Exploring the Stability of Genomic Imprinting and X-Chromosome Inactivation in the Aged Brain (RNA-Seq)

DNA methylation is a common mechanism regulating monoallelic expression of different genes, including imprinted genes. How age-induced alterations of methylation levels vary across alleles and impact on monoallelic expression is unknown. In the brain, this phenomenon may contribute mechanistically to neuronal dysfunction and disease. In order to investigate the allele specific transcriptional and epigenetic signatures of aging, we used key brain areas, hippocampus and cerebellum, of juvenile and old hybrid mice obtained from BL6CAST/EiJ (CAST) reciprocal crosses. We set out to explore whether epigenetic drift affects global DNA methylation machinery by investigating 5- methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels. Mass spectrometry analysis showed a significant increase of 5hmC levels in hippocampus of old mice, but this was not accompanied by measurable differences in methylating and demethylating enzymes. The expression of several repetitive elements (LINEs, SINEs, IAP) was also analysed, strictly dependent on 5mC and 5hmC levels, and reporting a reduction of the intracisternal A-particle (IAP) retrotransposons in HCP of old mice. We uncovered the allelic-specific DNA methylation profile of aging using genomic imprinting as a read-out by IMPLICON and allelic expression by RNA sequencing. Here, imprinting is not substantially changed during aging, not affecting their normal monoallelic expression. This work provides the first epigenetic and transcriptional landscape of aging with allelic resolution and confirms that genomic imprinting is a stable epigenetic phenomenon across the whole lifespan. We used key brain areas, mostly focused in hippocampus (HCP) and cerebellum (CB), of juvenile and old hybrid mice obtained from BL6CAST reciprocal crosses. We set out to explore whether epigenetic drift affects global DNA methylation machinery by investigating 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels by Liquid Chromatography with tandem mass spectrometry and the genes enconding for enzymes implicated in this process by RT-PCR and RNAseq, the later in HCP only . The expression of several repetitive elements (LINEs, SINEs, IAP) was also analysed by RTq-PCR and also by RNAseq in HCP . We uncovered the allelic-specific DNA methylation profile of aging using genomic imprinting as a read-out by IMPLICON in several brain areas such as hippocampus (HCP), cerebellum (CB), prefrontal cortex (PFC), hypothalamus (HY), nucleus accumbens (NA) and a control region such as lung. Allelic expression analysis by RNA sequencing was performed in HCP.DNA甲基化是调控包括印记基因(imprinted genes)在内的多种基因单等位基因表达的常见表观遗传机制。年龄诱导的甲基化水平改变如何在不同等位基因间产生差异，以及其对单等位基因表达的影响尚不明确。在大脑中，这一现象可能从机制层面参与神经元功能障碍与疾病的发生发展。为探究衰老的等位基因特异性转录组与表观遗传特征，我们使用了源自BL6CAST/EiJ(CAST)正反交的幼年与老年杂交小鼠的关键脑区——海马体(hippocampus, HCP)与小脑(cerebellum, CB)。我们旨在通过检测5-甲基胞嘧啶(5-methylcytosine, 5-mC)与5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5-hmC)水平，探究表观遗传漂移是否会影响全局DNA甲基化调控机制。质谱分析结果显示，老年小鼠海马体中的5-hmC水平显著升高，但这一变化并未伴随甲基化与去甲基化酶水平的可检测差异。我们还分析了多种重复元件：长散在核元件(LINEs)、短散在核元件(SINEs)、内质网A型颗粒(IAP)的表达，这类元件的表达严格依赖于5-mC与5-hmC水平；分析结果显示，老年小鼠海马体中的内质网A型颗粒(IAP)逆转录转座子表达水平出现降低。我们通过IMPLICON技术以基因组印记(genomic imprinting)作为读出指标，结合RNA测序分析等位基因表达情况，揭示了衰老的等位基因特异性DNA甲基化特征。本研究中，基因组印记在衰老过程中并未发生显著改变，未影响其正常的单等位基因表达模式。本工作首次提供了具有等位基因分辨率的衰老表观遗传与转录组图谱，并证实基因组印记是贯穿整个生命周期的稳定表观遗传现象。我们使用了源自BL6CAST正反交的幼年与老年杂交小鼠的关键脑区，主要聚焦于海马体(hippocampus, HCP)与小脑(cerebellum, CB)。我们通过液相色谱-串联质谱法检测5-mC与5-hmC水平，并通过逆转录PCR(RT-PCR)与RNA测序(仅在海马体中开展)分析参与该调控过程的酶编码基因，以此探究表观遗传漂移是否影响全局DNA甲基化调控机制。我们还通过实时定量逆转录PCR(RT-qPCR)与RNA测序(仅在海马体中开展)分析了多种重复元件：长散在核元件(LINEs)、短散在核元件(SINEs)、内质网A型颗粒(IAP)的表达。我们通过IMPLICON技术在多个脑区——包括海马体(HCP)、小脑(CB)、前额叶皮层(prefrontal cortex, PFC)、下丘脑(hypothalamus, HY)、伏隔核(nucleus accumbens, NA)以及对照组织肺——中，以基因组印记作为读出指标，揭示了衰老的等位基因特异性DNA甲基化特征。同时，我们在海马体中通过RNA测序完成了等位基因表达分析。