Epigenetic regulation of kappa opioid receptor gene in neuronal differentiation

Abstract

The gene of mouse kappa opioid receptor (KOR) utilizes two promoters, P1 and P2. P1 is active in various brain areas and constitutively in P19 mouse embryonal carcinoma cells. P2 is active in limited brain stem areas of adult animals and only in late differentiated cells of P19 induced for neuronal differentiation in the presence of nerve growth factor (NGF). NGF response of P2 was found to be mediated by a specific binding site for transcription factor activation protein 2 (AP2) located in P2. Electrophoretic gel shift assay showed specific binding of this AP2 site by AP2beta, but not AP2alpha. Knockdown of endogenous AP2beta with siRNA abolished the stimulating effect of NGF on the expression of transcripts driven by P2. Binding of endogenous AP2beta on the endogenous KOR P2 chromatin region was also confirmed by chromatin immunoprecipitation. The effect of NGF was inhibited by LY2942002 (phosphatidylinositol 3-kinase, PI3K inhibitor), suggesting that PI3K was involved in signaling pathway mediating the effect of NGF stimulation on KOR P2. The chromatin of P2 in P19 was found to be specifically modified following NGF stimulation, which included demethylation at Lys9 and dimethylation at Lys4 of histone H3 and was consistent with the increased recruitment of RNA polymerase II to this promoter. This study presents the first evidence for epigenetic changes occurred on a specific KOR promoter triggered by NGF in cells undergoing neuronal differentiation. This epigenetic change is mediated by recruited AP2beta to this promoter and involves the PI3K system.

Fig. 1

NGF induces expression of KOR c through P2 in RA-induced differentiating P19 cells. (A) The map of KOR gene promoter regions, including P1, P2, exons 1 & 2, and a transcription initiation site of P2 located in intron 1. Splicing variants of P1, isoforms a and b, and P2 transcript isoform c are shown under the map and specific 5′ primers for amplifying each isoform are labeled with A, B and C. The common reverse primer is specific to exon 3. (B) P19 cells were subjected to RA-induced differentiation. After dissociating embryoid bodies and plating onto tissue culture plates, NGF treatment was initiated on day 1 in the absence of RA and cells were harvested on day 3. Isolated RNAs were analyzed by RT-PCR followed by a semi-quantitative Southern blotting procedure using [³²P]-probe specific to KOR cDNA (upper left). NGF receptor p75 and neuronal differentiation marker BM88 were also examined on these samples as shown at the bottom. The images shown are the representative of two experiments. UD: Undifferentiated.

Fig. 2

NGF activates P2 through an AP2 site. (A) AP2 site is a functional positive element. The maps of reporter constructs are shown on top: K18 for the full-length P2, K18m for the specific AP2 site mutant of K18, and Kd50 for deletion mutant carrying only the AP2 site. Reporter genes were introduced into P19 cells and luciferase activity as well as internal controls was measured. (B) AP2 site mediates the stimulating effects of NGF. Reporter genes were introduced into P19 cells and luciferase activity as well as internal controls was measured. NGF effect was determined by normalizing the luciferase reading over the internal control for each reporter construct. The data of averages and SDs were collected from three independent experiments. Column NGF *p<0.01, versus No NGF for both constructs.

Fig. 3

NGF enhanced P19 nuclear extract binding to the AP2β site on P2. (A) P19 cells were induced with RA for 3 days and followed by NGF treatment for 1 day. Nuclear extracts were subjected to EMSA using probes prepared from the P2 DNA segment carrying the AP2β site. Arrowhead marks the dimer-binding complex and arrow depicts the monomer-binding complex. Underlined sequences represent core binding sites for the transcription factors. Competition and supershift assays using unlabeled cold probes and antibodies against AP2α, AP2β and HA are shown in lanes 5-10. Experiments were repeated two times. (B) P19 cells were transfected with siRNAs for Tcfap2b and treated with RA and NGF as described above. Isolated RNAs were analyzed by RT-PCR to assess the effectiveness of RNA interference of Tcfap2b. (C) The expression of endogenous KOR c and actin in P19 cells transfected with the Tcfap2b siRNA. Amplified KOR c was detected by Southern blot. siRNA experiments were performed two times.

Fig. 4

NGF enhances recruitment of AP2β to the chromatin of endogenous KOR P2. P19 cells were treated with RA for 3d and followed by NGF stimulation for the indicated periods of time, and were subjected to Western blot analyses to monitor protein expression levels (A), and in ChIP assays (B). DNA precipitated with antibodies against AP2β was amplified with primers specific to KOR P2, depicted with arrows (B, top panel). The images shown were the representative of three experiments.

Fig. 5

NGF signals are transmitted through PI3K to enhance AP2 reporter activity. PC12 cells were transfected with reporter constructs of the full length P2 K18 (A) and AP2 Kd50 (B), and treated with NGF for 24 h, or NGF in the presence of U73122 (U73) or LY294002 (LY29). Each luciferase reading was normalized to the internal control reading. The value of the control (con) group was arbitrarily set at 1 and the value of each experimental group was plotted as fold induction and SD by comparing to the control. The data were collected from three independent experiments. Column NGF *p<0.01 versus control, column LY294002 **p<0.05 versus NGF for both constructs.

Fig. 6

NGF triggers epigenetic changes on P2. P19 cells were induced by RA for 3 days, followed by NGF treatment for 24 h. Cells were subjected to ChIP assays with antibodies against dimethyl Lys9 of histone H3 (H3-K9-me2), dimethyl Lys4 of histone H3 (H3-K4-me2), acetylated histone H4 (AcH4), heterochromatin protein 1α (HP1α) and RNA polymerase II (RNA polII). The images shown were the representative of two experiments.

Fig. 7

A schematic illustration of NGF activation of KOR P2 during RA-induced neuronal differentiation of P19. In the absence of NGF, P2 is in a heterochromatin conformation marked by histone H3 K9 methylation (K9-me2). NGF can stimulate TrkA receptor and p75 NGFR. The p75 NGFR, but not TrkA, is induced in early differentiating P19, resulting in activation of intracellular signaling molecules such as Erk1/2, PLC, and PI3 kinase. In the P19 system, it is PI3 kinase pathway that is responsible for AP2β activation. Erk1/2 pathway is unclear, depicted with a question mark. PLC is not involved in AP2β. Activated AP2β increasingly binds to KOR P2, triggering epigenetic changes reflected by histone H3 K4 methylation (K4-me2), dissociation of HP1α and formation of RNA polII pre-initiation complex. KOR c is then actively transcribed in NGFR-expressing neurons.