Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease

Abstract

Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD). While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1) and beta-site amyloid precursor protein cleaving enzyme 1(BACE1). By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP) (N2a/APPswe) and wild-type APP (N2a/APPwt) as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected), respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT) adenoviral E1A-associated 300-kDa protein (p300) is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.

Figure 1. The expression levels of APP, PS1, BACE1 and Aβ were elevated in N2a/APPswe and N2a/APPwt cells.

(A) The expression of full length APP was increased in N2a/APPswe and N2a/APPwt cells compared to controls (vector-transfected). (B, C) mRNA and protein levels of PS1 and BACE1 were significantly elevated in N2a/APPswe and N2a/APPwt cells compared with control cells; (D) the downstream expression levels of Aβ1-42 and Aβ1-40 were significantly higher in N2a/APPswe and N2a/APPwt cells than control cells. *: P<0.05, as compared with N2a/APPwt cells, #: P<0.05, as compared with control cells (n = 3).

Figure 2. H3 in PS1 and BACE1 promoters and cellular H3 were hyperacetylated in N2a/APPswe cells.

(A) the binding of acetylation H3 (Ac-H3) to PS1 and BACE1 promoter regions was markedly higher in N2a/APPswe cells than N2a/APPwt and control cells; (B) the cellular Ac-H3 was increased in N2a/APPswe cells compared with N2a/APPwt and control cells. Ip represents amplification of input DNA from cells; Ac-H3 represents DNA bound to Ac-H3 in the sample; N represents DNA precipitated by normal mouse IgG as negative control. *: P<0.05, as compared with N2a/APPwt cells, #: P<0.05, as compared with control cells (n = 3).

Figure 3. Endogenous p300 was enhanced in N2a/APPswe cells.

(A) compared to N2a/APPwt and control cells, the p300 expression was dramatically enhanced in N2a/APPswe cells; (B) overexpression of p300 protein in N2a/APPswe cells was further confirmed by immunocytochemistry, Magnification 400-fold, Scale bar: 50 µm. (C) the HATs activity was significantly higher in N2a/APPswe cells compared to N2a/APPwt and control cells; (D) the level of p300 was higher in the promoter regions of PS1 and BACE1 in N2a/APPswe cells but not in N2a/APPwt cells compared with the controls. *: P<0.05, as compared with N2a/APPwt cells, #: P<0.05, as compared with control cells (n = 3).

Figure 4. Hyperacetylation of PS1 and BACE1 was suppressed by p300 specific inhibitor curcumin in N2a/APPswe cells.

(A) the p300 protein was markedly decreased in N2a/APPswe cells after being treated with curcumin; (B) curcumin dramatically reduced the enhancement effect of p300 in N2a/APPswe cells by immunocytochemistry, Magnification 400-fold, Scale bar: 50 µm; (C) curcumin treatment significantly suppressed the HATs activity in N2a/APPswe cells; (D) the Ac-H3 to PS1 and BACE1 promoter regions was much lower in N2a/APPswe cells after being treated with curcumin compared to DMSO (placebo) group; (E) the cellular Ac-H3 in N2a/APPswe cells was statistically significantly decreased after curcumin treatment; (F) N2a/APPswe cells after being treated with curcumin showed reduced binding of p300 to PS1 and BACE1 in N2a/APPswe cells. (G, H, I) the PS1 and BACE1 expression, followed by their downstream products Aβ1-42 and Aβ1-40, were markedly reduced in N2a/APPswe cells after curcumin treatment. Ip represents amplification of input DNA from cells; Ac-H3 represents DNA bound to Ac-H3 in the sample; N represents DNA precipitated by normal mouse IgG as negative control. #: P<0.05, as compared with DMSO treatment cells (n = 3).

Figure 5. p300-dependent transcription activation of PS1 and BACE1.

p300 HAT activity facilitates the histone acetylation of the PS1 and BACE1, accompanied by a more open chromatin structure that further facilitates accessibility by transcription factors to DNA-sequence of the genes, which in turn, leads to enhancement of the transcriptional activation. HAT: acetyltransferase activity; TF: transcription factor.