Metabolism & Genetics Group, iMed, Faculty of Pharmacy, University of Lisbon, Portugal.
Mol Genet Metab. 2010 Apr;99(4):425-30. doi: 10.1016/j.ymgme.2009.11.002. Epub 2009 Nov 16.
DNA methylation is an important epigenetic modification that has profound roles in gene expression and, in particular, is thought to be crucial for regulation of tissue-specific genes in animal cells. The pivotal E(1)alpha subunit of human pyruvate dehydrogenase complex, an essential and rate-limiting enzyme system in energy metabolism, is encoded by two distinct genes: PDHA1 gene, located on chromosome X is expressed in somatic tissues, whereas PDHA2 gene, located on chromosome 4, is exclusively expressed in spermatogenic cells. The objective of this study is to elucidate the role of DNA methylation as an epigenetic mechanism controlling the regulation of PDHA2 gene expression in human tissues, namely its repression in somatic tissues and its activation in testicular germ cells. Genomic DNA was isolated from human somatic tissues (circulating lymphocytes and gastric cells) and from testis, including isolated fractions of haploid and diploid germ cells. After primer design with appropriate software, it was performed the sodium bisulfite PCR sequencing of the PDHA2 promoter and coding regions. Total RNA of the same tissues was isolated, reverse transcribed and PDHA1and PDHA2 transcripts were amplified with specific primers and analysed by agarose gel electrophoresis. The analysis of the genomic sequence of the PDHA2 gene revealed the presence of 61 CpG sites whose distribution matches the criteria for the presence of two CpG islands. Sequence analysis of both CpG islands upon bisulfite treatment displayed several differences, either between islands or among tissues. In particular, the methylation pattern of one of the CpG islands revealed a perfect correlation with transcriptional activity of the PDHA2 gene either in testis or in somatic tissues. Surprisingly, it is the full demethylation of the CpG island located in the coding region that seems to play a crucial role upon PDHA2 gene transcription in testis.
DNA 甲基化是一种重要的表观遗传修饰，在基因表达中具有深远的作用，特别是被认为在动物细胞中对组织特异性基因的调控至关重要。人丙酮酸脱氢酶复合物的关键 E(1)alpha 亚基是能量代谢中必需的限速酶系统，由两个不同的基因编码：位于 X 染色体上的 PDHA1 基因在体细胞组织中表达，而位于 4 号染色体上的 PDHA2 基因仅在生殖细胞中表达。本研究的目的是阐明 DNA 甲基化为表观遗传机制在调控人组织中 PDHA2 基因表达中的作用，即在体细胞组织中抑制其表达，在睾丸生殖细胞中激活其表达。从人体细胞组织（循环淋巴细胞和胃细胞）和睾丸中分离基因组 DNA，包括单倍体和二倍体生殖细胞的分离部分。用适当的软件设计引物后，对 PDHA2 启动子和编码区进行了亚硫酸氢盐 PCR 测序。从相同组织中分离总 RNA，反转录并使用特异性引物扩增 PDHA1 和 PDHA2 转录本，然后通过琼脂糖凝胶电泳进行分析。PDHA2 基因的基因组序列分析显示存在 61 个 CpG 位点，其分布符合存在两个 CpG 岛的标准。对亚硫酸氢盐处理后的两个 CpG 岛的序列分析显示，无论是在岛屿之间还是在组织之间，都存在一些差异。特别是，一个 CpG 岛的甲基化模式与 PDHA2 基因在睾丸或体细胞组织中的转录活性具有完美的相关性。令人惊讶的是，正是位于编码区的 CpG 岛的完全去甲基化似乎在 PDHA2 基因在睾丸中的转录中起着至关重要的作用。
