Investigation of Promoter Sequence-Function Relationships in a Model Retrovirus

描述(申请人提供)：真核基因表达受到多种机制的调控，包括启动子内关键转录因子结合位点(TFBs)的身份、精确序列和结构安排，以及它的基因组环境。例如，一旦逆转录病毒将它们的基因组整合到宿主细胞基因组的一个半随机位置，它们就利用高度多样化的启动子序列来整合特定整合部位的遗传和表观遗传输入，以启动病毒基因的表达和复制。然而，考虑到人类基因组中调控序列和基因组环境的多样性，理解这样一个启动子如何将一些调控输入转化为mRNA表达的时间模式是非常具有挑战性的。因此，我们建议应用系统生物学的方法来研究一个重要且高度可变的启动子--人类免疫缺陷病毒(HIV-1)长末端重复序列(LTR)的性质，以阐明启动子序列和基因组环境中的广泛多样性调节基因表达动态和复制的原理，这项工作将产生对转录调控的新的定量见解，并可能有助于未来增强治疗的设计。与许多病毒一样，艾滋病毒的两个基本特征使其难以治疗，一是它的进化，二是它有能力建立一个潜在的、不活跃的群体。在这项工作的第一阶段，我们开发了B亚型HIV基因表达和潜伏期的实验和计算模型，通过在单细胞和种群水平上的定量测量将其联系起来。特别是，我们发现，整合位置子集上的基因表达的随机效应可能会导致高度“嘈杂”的基因表达动态，这可能会影响病毒的复制和潜伏期。然而，虽然B亚型艾滋病毒的实验室毒株是研究最广泛的，但由于其进化速度非常快，艾滋病毒在单个患者体内产生高度可变的序列，这一过程在全球范围内积累了多年，产生了不同的艾滋病毒亚型，在结构上具有刻板印象的差异，包括在LTR中。很明显，LTR序列的变化会影响病毒生命周期的许多方面--包括基因表达、复制和可能的毒力--我们现在建议对这个非常重要的哺乳动物启动子的序列-功能关系有更深入的了解。特别是，我们假设宿主TFBS的不同结构和染色质环境以可预测的方式相互作用来控制病毒LTR的基因表达动态，并且能够做出这种预测的模型可以被用来预测含有合成启动子和自然/临床分离启动子的病毒的基因表达行为。因此，这项拟议的工作将对哺乳动物基因调控机制产生独特的、定量的见解，在一个对人类疾病至关重要的系统中。 公共卫生相关性：这项建议的中心目标是应用一种综合的实验和计算方法，以更深入地了解人类重要启动子--人类免疫缺陷病毒长末端重复序列的序列和结构之间的基本关系，以及它的基因表达特性和功能。这项工作对基因调控的基本机制以及潜在的下游生物医学应用具有重要意义。

DESCRIPTION (provided by applicant): Eukaryotic gene expression is regulated by numerous mechanisms, including the identities, precise sequences, and architectural arrangements of key transcription factor binding sites (TFBSs) within a promoter, as well as its genomic environment. For example, once retroviruses integrate their genomes into a semi- random location of the host cell genome, they utilize a highly diverse promoter sequence to integrate the genetic and epigenetic inputs at a particular integration site to initiate viral gene expression and replication. Given the diversity in regulatory sequences and genomic environments in the human genome, however, it is highly challenging to understand how such a promoter transforms a number of regulatory inputs into a temporal pattern of mRNA expression. We thus propose to apply a systems biology approach to investigate the properties of an important and highly variable promoter, the Human Immunodeficiency Virus (HIV-1) long terminal repeat (LTR), to elucidate principles by which broad diversity in promoter sequence and genomic environment regulate gene expression dynamics and replication, work that will yield new quantitative insights into transcriptional regulation and that may aid in the future design of enhanced therapeutics. The two fundamental features of HIV that render it difficult to treat are, like many viruses, its evolution and it ability to establish a latent, inactive population. In the first phase of this work, we developed experimental and computational models of subtype B HIV gene expression and latency, linked through quantitative measurements at the single cell and population level. In particular, we found that stochastic effects in gene expression at a subset of integration positions could lead to highly "noisy" gene expression dynamics that may influence viral replication and latency. However, while laboratory strains of subtype B HIV are the most broadly studied, due to its very rapid rate of evolution, HIV generates highly variable sequences within an individual patient, and this process has accumulated over years at a global scale to yield diverse HIV subtypes with stereotypical differences in architecture, including in the LTR. It is clear that changes in LTR sequence impact numerous aspects of the viral life cycle - including gene expression, replication, and likely virulence - and we now propose to develop deeper insights into the sequence-function relationships of this highly important mammalian promoter. In particular, we hypothesize that different architectures of host TFBSs and chromatin environment interact in predictable ways to control gene expression dynamics of the viral LTR and that models capable of making such predictions can be formulated to predict gene expression behavior of virus containing both synthetic and natural/clinically isolated promoters. The proposed work will thus yield unique, quantitative insights into mechanisms of mammalian gene regulation, in a system that is of fundamental importance to human disease. PUBLIC HEALTH RELEVANCE: The central goal of this proposal is to apply an integrated experimental and computational approach to gain deeper insights into the basic relationship between the sequence and architecture of an important human promoter, the Human Immunodeficiency Virus Long Terminal Repeat, and its gene expression properties and functions. This work has implications for basic mechanisms of gene regulation, as well as potential downstream biomedical applications.