Identification of BERP (brain-expressed RING finger protein) as a p53 target gene that modulates seizure susceptibility through interacting with GABA(A) receptors

Abstract

p53 is a central player in responses to cellular stresses and a major tumor suppressor. The identification of unique molecules within the p53 signaling network can reveal functions of this important transcription factor. Here, we show that brain-expressed RING finger protein (BERP) is a gene whose expression is up-regulated in a p53-dependent manner in human cells and in mice. We generated BERP-deficient mice by gene targeting and demonstrated that they exhibit increased resistance to pentylenetetrazol-induced seizures. Electrophysiological and biochemical studies of cultured cortical neurons of BERP-deficient mice showed a decrease in the amplitude of GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents as well as reduced surface protein expression of GABA(A)Rs containing the gamma2-subunit. However, BERP deficiency did not decrease GABA(A)Rgamma2 mRNA levels, raising the possibility that BERP may act at a posttranscriptional level to regulate the intracellular trafficking of GABA(A)Rs. Our results indicate that BERP is a unique p53-regulated gene and suggest a role for p53 within the central nervous system.

Fig. 1.

p53 binding and transcriptional competence of predicted BERP p53REs. (A) Human BERP p53REs. Schematic localization of four putative p53REs (gray ovals labeled B, E, F, and G) in the human genomic BERP locus, as indicated. ATG, BERP translation start site; Chrom., chromosome. (B) Nucleotide sequences of consensus p53RE of the four putative human BERP (B, E, F, and G) p53REs, and the putative mouse BERP p53RE. Pu = A/G, Py = C/T, n = A/G/C/T; uppercase, half-site nucleotides; lowercase, spacer nucleotides. *Mismatches from p53RE consensus sequence. Residue positions are expressed relative to the “A” of ATG (+1). Numbers of residues matching the p53RE consensus sequence are indicated. (C) Binding of p53 to putative human BERP p53REs. ChIP assays were performed in HCT116 p53^+/+ and HCT116 p53^−/− cells to detect binding of p53 to BERP B and EFG sites (anti-p53 lanes). The p21 p53RE was used as a positive control (p21). Cells were treated with DMSO (“untreated” control) or 5FU to induce p53. IgG, nonspecific mouse Ig (negative control); Input, 1% of total sonicated chromatin was reserved before IP; H₂O, water control. Results shown are representative of three trials. (D) BERP E is a functional p53RE. HCT116 p53^−/− cells were cotransfected with a vector expressing either p53wt or p53mut plus a luciferase reporter construct containing putative BERP p53REs (pGL3-BerpB, pGL3-BerpE, pGL3-BerpF, or pGL3-BerpG). pGL3-p21, positive control; pGL3-TATA, empty vector. Luciferase activity was normalized to β-gal activity and expressed relative to pGL3-TATA levels. Results shown are mean fold change in luciferase activity ± SEM of four independent experiments performed in triplicate. (E) Mutation of BERP E abrogates transactivation. HCT116 p53^−/− cells were cotransfected with a vector expressing p53wt or p53mut as in D plus a luciferase construct containing either WT BERP E (pGL3-BerpE) or BERP E with a C→A point mutation (pGL3BerpE* = GGGAAGGCCCtGGGCTTGTTC). Results shown are the mean fold change in luciferase activity ± SEM of three independent experiments performed in quadruplicate. (F) Berp M is a functional p53RE, and mutation of Berp M abrogates transactivation. The functionality of Berp M was analyzed as in D. pGL3-BerpM, murine site Berp M; pGL3BerpM*, Berp M with a C→A point mutation (AGGAAGGCCCtGTGCATGTTC); pGL3-p21, positive control; pGL3-TATA, empty vector. Results shown are mean fold change in luciferase activity ± SEM of three independent experiments performed in triplicate.

Fig. 2.

BERP is specifically up-regulated by p53 in human cells. (A) Induction. HCT116 p53^+/+ and HCT116 p53^−/− cells were treated for 48 h with the indicated doses of 5FU to activate p53, and relative expression levels of BERP and p21 (control) mRNAs were analyzed by real-time RT-PCR. Values were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) and expressed relative to untreated controls. Results shown are the mean fold change in mRNA ± SEM of three independent experiments performed in triplicate. (B) Specificity. HCT116 p53^+/+ and HCT116 p53^−/− cells were treated for 24 h with the indicated doses of 5FU, and relative expression levels of BERP, NARF, HT2A, and p21 (control) mRNAs were analyzed by real-time RT-PCR as for A. Results shown are the mean fold change ± SEM of three independent experiments performed in triplicate.

Fig. 3.

PTZ up-regulates BERP expression in a p53-dependent fashion. p53^+/+ and p53^−/− mice were sham-injected or injected with 45 mg/kg of PTZ, and histological sections of the cerebellum and hippocampus were subjected to in situ hybridization to detect BERP mRNA. BERP mRNA expression was up-regulated in p53^+/+ mouse brains (B and D), whereas levels in p53^−/− mouse brains (A and C) were comparable to the endogenous levels present in sham-injected p53^−/− and p53^+/+ mice (E and F). Results shown are representative of five mice per genotype. (Scale bar: 200 μm.)

Fig. 4.

Altered PTZ seizure susceptibility and electrophysiology in BERP-deficient mice. (A and B) Reduced seizure susceptibility. PTZ was administered s.c. to mice at doses of 40 mg/kg (n = 21 for BERP^+/+, n = 19 for BERP^−/−), 60 mg/kg (n = 15 for BERP^+/+, n = 13 for BERP^−/−), 80 mg/kg (n = 11 for BERP^+/+, n = 9 for BERP^−/−), 90 mg/kg (n = 10 for BERP^+/+, n = 10 for BERP^−/−), and 100 mg/kg (n = 7 for BERP ^+/+, n = 7 for BERP^−/−). (A) Latency. Each data point represents the mean latency ± SEM to a PTZ-induced generalized seizure (GS) in BERP^+/+ and BERP^−/− mice. *P < 0.05, Student's t test. (B) Dose–response curves. Each data point represents the percentage of BERP^+/+ and BERP^−/− mice exhibiting GSs at the indicated dose of PTZ. *P < 0.05, Fisher's exact test and χ² test. (C and D) Reduced neuronal mean mIPSC amplitude. Cortical neurons from untreated BERP^+/+ (n = 11) and BERP^−/− (n = 13) mice were analyzed by the whole-cell patch-clamp method to determine mean mIPSC frequency (C) and amplitude (D). **P < 0.01, Student's t test.

Fig. 5.

BERP deficiency decreases surface expression of GABA_ARs. Cortical neuron cultures were established from BERP^+/+, BERP^+/−, and BERP^−/− E17.5 embryos. (A) Biotinylation of surface proteins was carried out, and extracts of pooled cultures were subjected to Western blot analysis to detect surface GABA_ARγ2 and GluR2 subunits. GABA_ARγ2 protein was reduced in the absence of BERP, but GluR2 levels were comparable in all genotypes. Results shown are a single trial representative of four independent experiments. (B) Densitometry analysis of surface GABA_ARγ2 expression in cortical neuron cultures from BERP^+/+, BERP^+/−, and BERP^−/− embryos expressed in arbitrary units. Results shown are the mean expression ± SEM from four independent experiments. **P < 0.01, Student's t test.