Understanding epigenetic mechanisms in tissue-specific gene expression

在人类和小鼠基因组中已经鉴定出20-25,000个蛋白质编码基因，这些基因在组织和发育阶段特异性组合中产生约200,000个转录本。这些转录本可以通过包括选择性剪接（AS）和选择性聚腺苷酸化（APA）在内的共转录前mrna加工机制产生。基于转录组分析的估计显示，约90%的人类转录本经历AS，而至少70%的哺乳动物pre- mrna中存在APA。AS涉及外显子的差异包涵，有时包括内含子，以形成成熟的mRNA。APA是指源自同一基因但在3′端不同的转录本的聚腺苷化。AS和APA都依赖于细胞机制识别的特定序列。APA事件可以发生在3'非翻译区（utr）或基因内位置，这里我们考虑基因内APA。基因内聚腺苷化（IPA）的发生率在不同的组织和细胞类型中有所不同，这为转录组和蛋白质组的多样化提供了一种方法。在全基因组范围内，我们已经鉴定出超过4000个宿主基因在哺乳动物基因组中含有（基因内CpG岛）iCGI，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异性的，我们首次证明了iCGI上游宿主基因转录物的过早终止与iCGI转录水平以dna甲基化独立的方式密切相关。这些研究表明，iCGI转录，而不是组蛋白修饰（如H3K36me3）或DNA甲基化，在全基因组范围内干扰宿主基因转录和前mrna加工，并有助于转录组的时空多样化和调控，从而影响蛋白质组。文献中有证据表明，正确的AS和APA的失败会导致病理状况和癌症。印迹基因是解剖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是由印迹控制区的等位基因特异性DNA甲基化协调的。印迹基因的表达是由遗传等位基因决定的。印迹基因的活性等位基因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位点是研究iCGI活性和活性和沉默等位基因染色质背景的理想模型，本研究将探讨iCGI依赖的内含子APA选择机制。在这里，Mcts2/H13基因座将被荧光标记在神经干细胞中产生一个报告系统。一旦该系统被表征，它将暴露于CRISPR KO筛选，以确定iCGI依赖性内含子APA的调节因子。潜在的候选细胞将在体外神经发生模型中进行验证和敲除，以评估它们在分化过程中的重要性。除了这种实验方法外，该项目的计算链将利用来自特定大脑区域的广泛单细胞RNA-seq数据，利用一个经过充分研究的神经发生模型，专门关注转录物水平，更重要的是，提供同源型特异性分辨率，以进一步了解大脑中AS和APA的转录物多样性潜力和特异性位点鉴定。

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.