Epigenetic regulation of caspase-3 gene expression in rat brain development

Abstract

The expression levels of caspase-3, a major contributor to the execution of neuronal apoptosis, markedly decrease in the process of brain maturation. We have previously cloned the rat caspase-3 gene promoter and identified its essential regulatory elements. In the present study, we extended previous findings by examining transcriptional regulation of caspase-3 expression in the rat brain of two different ages, corresponding to the immature and mature brain. In particular, we determined that the rate of transcription initiation substantially declines during brain maturation. Furthermore, we established that mRNA levels of Ets1, Ets2, and Sp1 do not change in the brain with maturation, suggesting that these transcription factors do not contribute to age-dependent caspase-3 down-regulation. Hence, we examined a role of DNA methylation and histone modification in this process. Utilizing bisulfite DNA sequencing, we determined the presence of age-dependent differentially methylated fragments within the caspase-3 promoter region. Strikingly, differentially methylated CpG sites correspond to the predicted binding sites for a number of transcription factors that have been previously shown to be involved in neuronal development and differentiation. Moreover, using chromatin immunoprecipitation, we found that mature brains displayed significantly lower levels of histone 3 acetylated Lys14 and histone 4 acetylated Lys5, 8, 12, and 16. This observation is consistent with the decreased level of expression of caspase-3 in the mature brain. Together with our observation that histone deacetylase inhibitor, trichostatin A, increased the level of caspase-3 mRNA in cortical neurons in vitro, these results further indicate an important role of epigenetic factors in the regulation of caspase-3 gene expression.

Figure 1

Transcription rate of caspase-3 in the rat brain at P2 and P20. Transcription initiation was measured using the nuclear run-on assay as described in Methods. A. Agarose gel electrophoresis of the PCR products for caspase-3 and GAPDH from individual samples (n=3 for each age group). B. Quantification of caspase-3 RNA synthesis at P2 and P60. Arbitrary units (Arb.U.) represent mean ratios of caspase-3-to-GAPDH electrophoretic band optical densities ± SEM (n=3).

Figure 2

Assessment of mRNA levels of Ets-1, Ets-2, and Sp1 at different stages of rat brain development: embryonic (E) day 17, postnatal (P) day 2, P7, P14, and P60. mRNA levels were measured by semi-quantitative RT-PCR and quantified as described in Methods. Arbitrary units represent mean ratios of gene-to-GAPDH electrophoretic band optical densities ± SEM (n=3).

Figure 3

A. Comparison of the frequency and distribution of methylated CpGs in the caspase-3 promoter from newborn (P2) and adult (P60) rat brain. Open circles indicate unmethylated CpG dinucleotides. Closed circles indicate methylated CpG. Asterisks indicate positions of the age-dependent differentially-methylated nucleotides within the caspase-3 promoter. B. Predicted transcription factor binding sites within the caspase-3 promoter are indicated in parentheses.

Figure 4

Acetylation and methylation status of histones 3 and 4 within the caspase-3 promoter at P2 and P60. ChIP was performed as described in Methods using antibodies against histone 3 trimethylated at lysine 4 (H3me3K4), dimethylated at Lys 4 (H3me2K4), dimethylated at Lys 9 (H3me2K9), acetylated at Lys9 and Lys14 (H3ac), acetylated at Lys14 (H3acK14) and against histone H4 acetylated at Lys5, Lys8, Lys12 and Lys16 (H4ac). Real-time PCR of the caspase-3 promoter region was performed as described in Methods. The ratio of PCR product obtained from immunoprecipitated DNA (ChIP DNA) to the amount of PCR product obtained from total chromatin before immunoprecipitation (input DNA) was calculated. Data are presented as percentage of the P2 levels. Each value represents the average of three chromatin preparations ± SEM. Asterisks indicate significant difference from P2 (p<0.05).