Understanding epigenetic mechanisms in tissue-specific gene expression

在人类和小鼠基因组中已经确定了20-25,000个蛋白质编码基因，这些基因在组织和发育阶段特定的组合中产生了大约200,000个转录本。这些转录本可以通过共转录的前mRNA处理机制产生，其中包括选择性剪接(AS)和选择性多聚腺苷酸化(APA)。基于转录组分析的估计表明，~90%的人类转录本经历了AS，而APA至少存在于70%的哺乳动物前mRNAs中。AS涉及外显子和有时内含子的差异包涵体，以形成成熟的mRNA。APA是指起源于相同基因但3‘端不同的转录本的聚腺苷酸化。AS和APA都依赖于细胞机制识别的特定序列。APA事件既可以发生在3‘非翻译区，也可以发生在基因内，这里我们称之为基因内APA。基因内多聚腺苷酸化(IPA)的发生率因组织和细胞类型而异，提供了一种使转录组和蛋白质组多样化的方法。在整个基因组范围内，我们已经在哺乳动物基因组中发现了4000多个含有ICGI(基因内CpG岛)的宿主基因，包括新的ICGI/宿主基因对。这些iCGI的转录活性是组织和发育阶段特有的，我们首次证明了iCGI上游宿主基因转录的提前终止与ICGI转录水平密切相关，这种关系不依赖于DNA甲基化。这些研究表明，ICGI转录，而不是组蛋白修饰(如H3K36me3)或DNA甲基化，干扰了宿主基因的转录和全基因组的前mRNA加工，并有助于转录组的时空多样化和调节，影响蛋白质组。文献中有证据表明，正确的AS和APA不能导致病理状况和癌症。印迹基因是剖析表观遗传基因表达调控的特别有用的模型。在小鼠和人类中，大约有130个基因受到基因组印记的影响。这些基因的单等位基因表达通过印记控制区的等位基因特异性DNA甲基化来协调。印记基因的表达由遗传等位基因决定。印迹基因的活跃和沉默等位基因共享相同的DNA序列，存在于相同的细胞环境中，因此基因表达的等位基因差异是等位基因之间表观遗传差异的结果。印迹Mcts2/H13基因座是研究ICGI活性和染色质背景对活跃和沉默等位基因的理想模型，本研究将剖析ICGI依赖内含子APA选择的机制。在这里，Mcts2/H13基因座将被标记在神经干细胞中，以产生一个报告系统。一旦该系统被表征，它将被暴露在CRISPR KO屏幕上，以识别ICGI依赖的内含子APA的调节器。潜在的候选者将在体外神经发生模型中得到验证和剔除，以评估他们在分化过程中的重要性。除了这种实验方法，该项目的一个计算链将被执行，以利用来自特定大脑区域的广泛的单细胞RNA-SEQ数据，以利用一个研究得很好的神经发生模型来专门关注转录水平，更重要的是，提供特定于异构体的分辨率，以进一步了解大脑中的AS和APA在转录多样性潜力和特定位点识别方面的作用。

Between 20-25,000 protein coding genes have been identified in the human and mouse genomes that give rise to ~200,000 transcripts in tissue- and developmental stage-specific combinations. These transcripts can be generated via co-transcriptional pre-mRNA processing mechanisms that include alternative splicing (AS) and alternative polyadenylation (APA). Estimates based on transcriptome analyses reveal that ~90% of human transcripts undergo AS and that APA occurs in at least 70% of mammalian pre-mRNAs. AS involves the differential inclusion of exons and sometimes introns to form the mature mRNA. APA refers to the polyadenylation of transcripts originating from the same gene but that differ in their 3' end. Both AS and APA are dependent on specific sequences recognised by the cellular machinery. APA events can occur either at 3' untranslated regions (UTRs) or intragenic locations, here we consider intragenic APA. The incidence of intragenic polyadenylation (IPA) varies across tissues and cell types providing a way to diversify both the transcriptome and the proteome. Genome-wide, we have identified over 4000 host genes that harbour an (intragenic CpG island) iCGI in the mammalian genome, including novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrated that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than histone modification (eg H3K36me3) or DNA methylation, interfere with host gene transcription and pre-mRNA processing genome-wide and contribute to the spatiotemporal diversification and regulation of the transcriptome, impacting proteome. There is evidence in the literature for the failure of correct AS and APA to result in pathological conditions and cancer. Imprinted genes are particularly useful models for the dissection of epigenetic gene expression regulation. There are ~130 genes in mouse and human that are subject to genomic imprinting. Monoallelic expression of these genes is coordinated by allele-specific DNA methylation of imprinting control regions. The expression of imprinted genes is determined by the inherited allele. The active and silent alleles of imprinted genes share the same DNA sequence and are present within the same cellular environment, so that allelic differences in gene expression are the consequence of epigenetic differences between the alleles. The imprinted Mcts2/H13 locus is an ideal model in which to study iCGI activity and chromatin context on active and silent alleles and this study will dissect the mechanism of iCGI dependent intronic APA choice. Here, the Mcts2/H13 locus will be tagged in neural stem cells with fluorescent markers to generate a reporter system. Once this system has been characterised, it will be exposed to a CRISPR KO screen to identify regulators of iCGI dependent intronic APA. Potential candidates will be validated and knocked out in an in vitro neurogenesis model to assess their essentiality during differentiation. Alongside this experimental approach, a computational strand of the project will be performed to take advantage of extensive single cell RNA-seq data from specific brain regions to leverage a well-studied neurogenesis model to specifically focus on transcript levels and more importantly, provide isoform-specific resolution to further understand AS and APA in the brain in terms of transcript diversity potential and specific locus identification.