Figure 1.
APP regulates IEGs expression in mouse PF cortex.
A) Egr1 mRNA levels were quantified by q-RT PCR in APP+/+ and APP−/− PF cortex (n = 9), and in the hippocampus (n = 6). B) q-RT PCR method was used to quantify mRNA levels of c-Fos, Arc and Bdnf in APP+/+ and APP−/− PF cortex (n = 6). Values were normalized to the GAPDH mRNA, and expressed as percentage of APP+/+, mean ± SD. Student’s t-test: ***p<0.001,**p<0.01, *p<0.05.
Figure 2.
Analysis of histone acetylation by ChIP assays on IEGs promoters.
ChIP experiments were performed with chromatin obtained from APP+/+ and APP−/− PF cortex. Immunoprecipitation was completed with antibody recognizing normal mouse IgG as negative control, or anti- H3Ac, H2BAc and H4Ac antibodies. The quantification of immunoprecipitated chromatin and the normalization versus total chromatin (input) was assessed by real-time qPCR with primers designed on A) Egr1, B) c-Fos, C) Arc and D) Bdnf exon IV promoters; *p<0.05. All results were obtained from at least 3 mice per group and per antibody, and are expressed as mean ± SD.
Figure 3.
H4K5 and H4K12 are enriched at Egr1 and c-Fos gene promoter in APP−/− mice.
ChIP method was used to evaluate the enrichment of H4K12Ac, H4K5Ac, and H4K16Ac. A) Egr1 promoter. B) c-Fos promoter. Data represent the level of enrichment normalized as percentage of APP+/+. ***p<0.001. All results derive from at least 3 mice per group and per antibody, and are expressed as mean ± SD.
Figure 4.
CREB is better recruited on the Egr1 gene promoter in APP−/− mice.
A) Schematic representation of the structure of Egr1 gene promoter containing several binding sites for transcription factors and localization of primers utilized for q-PCR (CREB: cAMP response element, CRE: cAMP response elements SRF: Serum Response factor, SRE: Serum Response Element, SP1: specificity protein). B) Tip60 binding to Egr1 gene promoter in APP+/+ and APP−/− PF cortex was assessed by ChIP using anti-Tip60 antibody. C) CREB binding to Egr1 promoter in APP+/ and APP−/− mice. IgG was used as negative control. Equal amounts of ChIP and input DNA were used for qRT-PCR analysis on the Egr1 gene promoter. Results show a significantly lower enrichment of CREB in APP+/+ mice, *p<0.05. Enrichment values were normalized to input values and represented the average of three or more experiments per group. Results are expressed as mean ± SD. D) Ratio between total and phosphorylated CREB was detected by western blot analysis of nuclear extracts from PF cortex of APP+/+ and APP−/− mice, 5 months of age (n = 5). Typical blot is shown, CREB/P-CREB ratio were quantified, and expressed as percentage of the APP+/+ mice. Data are expressed as mean ± SD. E) ChIP assay on Egr1 promoter using HDAC2 antibody.
Figure 5.
HDAC2 is enriched on the c-Fos gene promoter in APP−/− mice.
A) Schematic representation of the structure of c-Fos promoter containing only one CREB binding site and the localization of the primers utilized for q-PCR. (CREB: cAMP response element, CRE: cAMP response elements SRF: Serum Response factor, SRE: Serum Response Element, AP1: Activator protein 1, SIE: sis-inducible element, STAT: Signal Transducer and Activator of Transcription). B) Study of CREB binding to c-Fos gene promoter in APP+/and APP−/− mice. C) ChIP assay on c-Fos gene promoter using anti-HDAC2 antibody. Results show a significant lower enrichment of HDAC 2 in PF cortex of APP−/− mouse, Student’s t-test: **p<0.01. IgG was used as negative control. Equal amounts of ChIP and input chromatin were used for qRT-PCR analysis on the c-Fos gene promoter. Enrichment values were normalized to input values and represented the average of three or more mice per experiment. Results are expressed as mean ± SD. D) HDAC2 protein expression was detected by western blot analysis of nuclear (N) or cytoplasmic (C) extracts obtained from PF cortex of APP+/+ and APP−/− mice, 5 months of age (n = 5). Typical blot is shown with anti-histone H3 and anti-tubulin used as a loading control. Data are normalized against histone H3 to assess the level of nuclear HDAC2 and expressed as mean ± SD.
Figure 6.
APP+/+ but not APP−/− mice induce Egr1 and c-Fos expression after exposure to novelty.
A) Effect of exposure to novelty on the levels of Egr1 and c-Fos transcripts. qRT PCR were performed on mRNA extracted from APP+/+ and APP−/− mice exposed (Novelty) or not (Home cage) to the open field (n = 6 or more per group). All values were normalized to the GAPDH mRNA, and expressed as percentage of the APP+/+. Results are expressed as mean ± SD. Student’s t-test. (***p<0.001).