Using Single-Cell Data to Decipher Mechanisms of NF-kB-chromatin-mediated HIV Transcriptional Regulation

遗传上相同的细胞由于基因表达的随机波动（噪声）而表现出显著的表型异质性，这是由转录因子结合和每个基因启动子上的染色质修饰所调节的。染色质和转录因子的相互作用在哺乳动物逆转录病毒中尤其明显，如人类免疫缺陷病毒1 (HIV)，它们半随机地整合到宿主DNA的基因组中，并依赖宿主调节因子启动病毒基因表达和完成复制。在HIV的情况下，嘈杂的病毒基因表达导致转录延迟，这可能是潜伏感染的前兆。潜伏的HIV目前阻碍了患者体内病毒的完全根除，通过激活潜伏的HIV储存库来逆转病毒潜伏期是一种很有前途的治疗策略。然而，对染色质和转录因子介导的HIV调控的复杂机制的不完全理解阻碍了进展。为了解决这一知识缺口，该项目将实验和计算方法结合起来，研究经典转录因子NF-kB和病毒整合位点的局部染色质环境如何共同调节潜伏HIV LTR启动子的激活。无法靶向和清除潜伏（即沉默）的艾滋病毒感染细胞是目前治疗的最大障碍，本提案旨在解决这一重大的公共卫生问题。通过整合单细胞实验和计算模型，这项研究将提高对蛋白质- dna相互作用（称为染色质）如何调节HIV-1转录的理解，并将进一步朝着根除潜伏病毒库的方向发展。这项工作也可能更广泛地推进染色质介导的转录调节机制的知识，具有影响免疫学、发育和癌症的潜力。此外，还提出了两项教育举措，通过提供暑期研究金，扩大代表性不足的少数族裔工程专业学生参与跨学科研究；并通过设计跨学科研究生课程的新讨论环节，为生物和物理科学界面的变革教学方法做出贡献。

1264246Miller-Jensen, Kathryn Genetically identical cells can display significant phenotypic heterogeneity as a result of random fluctuations (noise) in gene expression, which is modulated by transcription factor binding and chromatin modifications at each gene promoter. The interaction of chromatin and transcription factors is especially evident among mammalian retroviruses, such as the human immunodeficiency virus-1 (HIV), which integrate semi-randomly into the genome of their host's DNA and depend on host regulatory factors to initiate viral gene expression and complete replication. In the case of HIV, noisy viral gene expression results in transcriptional delays that may be a precursor for latent infections. Latent HIV currently prevents complete eradication of the virus in a patient, and reversing viral latency via activation of the latent HIV reservoir is a promising therapeutic strategy. However, an incomplete understanding of the complex mechanisms underlying chromatin- and transcription factor-mediated regulation of HIV hinders progress. To address this knowledge gap, the proposed project integrates experimental and computational approaches to study how the canonical transcription factor NF-kB and the local chromatin environment at the viral integration site together regulate activation of the latent HIV LTR promoter. The inability to target and purge latent (i.e., silent) HIV-infected cells is currently the biggest obstacle to a cure, and this proposal seeks to address this significant public health problem. By integrating single-cell experiments and computational modeling, this research will improve understanding of how protein-DNA interactions called chromatin regulate HIV-1 transcription, and will further progress towards eradication of the latent viral reservoir. This work may also advance knowledge of mechanisms of chromatin-mediated transcriptional regulation more broadly, with the potential to impact immunology, development, and cancer. In addition, two educational initiatives are proposed to broaden participation of under-represented minority engineering students in interdisciplinary research through summer research fellowships; and to contribute to transformative teaching approaches at the interface of the biological and physical sciences through design of a new discussion session for an interdisciplinary graduate course.