Transcription, Chromatin and DNA repair

转录、染色质和 DNA 修复

• 批准号: 8157146
• 负责人: rafael c casellas
• 金额: $ 116.72万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157146
• 关键词: Chromatin; DNA Repair; Genetic Transcription

The initiator factor of hypermutation and recombination is AID, a B cell specific enzyme that is only expressed in B lymphocytes directly involved in the immune response. Because of AIDs link to tumor development, B cells have developed a variety of mechanisms that curtail its activity in vivo. One prominent mechanism by which AID is regulated is via microRNA activity. microRNAs (miRNAs) are non-coding small RNAs that modulate the cells proteome by annealing to 3-unstranslated regions of cognate mRNAs and inhibiting protein translation and/or promoting mRNA instability. Since their discovery in C. elegans 16 years ago, miRNA orthologs and paralogs have been described in a variety of species, suggesting these regulatory RNAs are involved in basic cellular functions across the phyla. In the mammalian genome, microRNAs are encoded within introns of protein-coding genes or as independent entities transcribed either by RNA polymerase II or RNA polymerase III. In some instances, groups of miRNAs are also clustered and processed from a single transcript. Due to the their palindromic nature, miRNAs in nascent primary transcripts (pri-miRNAs) display a characteristic stem-loop structure that in the nucleus is recognized and cleaved by the Drosha/DGCR8 complex into 60-70 nt precursor (pre) miRNAs. Once in the cytoplasm, pre-miRNAs are further processed by the RNase III endonuclease DICER into mature RNA fragments of 22 nt in length, which are loaded into the RNA-silencing complex RISC. Partial sequence complementary between the 5 end of the mature miRNA (6-8 nt seed region) and its target mRNA leads to downregulation of protein expression. As is the case for non-hematopoietic tissues, lymphocytes and other cells of the immune system rely on miRNAs to effect lineage commitment, proliferation, migration, and differentiation. In most cases, these activities are orchestrated by both ubiquitously expressed and hematopoietic specific miRNA species, which upon deletion or overexpression impair the immune system at various levels. In like manner, conditional ablation of DICER or other miRNA processing factors results in a profound block of both B and T cell development. In apparent contrast to these striking phenotypes, it is notable that miRNAs control hematopoiesis through small changes in the cellular concentration of key factors. In the B cell compartment for instance, mir150 curtails c-Myb activity in a dose-dependent fashion over a narrow range of miRNA and c-Myb concentrations. Similarly, mass action seems to be the underlying principle behind mir155 regulation of AID, or mir17-92-mediated inhibition of PTEN and Bim proteins. In retrospect, the graded miRNA activity shown in these examples provides a rationale to the happloinsufficiency previously observed in AID, cMyb, PTEN, and Bim heterozygous mice. This basic principle however is not restricted to the mammalian immune system as data from a wide variety of experimental systems increasingly demonstrate that the absolute concentration of miRNAs is crucial at managing a cells proteome. Yet, how miRNA levels are controlled upon differentiation of specific cell lineages remains to be explored. One major limitation in measuring miRNA abundance during ontogeny has been the lack of quantitative, systematic approaches to profile small RNAs with high accuracy. In a manuscript in preparetion, we have used massive parallel sequencing to monitor miRNA changes in a large number of B and T cell developmental stages. To define lymphocyte-specific miRNA signatures, the microRNome of other hematopoietic lineages and samples representing diverse mouse cells and tissues was also characterized. Furthermore, by means of genome-wide chromatin immunoprecipitation (ChIP-seq) and high-throughput mRNA sequencing (RNA-seq), we have assessed the epigenetic status of miRNA genes concomitant with pri-miRNA expression during lymphopoiesis. This approach provided a comprehensive view of the epigenetic, transcriptional, and indirectly, posttranscriptional mechanisms shaping miRNA cellular concentration. The data partitioned miRNAs into distinct subsets. Epigenetically, the great majority of lymphocyte-specific miRNAs, which are silenced outside the immune system, were found to be H3K27 demethylated in hematopoietic stem cells (HSCs). Demethylation however did not necessarily promote pri-miRNA gene transcription, which in most cases was only fully induced in subsequent T or B cell developmental stages along with dramatic increases in acetylating of histone H3K36 and H3K14, and methylation of H3K36 and H3K79. In contrast to this large group, expression of a small minority of miRNAs were more tightly regulated as they maintained H3K27me3 up until full gene transcription was induced. In the context of gene expression per se, our data shows that up to one quarter of mature miRNAs mirror the relative expression profiles of pri-miRNA transcripts during differentiation. Finally, we provide evidence suggesting deviations in pri-miRNA/miRNA expression profiles can be attributed at least in part to target mRNA levels, which seem to directly influence cellular concentration of cognate miRNAs. Altogether, our findings shed light on the mechanisms that fine-tune expression and cellular abundance of miRNAs during lymphocyte differentiation. The quantitative microRNome profiles from a large number of mouse cells and tissues, the discovery of 19 novel, phylogenetically conserved miRNAs, and the systematic classification of miRNAs based on their epigenetic and transcriptional regulation constitute an invaluable resource for a wide range of future miRNA studies.

超突变和重组的起始因子是AID，一种仅在直接参与免疫应答的B淋巴细胞中表达的B细胞特异性酶。由于艾滋病与肿瘤的发展有关，B细胞已经发展出多种机制来限制其在体内的活性。调节AID的一个突出机制是通过microRNA活性。 microRNA（miRNAs）是一类非编码小分子RNA，通过与同源mRNA的3-未翻译区退火，抑制蛋白质翻译和/或促进mRNA不稳定性，从而调节细胞蛋白质组。自从C. 16年前，在许多物种中已经描述了miRNA的直系同源物和旁系同源物，这表明这些调控RNA参与了整个门的基本细胞功能。 在哺乳动物基因组中，微小RNA编码在蛋白质编码基因的内含子内，或作为由RNA聚合酶II或RNA聚合酶III转录的独立实体。在一些情况下，miRNA组也从单个转录物成簇和加工。由于它们的回文性质，新生初级转录物（pri-miRNAs）中的miRNAs显示出特征性的茎环结构，其在细胞核中被Drosha/DGCR 8复合物识别并切割成60-70 nt前体（pre）miRNAs。一旦在细胞质中，pre-miRNA被RNase III内切核酸酶DICER进一步加工成长度为22 nt的成熟RNA片段，其被加载到RNA沉默复合物RISC中。成熟miRNA的5端（6-8 nt种子区）与其靶mRNA之间的部分序列互补导致蛋白质表达下调。 与非造血组织的情况一样，免疫系统的淋巴细胞和其他细胞依赖于miRNA来实现谱系定型、增殖、迁移和分化。在大多数情况下，这些活动是由普遍表达的和造血特异性的miRNA种类协调的，其在缺失或过表达时在不同水平上损害免疫系统。以类似的方式，DICER或其他miRNA加工因子的条件性切除导致B和T细胞发育的深度阻断。与这些引人注目的表型形成明显对比的是，值得注意的是，miRNA通过关键因子的细胞浓度的微小变化来控制造血。例如，在B细胞区室中，mir 150在小范围的miRNA和c-MyB浓度内以剂量依赖性方式减少c-MyB活性。类似地，质量作用似乎是mir 155调节AID或mir 17 -92介导的抑制PTEN和Bim蛋白背后的基本原理。回顾过去，这些实施例中显示的分级的miRNA活性为先前在AID、cMyb、PTEN和Bim杂合小鼠中观察到的突变不足提供了理论基础。然而，这一基本原理并不局限于哺乳动物免疫系统，因为来自各种实验系统的数据越来越多地表明，miRNA的绝对浓度在管理细胞蛋白质组方面至关重要。然而，miRNA水平如何在特定细胞谱系分化时被控制仍有待探索。 在个体发育过程中测量miRNA丰度的一个主要限制是缺乏定量的、系统的方法来高精度地分析小RNA。在《自然》杂志的一篇文章中，我们使用大规模平行测序来监测大量B和T细胞发育阶段中miRNA的变化。为了定义淋巴细胞特异性miRNA特征，还表征了代表不同小鼠细胞和组织的其他造血谱系和样品的microRNome。此外，通过全基因组染色质免疫沉淀（ChIP-seq）和高通量mRNA测序（RNA-seq），我们评估了淋巴细胞生成过程中伴随pri-miRNA表达的miRNA基因的表观遗传状态。这种方法提供了一个全面的观点的表观遗传，转录，并间接，转录后机制塑造miRNA细胞浓度。这些数据将miRNAs划分为不同的子集。在表观遗传学上，发现绝大多数在免疫系统外沉默的淋巴细胞特异性miRNA在造血干细胞（HSC）中被H3 K27去甲基化。然而，去甲基化不一定促进pri-miRNA基因转录，在大多数情况下，其仅在随后的T或B细胞发育阶段沿着组蛋白H3 K36和H3 K14的乙酰化以及H3 K36和H3 K79的甲基化的显著增加而被完全诱导。与这一大组相比，一小部分miRNA的表达受到更严格的调控，因为它们维持H3 K27 me 3直到诱导完整的基因转录。在基因表达本身的背景下，我们的数据显示，多达四分之一的成熟miRNA反映了分化期间pri-miRNA转录本的相对表达谱。最后，我们提供的证据表明pri-miRNA/miRNA表达谱的偏差至少部分归因于靶mRNA水平，这似乎直接影响同源miRNA的细胞浓度。 总之，我们的发现揭示了淋巴细胞分化过程中微调miRNAs表达和细胞丰度的机制。来自大量小鼠细胞和组织的定量microRNome图谱，19种新的、遗传学上保守的miRNA的发现，以及基于其表观遗传和转录调控的miRNA的系统分类，构成了未来广泛的miRNA研究的宝贵资源。