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. 2012 Feb 3;287(6):4000-13.
doi: 10.1074/jbc.M111.300194. Epub 2011 Dec 15.

Selective roles for cAMP response element-binding protein binding protein and p300 protein as coregulators for androgen-regulated gene expression in advanced prostate cancer cells

Irina Ianculescu  1 , Dai-Ying Wu, Kimberly D Siegmund, Michael R Stallcup
Affiliations

Selective roles for cAMP response element-binding protein binding protein and p300 protein as coregulators for androgen-regulated gene expression in advanced prostate cancer cells

Irina Ianculescu et al. J Biol Chem. .

Erratum in

  • J Biol Chem. 2012 Oct 19;287(43):35985

Abstract

The protein acetyltransferases p300 and cAMP response element-binding protein binding protein (CBP) are homologous, ubiquitously expressed proteins that interact with hundreds of proteins involved in transcriptional regulation and are involved globally as transcriptional coregulators. Although these two proteins acetylate and interact with overlapping sets of proteins, we found that p300 and CBP contribute to androgen-induced regulation of distinct sets of genes in C4-2B prostate cancer cells, a model of advanced prostate cancer. CBP cannot compensate for the loss of p300 to support androgen-induced expression of many genes, such as TMPRSS2 and PSA. Global gene expression analysis indicated that 47% of androgen-regulated genes are p300-dependent in these cells, whereas, surprisingly, only 0.3% of them are CBP-dependent. Chromatin immunoprecipitation analysis after depletion of cellular p300 indicated that p300 is required for androgen-induced acetylation of histones H3 and H4, methylation of histone H3 at Lys-4, and recruitment of TATA box binding protein (TBP) and RNA polymerase II, but not recruitment of the androgen receptor, on the TMPRSS2 gene in response to androgen. Thus, p300 is the dominant coregulator of the CBP/p300 pair for androgen-regulated gene expression in C4-2B cells. p300 is required at an early stage of chromatin remodeling and transcription complex assembly after binding of androgen receptor to the gene but before many critical histone modifications occur.

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Figures

FIGURE 1.
FIGURE 1.
Effects of p300 and CBP depletion on global and DHT-induced gene expression in C4-2B cells. A, qRT-PCR was performed on RNA extracted from cells transfected with siRNA against p300, CBP, or a nonspecific (NS) sequence. Cells were treated with ethanol (EtOH) or 10 nm DHT for 16 h before harvest. Levels of mRNA are shown relative to 18 S rRNA and represent the mean ± S.D. of three technical PCR replicates. B, whole cell extracts from cells transfected with siRNA were analyzed by immunoblot using antibodies against p300, CBP, or tubulin as a control. C, total RNA was extracted from cells transfected with siRNA and treated with DHT or ethanol as in A. Four independent experiments of this type were conducted on different days, and the 24 resulting RNA samples were analyzed on Illumina microarrays. The Venn diagram for the hormone-regulated gene set shows the number of genes with significantly different expression (q ≤ 0.05) for RNA from cells transfected with nonspecific siRNA and then treated with ethanol versus DHT. The Venn diagram for the p300-regulated gene set shows the number of genes with significantly different expression (q ≤ 0.05) for RNA from DHT-treated cells that had been transfected with nonspecific siRNA versus siRNA against p300. The Venn diagram for the CBP-regulated gene set shows the number of genes with significantly different expression (q ≤ 0.1) for RNA from DHT-treated cells that had been transfected with nonspecific siRNA versus siRNA against CBP. Overlap areas indicate the number of genes belonging to two or all three of the three gene sets. The table on the right depicts the number of genes up- or down-regulated with DHT and the number that are either up-regulated (↑), down-regulated (↓), or unchanged ( =) with p300 knockdown. The complete lists of genes for the three major sets in the Venn diagram are found in supplemental Tables 3–5. D, Illumina microarray results for the genes which were DHT-regulated and CBP-dependent. For each gene, expression is given relative to cells transfected with siNS and treated with ethanol and is plotted as the mean ± S.D. of four independent experiments.
FIGURE 2.
FIGURE 2.
Effect of p300 and CBP depletion on androgen-regulated expression of specific genes. A–E, qRT-PCR was performed on total RNA extracted from C4-2B cells transfected with siRNA against p300, CBP, both p300 and CBP, or a nonspecific (NS) sequence. Cells were treated either with ethanol (EtOH) or 10 nm DHT for 16 h. Levels of mRNA are shown relative to 18 S rRNA. Results shown are mean ± S.D. of three replicate PCR analyses of RNA samples from a single experiment. *, p < 0.05 compared with the siNS/DHT sample in the same graph, calculated using a paired Student's t test from at least three biological replicates.
FIGURE 3.
FIGURE 3.
Effect of p300 and CBP depletion on the time course of DHT-induced increases in pre-mRNA and mRNA from AR target genes. A–C, qRT-PCR with primers specific for pre-mRNA was performed on total RNA extracted from C4-2B cells transfected with siRNA against nonspecific, p300, or CBP and then treated for 0, 4, 8, or 16 h with 10 nm DHT before harvesting. cDNA was made with random hexamers as primers. The pre-mRNA levels are calculated relative to 18 S rRNA, and each value is the mean ± S.E. for three biological replicates. D–F, the same cDNA samples described in A–C were analyzed by qPCR with primers specific for mature mRNA. The mRNA levels are calculated relative to 18 S rRNA, and each value is the mean ± S.E. for three biological replicates.
FIGURE 4.
FIGURE 4.
DHT-induced histone modifications and transcription complex assembly on the TMPRSS2 gene. A, diagram of the regulatory region of the TMPRSS2 gene showing five putative AREs (circles) and their locations measured in bp relative to the TSS (arrow). ARE V is the main site of AR recruitment. B–E, after growth in hormone-free media for 3 days, C4-2B cells were treated with ethanol (EtOH) or 10 nm DHT for 4 h, and ChIP was performed with the indicated antibodies or no antibody (-ab) as a control, using primers for the indicated region of the TMPRSS2 gene. Each graph shows a single biological experiment that is representative of at least two independent biological experiments. Immunoprecipitated DNA was analyzed relative to input DNA (before immunoprecipitation), and values shown are mean ± S.D. of three technical qPCR replicates, using primers specific for the indicated region.
FIGURE 5.
FIGURE 5.
DHT-induced histone modifications and transcription complex assembly on the FKBP5 gene. A, diagram of the regulatory region of the FKBP5 gene showing putative AREs (circles) and their locations measured in bp relative to the TSS (arrow). ARE VIII/IX is the main site of AR recruitment. B–C, ChIP was performed with C4-2B cells treated with ethanol or DHT for 4 h as in Fig. 4.
FIGURE 6.
FIGURE 6.
Effect of p300 and CBP depletion on DHT-induced histone modifications and transcription complex assembly on the TMPRSS2 gene. A–D, C4-2B cells were transfected with siRNA against p300 (A and C) or CBP (B and D), and cells were treated with ethanol or DHT for 4 h, prior to ChIP analysis performed as in Fig. 4. Each graph shows a single biological experiment that is representative of at least two independent biological experiments. Immunoprecipitated DNA was analyzed relative to input DNA (before immunoprecipitation), and values shown are mean ± S.D. of three technical qPCR replicates, using primers specific for the indicated region.
FIGURE 7.
FIGURE 7.
Effect of p300 depletion on DHT-induced histone modifications and transcription complex assembly on the FKBP5 gene. A–B, C4-2B cells were transfected with siRNA against p300, and cells were treated with ethanol or DHT for 4 h, prior to ChIP analysis performed as in Fig. 4. Each graph shows a single biological experiment that is representative of at least two independent biological experiments. Immunoprecipitated DNA was analyzed relative to input DNA (before immunoprecipitation), and values shown are mean ± S.D. of three technical qPCR replicates, using primers specific for the indicated region.
FIGURE 8.
FIGURE 8.
Model of a p300-dependent AR target gene in intact and p300-depleted conditions. A, in intact cells DHT causes binding of AR to the ARE, which results in recruitment of p300 and a variety of other coactivators including p160 (or SRC) coactivators. p300 and other recruited histone acetyltransferases increase the acetylation levels of histones H3 and H4 (ac) near the AR binding site and the TSS. Subsequent events include increases in the level of H3K4me1 (K4me1) near the AR binding site and in the level of H3K4me3 (K4me3) near the TSS, recruitment of the basal transcription machinery including TBP and RNA polymerase II (RNA pol II), and enhanced transcription from the TSS (arrow). B, in the absence of p300, DHT still induces binding of AR to the ARE, and some coactivators (represented here by the p160 coactivator) may still be recruited normally to the promoter. However, acetylation of histones H3 and H4 and methylation of H3K4 are not enhanced by DHT; TBP and RNA polymerase II are not recruited to the TSS; and transcription enhancement by DHT is inhibited or eliminated (red barred circle covering the transcription arrow at the TSS).
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