Bdnf expression was induced during both heat conditioning and heat challenge of previously conditioned chicks
Plastic events in the hypothalamus during the critical period of temperature control establishment are responsible for long-term changes in the thermal control set-point. That Bdnf is crucial for synaptic plasticity and maintenance of long-term memory (Kang & Schuman, 1995; Patterson et al., 1996; Barco et al., 2005), that it plays an important role in critical periods of sensory development (Huang et al., 1999; Katz & Meiri, 2006), and that we have previously demonstrated its critical role in thermal control establishment (Katz & Meiri, 2006), led us to investigate changes in the expression of this protein during the time window known to be most effective for heat conditioning in chicks, i.e. on the third day of life. As revealed by Western blot analysis, Bdnf expression was induced during heat conditioning (heat exposure of the chicks to 37.5°C): the amplification began 2 h into the treatment and peaked 6–12 h into the heat treatment, at a level that was ∼60% higher than in naïve chicks. At 24 h, the expression level had decreased back to that in naïve chicks (Fig. 1A and B; Bdnf expression relative to that in naïve chicks was 1.58 ± 0.19 after 6 h, F1,27 = 6.33, P < 0.02; 1.63 ± 0.23 after 12 h, F1,26 = 5.65, P < 0.03).
Figure 1. Induction of Bdnf protein expression in chick PO/AH during heat conditioning on day 3. (A) Representative Western blot analysis of heat treatment. Each time point represents Bdnf protein expression from a single chick. (B) Densitometry of Western blot analysis evaluated by imagej 1.30 image-analysis software. Each time point represents the level of Bdnf protein relative to naïve age-matched chicks (to minimize loading or exposure differences, Bdnf was compared with β-actin in each gel lane; n = 15 at each time point; bars are + SEM). *P < 0.05 between conditioned and naïve chicks. (C) Representative immunofluorescent staining of the PO/AH in sagittal sections reacted with antibody to Bdnf. Immunohistochemistry was performed with polyclonal antibody to Bdnf (Santa Cruz Biotechnology Inc.) and with Cy3-conjugated donkey antirabbit IgG. Enlargements (100×) of the PO/AHs from 6-h-conditioned and naïve chicks are presented in the left panels. The same fields counterstained with DAPI, which stains nuclei, are presented in the right panel. Coordinates of the section according to Kuenzel & Masson (1988), lateral distance from midline, L = 0.5 mm.
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To visualize the localization of Bdnf protein during heat exposure, sagittal sections prepared from naïve and 6-h heat-treated chicks were immunofluorescently stained. The cellular distributions on the two representative slides were similar, as demonstrated by DAPI staining of the nuclei (Fig. 1C). Bdnf signal was visualized in more cells in the PO/AH area after 6 h of heat treatment than in naïve untreated chicks (Fig. 1C). It should be noted that this method is not quantitative.
To correlate the timing of Bdnf expression with the critical period of thermal control establishment, its expression was also evaluated in the PO/AH of two groups of 10-day-old chicks, one group that had been heat-exposed on day 3 (37.5°C for 24 h; the heat re-exposed group) and a group of naïve 10-day-old untreated chicks. Using real-time PCR, we found that Bdnf mRNA expression was not altered by heat treatment in chicks that had passed the critical age for thermal control establishment (Fig. 2A). However, Bdnf mRNA expression was significantly induced during heat challenge in chicks that had previously undergone heat exposure (Fig. 2A). A significant increment relative to the respective controls was observed 6 h into the challenge, reaching a maximum level 12 h into the treatment (2.51 ± 0.52 after 6 h, F1,15 = 6.99, P < 0.05; 4.27 ± 1.04 after 12 h, F1,13 = 11.21, P < 0.01). At 24 h, the level of expression had declined to that observed at 6 h (3.04 ± 0.66, F1,15 = 8.06, P < 0.02; Fig. 2A). The induction of Bdnf expression in the challenged (re-exposed) group relative to that in their naive age-matched counterparts was significant throughout the experiment (P < 0.05).
Figure 2. Bdnf mRNA expression pattern during heat exposure of naive chicks and heat re-exposure (challenge) of previously conditioned chicks on day 10 of age. (A) Real-time PCR quantification of Bdnf mRNA expression in the PO/AH of two groups of 10-day-old chicks: a group that was conditioned on day 3 by exposure to 37.5°C for 24 h (heat re-exposed), and a group of naïve untreated chicks. Bdnf expression level was compared to that of 18S using the SYBR green method. Each time point is an average + SEM of six naive chicks or nine re-exposed chicks. (B) Densitometry of Western blot analysis of Bdnf expression in the PO/AH of the two groups of 10-day-old chicks: naive and heat re-exposed chicks, evaluated by imagej 1.30 image analysis software. Each time point represents the levels of Bdnf protein relative to naïve age-matched chicks (to minimize loading or exposure differences, Bdnf was compared with β-actin in each gel lane; n = 6 naive and 8–9 re-exposed chicks at each time point; bars are + SEM). In the figure, aP < 0.05 within the heat re-exposed group and *P < 0.05 between naive and heat re-exposed chicks.
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As induction of a particular gene does not always reflect a change in the expression of the protein it encodes, the expression of Bdnf protein was evaluated during heat challenge of previously heat-conditioned chicks and of age-matched naive untreated chicks by Western blot analysis. Bdnf protein level was consistent with its mRNA expression: it was not altered in naive chicks during heat exposure on day 10 (Fig. 2B). However, its expression was induced upon heat challenge in previously heat-conditioned chicks. The Bdnf protein levels in the PO/AH from 10-day-old heat-challenged, previously conditioned, chicks was compared to that from age-matched naive heat-exposed chicks: as a result of the re-exposure, Bdnf levels were induced from 2 to 12 h into the heat challenge, with peak induction at 12 h of 2.23 ± 0.38 (F1,11 = 48.48 and F1,14 = 9.54, P < 0.01 after 6 and 12 h respectively, and F1,11 = 5.72, P < 0.05 after 2 h (Fig. 2B). Similar to the expression of the Bdnf mRNA, there was a decline in Bdnf protein expression in the heat-conditioned chicks after 24 h of heat re-exposure.
DNA methylation was dynamically altered at the Bdnf promoter during heat exposure
As Bdnf was induced during heat exposure of 3-day-old chicks, we investigated the regulation mechanism which might underlie this induction. The reading frame of the chick Bdnf gene is delineated on one exon. Upstream of the Bdnf coding region’s initiation site there are several CpG dinucleotides which can be methylated and therefore are potentially involved in the regulation of Bdnf expression (Fig. 3A). Methylation of DNA in the PO/AH was measured at six CpG positions detected in silico during heat exposure of 3-day-old chicks (Fig. 3B–G). A complete sequence of the Bdnf promoter region was performed in order to control for a complete bisulfite conversion processes. Analyzing the DNA sequence in which these methylation sites reside we found that M1, located 186 bp upstream of the coding region (−186), is a potential CEB/P site, which is known to be involved in energy metabolism; M2 (−251) resides within an AP1 site which is the binding site of Jun; M3 (−370) is not located within a known transcription-regulation site; M4 (−474) is located in an ENKCRE and c-Myc site; M9 (−1082) is a CREB site, and M12 (−1363) is a c-Myb site (Fig. 3A).
Figure 3. Alteration of CpG methylation upstream of the Bdnf coding sequence during heat conditioning. Chicks were thermally conditioned on day 3 of life. At 2, 6, 12 or 24 h into conditioning, their PO/AH was dissected, the DNA extracted and modified using a CpGenome DNA modification kit, and the amount of methylated CpG was determined using PCR with specific primers. Methylation was determined by comparing the PCR product from the CpG site with that from a site in the coding region. (A) A schematic chart depicting the CpG locations upstream of the Bdnf coding region. The analyzed methylated sites are marked in black and their exact location is delineated. (B–G) Methylation levels at positions M1, 2, 3, 4, 9 and 12, respectively. Each time point represents the methylation levels in 8–11 chicks and is + SEM. (B, D and F) *P < 0.05 between conditioned and naïve chicks.
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Following heat exposure of 3-day-old chicks, there was a significant increase in the level of methylation in heat-exposed chicks compared to naïve chicks at positions M1 and M3 (Fig. 3B and D). There was a significant decrease in methylation in heat-exposed chicks compared to naïve chicks at position M9 (Fig. 3F). Positions M2, M4 and M12 did not show any significant changes in methylation during heat exposure (Fig. 3C, E and G). Methylation induction at the first CpG location upstream of the ATG initiation site (M1) began after 2 h of heat exposure (2.07 ± 0.37-fold increase compared to the level in naïve age-matched chicks; F1,17 = 6.13, P < 0.02; Fig. 3B) and remained high throughout the heat treatment (2.38 ± 0.35 after 6 h, F1,17 = 11.24, P < 0.01; 2.61 ± 0.41 after 12 h, F1,17 = 11.97, P < 0.01; and 2.52 ± 0.41 after 24 h of heat treatment, F1,16 = 11.56, P < 0.01; Fig. 3B). The methylation level at the third CpG location (M3) was only induced 12 h after the beginning of heat treatment, at which time it was 1.65 ± 0.15-fold that in naïve chicks (F1,18 = 9.83, P < 0.01; Fig. 3D). The level of methylation at M3 was further elevated after 24 h (2.29 ± 0.23-fold higher than in naïve chicks, F1,18 = 28.32, P < 0.01; Fig. 3D). In contrast, on M9 (−1082 bp) there was a significant decrease in the level of methylation, which started 6 h into the heat exposure and lasted for an additional 6 h (the level of methylation relative to that in naive chicks was 0.52 ± 0.05 after 6 h and 0.62 ± 0.17 after 12 h; F1,12 = 12.79, P < 0.01 at 6 h, F1,15 = 9.01, P < 0.01 at 12 h; Fig. 3F). However, after 24 h the expression level had returned to that in naïve chicks (Fig. 3F). At M2, M4 and M12 there was no change in methylation level during heat treatment (Fig. 3C, E and G).
Long-term effect of heat conditioning on Bdnf promoter methylation pattern
Heat conditioning has a long-term phenotypic effect on heat tolerance (Yahav & McMurtry, 2001; Labunskay & Meiri, 2006), which can be best evaluated by heat challenge later in life. In correlation with this phenotypic change, the expression pattern of Bdnf differed between heat-conditioned and naive age-matched chicks during heat challenge on day 10 (Fig. 2). We therefore evaluated the methylation pattern in the Bdnf promoter in two groups of 10-day-old chicks, a group that had been heat-conditioned on day 3 by exposure to 37.5°C for 24 h (the heat re-exposed group) and a group of naïve untreated chicks. As there was a slight but nonsignificant change in the baseline of Bdnf methylation between previously heat-conditioned and naïve chicks on day 10, the methylation level of the naive 10-day-old chicks was arbitrarily set to 1.
The DNA methylation pattern clearly differed in previously heat-conditioned vs. naïve 10-day-old chicks (Fig. 4). While CpG methylation was dynamically induced at all CpG positions on the Bdnf promoter during heat exposure of naive 10-day-old chicks, there were only minor changes in the Bdnf promoter during heat re-exposure of previously heat-conditioned chicks. These changes included a significant reduction in the amount of methylation at position M9 (Fig. 4E) and a single increment at position M4 (Fig. 4D). The difference in CpG methylation between conditioned and naïve heat-exposed chicks during heat re-exposure was significant in five out of the six positions checked (at M1 after 12 and 24 h, F1,11 = 10.73, P < 0.01 and F1,18 = 6.72, P < 0.02, respectively; at M2 after 2 h, F1,13 = 7.50, P < 0.02, after 6 h, F1,13 = 6.58, P < 0.02, after 12 h, F1,11 = 11.09, P < 0.01 and after 24 h, F1,13 = 5.22, P < 0.04; at M3 after 6 h, F1,8 = 5.77, P < 0.04 and after 12 and 24 h, F1,11 = 9.00 and F1,9 = 15.21, respectively, P < 0.01; at M9, after 2 h, F1,21 = 6.37, P < 0.02, after 6 h, F1,18 = 5.76, P < 0.03, after 12 h F1,15 = 9.22, P < 0.01 and after 24 h, F1,17 = 15.23, P < 0.01, and at M12 after 12 and 24 h, F1,12 = 4.94 and F1,15 = 4.89, respectively, P < 0.05). There was no difference in CpG methylation patterns between previously heat-conditioned and naive chicks at position M4.
Figure 4. Comparison between CpG methylation patterns upstream of the Bdnf coding sequence of previously conditioned chicks and aged-matched naive chicks during heat exposure on day 10 of life. Methylation in the Bdnf promoter was evaluated in two groups of 10-day-old chicks, a group that had been conditioned on day 3 by exposure to 37.5°C for 24 h (heat re-exposed) and a group of naïve untreated chicks, by exposing them to 37°C for 24 h. At 2, 6, 12 or 24 h into heat exposure their PO/AH was dissected, the DNA extracted and modified using a CpGenome DNA modification kit, and the amount of methylated CpG was determined using PCR with specific primers. (A–F) The amounts of methylation at M1, 2, 3, 4, 9 and 12, respectively. Each time point represents the methylation in 8–11 chicks and is + SEM.
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In the first position upstream of the ATG initiation site (M1) there was a significant elevation in the methylation of the CpG dinucleotide throughout heat exposure of naïve 10-day-old chicks (Fig. 4A). The induction started 2 h into the heat exposure, at which time the methylation level was two-fold that in naive chicks; at 6 h, it had risen to 2.5-fold; the induction peaked at 12 h, at ∼3.5-fold the value in naive chicks and after 24 h the induction was 2.7 times that in naïve chicks (P < 0.01 at all time points for heat-exposed vs. naive chicks, F1,16 = 6.76, F1,16 = 11.22, F1,13 = 13.95 and F1,16 = 12.19 at 2, 6, 12 and 24 h, respectively). The methylation of CpG at M1 in previously conditioned chicks was not altered during heat challenge (Fig. 4A).
At M2, the methylation induction in naïve 10-day-old chicks started 2 h into the heat exposure, at which time the methylation level was 1.3-fold that in naïve untreated chicks; at 6 h it was 1.6-fold; the level peaked at 12 h and was 2.5 times higher than in naive chicks, and after 24 h it was 1.5 times higher (F1,16 = 4.64, P < 0.05 at 6 h and F1,13 = 17.29, P < 0.01 after 12 h; Fig. 4B).
Whereas in naïve 10-day-old chicks there was a clear induction in methylation at M3 which stayed elevated throughout the heat exposure, methylation level in the age-matched heat-challenged group did not change (Fig. 4C; the amount of methylation in naïve 10-day-old heat-exposed chicks vs. naive age-matched chicks was 2.43 ± 0.45, F1,10 = 8.76, P < 0.01 after 6 h, 2.31 ± 0.36, F1,10 = 10.61, P < 0.01 after 12 h, and 2.72 ± 0.33, F1,10 = 20.72, P < 0.01 after 24 h; the amount of methylation in heat re-exposed chicks remained the same throughout the heat challenge).
As mentioned above, the only position at which both heat-exposed naïve 10-day-old chicks and challenged, previously exposed, chicks’ methylation levels changed with time was M4. Both methylation levels were elevated by 40% compared to their respective controls (Fig. 4D).
At M9, the amount of methylation in previously heat-conditioned chicks was significantly reduced throughout heat re-exposure on day 10 (Fig. 4E). In these chicks, the amount of methylation was reduced by 40%, after 2, 6, 12 and 24 h (F1,24 = 4.37, F1,21 = 5.83, F1,18 = 5.31 and F1,20 = 7.54 after 2, 6, 12 and 24 h respectively; P < 0.05 in all groups). In addition, the methylation in this challenged group was significantly lower than that in its counterpart aged-matched naïve 10-day-old group.
At M12, whereas in heat exposure of naive 10-day-old chicks there was a clear induction in methylation which stayed elevated throughout the heat exposure, methylation level in the heat-challenged group (chicks that were heat-exposed on day 3) did not change (Fig. 4F). The amount of methylation in naïve heat-exposed chicks compared to age-matched nonexposed counterparts was 1.42 ± 0.24, 2.16 ± 0.52, 2.54 ± 0.64 and 2.18 ± 0.47 after 2, 6, 12 and 24 h, respectively (F1,14 = 7.17, P < 0.05 after 12 h and F1,16 = 6.03, P < 0.03 after 24 h).
DNMT3a expression was induced during heat exposure of 3-days-old chicks and it may bind to specific sites at the Bdnf promoter region
DNMTs are a family of enzymes that catalyze the methylation of cytosine residues (Goll & Bestor, 2005). Given that we observed induction of methylation at two CpG sites during heat exposure of 3-day-old chicks at the Bdnf promoter (M1 and M3), we sought to corroborate these results by determining which DNMT is involved in this process. Therefore, we evaluated the protein expression of both DNMT3a and -3b in the PO/AH of 3-day-old chicks during heat exposure. Western blot analysis of DNMT3a expression indicated an induction in its expression, but there was no change in the expression level of DNMT3b (Fig. 5C and D, respectively). The expression of DNMT3a reached a peak induction of 30% at 6 and 12 h after heat-exposure (F1,25 = 4.85, P < 0.04 and F1,24 = 5.35, P < 0.03 respectively).
As CpG methylation was induced during heat exposure of 3-day-old chicks at two positions, M1 and M3, and reduced at position M9, we wanted to demonstrate that, indeed, DNMT3a but not -3b can bind to the two inducible sites and not to the site of reduced CpG methylation. Electrophoresis mobility shift assay was used to detect the binding abilities of DNMT3a and -3b to the DNA in the Bdnf promoter at the relevant CpG sites. PO/AH extracts were reacted with antibodies to DNMT3a or -3b, and then with DIG-labeled oligonucleotides, each containing one CpG position (corresponding to M1, M3 or M9); they were then separated on a polyacrylamide gel and visualized with anti-DIG antibody. As can be seen in Fig. 5E, there was a clear supershifting of the DIG-labeled oligonucleotide at positions M1 and M3 when reacted with DNMT3a antibody, whereas there was no binding of DNMT3a to the sequence in position M9. DNMT3b did not bind to any of these positions (Fig. 5E).