Figure 3 - uploaded by Yonghe Wu
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Schematic diagram of the human pituitary. The pituitary gland is located at the base of the skull between the optic nerves. The anterior pituitary comprises 5 main cell types that synthesize and release (into the blood stream) the adenohypophyseal hormones adrenocorticotrophic (ACTH) (target: adrenal cortex), luteinizing hormone (LH) and folliclestimulating hormone (FSH) (gonads), growth hormone (GH) (skeletal and metabolic tissues), prolactin (PRL) (mammary glands), and thyroid-stimulating hormone (TSH) (thyroid). AVP and oxytocin are released from hypothalamic nerve terminals contacting the posterior lobe and then into the general circulation. Rodent anterior pituitary (from now on, referred to simply as pituitary) cells, including corticotropes, proliferate and differentiate during postnatal development.
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Early life stress produced long-lasting alterations of the HPA axis characterized by elevated corticosterone level, increased Pomc mRNA levels in the pituitary as well as hypersecretion of ACTH into the blood. Epigenetic mechanisms, especially DNA methylation, appear to be responsible for controlling Pomc gene expression by recruiting MeCP2 to sile...
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... address this question, pituitary primary cells were treated with AVP (10 -7 M) for 2 hours, the activity of phospho-CaMKII was monitored by immunostaining. As shown in Figure 30A, the phosphorylated form of CaMKII was significantly increased after AVP treatment without influencing the total levels of CaMKII. As we have already shown that MeCP2 phosphorylation is mediated by CaMKII activity, we hypothesized that MeCP2 could be phosphorylated when primary pituitary cells were treated with AVP peptide. ...
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... results showed that this was indeed the case. Interestingly, most of the pS438-MeCP2 positive cells colocalized well with ACTH suggesting that MeCP2 phosphorylation at serine 438 occurred predominantly in corticotrope cells ( Figure 30B). The reason for this result could be explained by the fact that the AVP v1b receptor only exists in corticotrope cells in the pituitary. ...
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... of AtT20 cells with SSR149415 completely blocked the dissociation of MeCP2 due to AVP treatment ( Figure 30C). ...
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... slides (8 sections/animal, 5 mice per group) from10-day and 3-month-old mice were used for this experiment ( Figure 33). The results revealed no difference in the ratio of the corticotrope cells with 4.56 ± 0.15 in the control group and 4.65 ± 0.31 in the ELS group at PND 10 and ratios of 3.93 ± 0.16 and 3.95 ± 0.14 at 3 months of age, respectively. ...
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... a control, the second primer pair was designed in the coding region (exon3) which lies 6 kb downstream ( Figure 24A). In vivo ChIP analysis revealed increased activated RNA pol II occupancy at the Pomc promoter of ELS mice, reflecting increased Pomc gene transcription ( Figure 34A). When we performed the ChIP experiment using anti-MeCP2 antibody, the results showed that total MeCP2 occupancy was reduced in the ELS group ( Figure 34B). ...
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... vivo ChIP analysis revealed increased activated RNA pol II occupancy at the Pomc promoter of ELS mice, reflecting increased Pomc gene transcription ( Figure 34A). When we performed the ChIP experiment using anti-MeCP2 antibody, the results showed that total MeCP2 occupancy was reduced in the ELS group ( Figure 34B). These results agree with the findings of less DNA methylation in the ELS group. ...
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... statistical analysis was performed and the results demonstrated a negative correlation between total MeCP2 antibody and RNA Pol II binding (p < 0.05, data not shown). Monitoring MeCP2 mRNA expression levels showed no differences between pituitaries from ELS mice and control mice in 6 weeks old mice ( Figure 34C). ...
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... mice also displayed reduced MeCP2 occupancy at the Pomc promoter region compared with control litters ( Figure 35A). Although Pomc mRNA level was markedly increased in 10 days old mice ( Figure 12, 35A), the DNA methylation level did not differ between control and ELS mice of this age. ...
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... that 10 days old control and ELS mice have similar methylation patterns, the differences in MeCP2 occupancy indicated that ELS-induced phosphorylation of MeCP2 at serine 438 leads to relief of MeCP2 occupancy from the Pomc promoter. There was no difference for MeCP2 mRNA expression levels between pituitaries from ELS mice and control mice in 10 days old mice ( Figure 35C). mRNA expression levels showed no difference between pituitaries from ELS mice and control mice in 10-days old mice. ...
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... address this question, a sequential ChIP experiment was performed using the first antibody against MeCP2 and the second antibody against Dnmt1 for comparing ELS and control pituitaries. When we performed the ChIP experiment using anti- MeCP2 antibody, the results showed that total MeCP2 occupancy was reduced in ELS group ( Figure 36A, 37A) as we showed previously by simple ChIP both in 10-day-old pituitary and in 6-week-old mice (Figure 34, 35). When we performed the second round ChIP with anti-Dnmt1, we recovered significantly more Pomc from chromatin from control mice than from ELS mice both in 10-day old and 6-weeks old mice pituitaries ( Figure 36B, 37B). ...
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... address this question, a sequential ChIP experiment was performed using the first antibody against MeCP2 and the second antibody against Dnmt1 for comparing ELS and control pituitaries. When we performed the ChIP experiment using anti- MeCP2 antibody, the results showed that total MeCP2 occupancy was reduced in ELS group ( Figure 36A, 37A) as we showed previously by simple ChIP both in 10-day-old pituitary and in 6-week-old mice (Figure 34, 35). When we performed the second round ChIP with anti-Dnmt1, we recovered significantly more Pomc from chromatin from control mice than from ELS mice both in 10-day old and 6-weeks old mice pituitaries ( Figure 36B, 37B). ...
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... we performed the ChIP experiment using anti- MeCP2 antibody, the results showed that total MeCP2 occupancy was reduced in ELS group ( Figure 36A, 37A) as we showed previously by simple ChIP both in 10-day-old pituitary and in 6-week-old mice (Figure 34, 35). When we performed the second round ChIP with anti-Dnmt1, we recovered significantly more Pomc from chromatin from control mice than from ELS mice both in 10-day old and 6-weeks old mice pituitaries ( Figure 36B, 37B). Monitoring the mRNA expression of Dnmt1 by qRT- PCR revealed no difference in basal Dnmt1 expression levels between pituitaries from ELS and control mice both in 10-day and 6-week-old mice ( Figure 36C, 37C). ...
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... we performed the second round ChIP with anti-Dnmt1, we recovered significantly more Pomc from chromatin from control mice than from ELS mice both in 10-day old and 6-weeks old mice pituitaries ( Figure 36B, 37B). Monitoring the mRNA expression of Dnmt1 by qRT- PCR revealed no difference in basal Dnmt1 expression levels between pituitaries from ELS and control mice both in 10-day and 6-week-old mice ( Figure 36C, 37C). ...
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... polyclonal antibody MeCP2 pS97 that recognizes the phosphorylated serine 97 was generated by injecting New Zealand White rabbits with the KLH-conjugated peptide NH2-EASA p SPKQR (phosphor-serine). The antiserum was purified by affinity chromatography on a column that was coupled to unphosphorylated MeCP2 S97 peptide ( Figure 38). ...
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... antibody from rabbit and Flag antibody from mouse were used for staining. As shown in Figure 43, the MeCP2 antibody detected expression of the wild type MeCP2 construct exclusively in the nuclei of transfected LLC-PK1 cells. By contrast, following expression of MeCP2∆C, no signal was detected, while the Flag antibody detected both MeCP2 proteins. ...
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... promoter. Interestingly, MeCP2 phosphorylation occurs predominantly in corticotrope cells expressing the AVP V1b receptor ( Figure 30B). Supporting this, it was found that AVP-induced MeCP2 phosphorylation can be blocked by pre-treatment with the AVP V1b receptor antagonist---SSRI 149415 ( Figure 30B). ...
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... MeCP2 phosphorylation occurs predominantly in corticotrope cells expressing the AVP V1b receptor ( Figure 30B). Supporting this, it was found that AVP-induced MeCP2 phosphorylation can be blocked by pre-treatment with the AVP V1b receptor antagonist---SSRI 149415 ( Figure 30B). Specificity of the effect was further demonstrated in an experiment in corticotrope AtT20 cells which had been stably transfected with an AVP V1b receptor since they lack expression of this receptor ( Figure 31). ...
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... this, it was found that AVP-induced MeCP2 phosphorylation can be blocked by pre-treatment with the AVP V1b receptor antagonist---SSRI 149415 ( Figure 30B). Specificity of the effect was further demonstrated in an experiment in corticotrope AtT20 cells which had been stably transfected with an AVP V1b receptor since they lack expression of this receptor ( Figure 31). ...
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... research in our laboratory has shown that ELS produces long-lasting elevations of the levels of AVP mRNA and peptide in the hypothalamus. The present results show that MeCP2 phosphorylation status can be dynamically regulated by AVP in pituitary cells ( Figure 30). Taken together, these findings suggest that ELS can trigger the dynamic modification of MeCP2 via AVP stimulation. ...
