed by Fisher's post-hoc test, P = 0.03) and C/EBP (10; P = 0.03 and P = 0.008 at C_15min and C1, respectively) sites are shown individually. Promoter III is demethylated after 15 min of conditioning, which is significantly different from naïve by one session of conditioning (C1 or 25 minutes; F_(3,40) = 3.96, P = 0.01). Data for the CREB binding site (5; one-way ANOVA followed by Fisher's post-hoc test, P = 0.007 and P = 0.002 at C_15min and C1, respectively) is also shown. (D) Treatment with DNMT inhibitors zebularine (100 µM) or RG108 (200 ng/µl) during conditioning suppressed the overall level of methylation of promoters and exons II and III. (E) Total methylation levels of promoter and exon II are moderately suppressed by zebularine and RG108 treatment during conditioning compared to normal conditioning (upper left). However, when specific CpG sites are examined (7, 8, 10, and 12), both drugs significantly inhibit methylation after one session of conditioning (F_(3,12) = 10.09, P = 0.001; upper right). For promoter and exon III, which are normally demethylated in conditioning, drug treatment results in reduced levels of methylation for naïve compared to normal naïve groups but did not affect the conditioned preparations already demethylated (F_(5,60) = 5.98, P = 0.0002).

Figure 3.

Conditioning-dependent binding of DNA regulatory proteins to tBDNF promoters II and III and inhibition by PKA and PDK1 blockers. Data are from ChIP-qPCR analysis of tissue samples from naïve (N), pseudoconditioned (Ps), or conditioned (C) preparations for 15 min, or those conditioned for 15 min in the presence of the PKA inhibitor Rp-cAMPs (Rp; 50 µM) or the PDK1 blocker BX-912 (BX; 0.3 µM; n = 5/group). Significant P values between N and C_15min are given in the text. Individual comparisons for promoters II and III between the C_15min and Rp treated groups were, promoter II: MeCP2, P < 0.001; Tet1, P < 0.01; BHLHB2, P = 0.001; and promoter III: MeCP2, P = 0.005; Tet1, P = 0.001; CREB, P < 0.0001; ERK1/2, P = 0.001. Comparisons between C_15min and BX treated groups were, promoter II: MeCP2, P < 0.001; Tet1, P < 0.0001; BHLHB2, P = 0.002; and promoter III: MeCP2, P = 0.07; Tet1, P = 0.001; CREB, P < 0.0001; ERK1/2, P < 0.001. *, Significant differences from naïve in normal saline; +, significant differences from normal conditioning. Representative agarose gels are also shown of the resulting PCR products from the different experimental conditions.

Figure 4.

Tet1 partners with MeCP2 and ERK1/2 but dissociates from CREB during conditioning. (A–C) Co-immunoprecipitation of the protein-protein interactions between (A) MeCP2, (B) CREB, and (C) ERK1/2 with Tet1 protein in naïve and conditioned preparations that were trained for 15 or 25 (C1) min. P values are given in the text (n = 5/group). Input lanes are whole cell lysates from naïve samples and control IgG lanes are shown. Loading controls are also shown, except for MeCP2 that is detected at the same molecular weight as IgG heavy chain.

Figure 5.

Inhibition of Tet1 or ERK1/2 suppresses regulatory DNA protein binding to tBDNF promoters as determined by ChIP assays. Preparations were treated with an siRNA directed against turtle Tet1 (24 hr incubation, 150 nM) or the MEK1/2 inhibitor PD0325901 (1 hr, 1 µM) during 15 min of conditioning. The Western blots show a substantial reduction in Tet1 protein expression by application of the siRNA to naïve preparations to a mean of 54% (n = 4). The marker for Tet1 indicates 250 kDa. Westerns for Tet2, Tet3, CREB, and ERK1/2 show no reduction in protein expression after siRNA treatment verifying the specificity of the Tet1 siRNA. Both the Tet1 siRNA and PD resulted in significant alterations in tBDNF binding by all of the proteins examined compared to normal conditioning. Data from the conditioned group shown in Figure 3 are repeated here for direct comparison. P and F values are given in the text (n = 5/group). Individual comparisons for promoters II and III between the C_15min and Tet1 siRNA treated groups were, promoter II: BHLHB2, P = 0.0002; and promoter III: MeCP2, P = 0.03; Tet1, P = 0.001; CREB, P < 0.001; ERK1/2, P = 0.003. Comparisons between C_15min and PD treated groups were, promoter II: BHLHB2, P = 0.02; and promoter III: MeCP2, P < 0.0001; Tet1, P < 0.05; CREB, P < 0.0001; ERK1/2, P < 0.001. *, Significant differences from naïve in normal saline; +, significant differences from normal conditioning.

Figure 6.

Histone modifications and RNAP II tBDNF binding during conditioning are regulated by Tet1 and ERK1/2. (A) Western blots showing dramatically increased protein levels for both H3K4me3 (P < 0.0001) and H3K27me3 (P < 0.0001; n = 5/group) after 15 min of conditioning but not after 5 min. These levels are maintained for at least 80 min after 2 pairing sessions of conditioning (C2). (B) ChIP analysis reveals significantly elevated H3K4me3 (P < 0.0001) and reduced H3K27me3 (P < 0.01; n = 5/group) tBDNF promoter III levels after 15 min of conditioning. The H3K4me3 and H3K27me3 marks for promoter II were both significantly reduced by conditioning (P < 0.0001). Treatment of preparations with Tet1 siRNA or the MEK1/2 inhibitor PD during conditioning interfered with these histone modifications such that they were either suppressed or reversed (P values in the text). ChIP assays of phosphorylated forms of RNAP II tBDNF binding showed markedly elevated levels for promoter III after 15 min of conditioning (Ser5, Ser2 promoter, Ser2 exon, all P < 0.0001; Ser5 and Ser2 promoter, n = 5/group; Ser2 exon, n = 3/group) compared to naïve. Tet1 siRNA or PD treatment inhibited promoter III RNAP II binding to naïve values (Ser5, F_(2,12) = 138.52, P < 0.0001; Ser2 promoter, F_(2,12) = 16.76, P = 0.0003; Ser2 exon, F_(2,6) = 52.30, P = 0.0002; comparison of C_15min, Tet1 siRNA, PD). RNAP II binding to promoter II after conditioning showed surprisingly high values of the Ser5 phosphorylated form compared to naïve. However, Ser2 at both promoter and exonic sites was significantly lower than naïve (Ser2 promoter and exon, P < 0.001; Ser5 and Ser2 promoter, n = 5/group; Ser2 exon, n = 3/group). Tet1 and PD treatment also interfered with the normal activity of RNAP II during conditioning (Ser5, F_(2,12) = 20.42, P = 0.0001; Ser2 promoter, F_(2,12) = 4.55, P < 0.05; Ser2 exon, F_(2,6) = 1.13, P = 0.38; comparison of C_15min, Tet1 siRNA, PD). *, Significant differences from naïve in normal saline; +, significant differences from normal conditioning.

Figure 7.

Disruption of tBDNF mRNA expression by Tet1 siRNA and an inhibitor of ERK1/2 blocks acquisition of conditioned responding. (A) During normal conditioning mRNA transcripts from promoter II are downregulated (F_(7,16) = 26.49, P < 0.0001; n = 3/group) while promoter III transcript 3b is significantly upregulated by threefold (P < 0.0001) compared to naïve. Tet1 siRNA treatment further lowered expression levels of promoter II transcripts (2c, P = 0.004; 2d, P = 0.001) compared to normal conditioning, while both Tet1 siRNA and PD blocked expression of promoter III transcript 3b (F_(2,6) = 65.23, P < 0.0001). *, Significant differences from naïve in normal saline; +, significant differences from normal conditioning. (B) Treatment of preparations with Tet1 siRNA (n = 5/group) or the MEK inhibitor PD (n = 3/group) completely blocks the acquisition of CRs during training for 2 pairing sessions. However, application of a negative control siRNA (NC siRNA; n = 4/group) resulted in robust conditioning that typically occurs in session 2. Electrophysiological records show extracellular recordings from the abducens nerve during application of the CS to the auditory nerve followed by the US to the trigeminal nerve which results in expression of a burst discharge characteristic of the unconditioned “blink” response. A CR, a burst discharge in response to the CS, is indicated by the arrow for a preparation treated with control siRNA.

Figure 8.

Model of tBDNF transcriptional regulation during classical conditioning. The naïve state is shown to the left and 15 min after conditioning onset (C_15min) is shown to the right of the arrows. The DNA segments illustrated represent the regions covered by primer pairs for ChIP assays flanking transcription factor binding sites (promoter II, −158 to −55 nt; promoter III, −66 to +22 nt). Promoter II is hypomethylated in the naïve state by Tet1 and bound by RNAPIISer5 that initiates active transcription. After conditioning, Tet1 is released and tBDNF II undergoes transcriptional repression by increased methylation and binding by MeCP2 and the transcription factor BHLHB2. While RNAP II remains phosphorylated at Ser5 and bound to tBDNF, the levels of RNAPIISer2 decline and transcription is suppressed. Promoter III is methylated and bound by MeCP2 in naïve preparations. It is transcriptionally activated during conditioning following demethylation by Tet1 and release of MeCP2, which allows access of ERK1/2 and transcription factor CREB. There is also dramatically increased binding of RNAPIISer5 and Ser2 required for successful initiation and elongation for mRNA transcription. Since a Tet1-MeCP2 interaction is sustained during conditioning as shown by co-IP, we speculate that these proteins rotate around one another to rapidly interact with DNA as illustrated.

Figure 9.

Specificity of the antibodies used in the present study. Antibodies were tested on naïve brain tissue from turtle (T) and rat (R). Bands appeared at the appropriate molecular weights in both species.