ZIA BC 011383 (ZIA) | |||
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Title | Transcription and Splicing Dynamics in Single Cells | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Larson, Daniel | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $964,491 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Chronic Myeloproliferative Disorders (10.0%) |
Breast (70.0%) Leukemia (10.0%) Multiple Myeloma (10.0%) Prostate (10.0%) |
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Research Type | |||
Normal Functioning Cancer Initiation: Oncogenes & Tumor Suppressor Genes |
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Abstract | |||
Cancer is a disease of rare events in single cells, and heterogeneity of cancer cells contributes to disease progression, treatment resistance, and relapse. In recent years, there has been tremendous progress in the development of methodologies to interrogate single cancer cells, including in situ imaging and single-cell DNA and RNA sequencing. Heterogeneity can be both genetic and non-genetic. Genetic heterogeneity - rare mutations in somatic cells which lead to the initiation and pathogenesis of cancer - is well-appreciated. Non-genetic heterogeneity - changes in the phenotype of the cell which may be unrelated to genetic mutations - is only beginning to be understood and is the subject of the investigations carried out in the Systems Biology of Gene Expression section. Although heterogeneity or variability plays an important role in cancer progression, it is also a window into the mechanisms of gene regulation. Thus, even without addressing the role of heterogeneity in a particular phenotype, one can use variations or fluctuations to understand the mechanisms of RNA synthesis and processing in single cells. This principle of using dynamic observations to understand molecular mechanism in living cells is the motivation behind the experiments carried out in the lab. I. Transcription dynamics in living cells: the causes and consequence of expression heterogeneity. One paradigm for genetic control in the metazoan nucleus is steroid receptor-mediated transcription. Steroid receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases. Moreover, the ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic intervention, necessitating a quantitative understanding of how receptors control output from target genes. In addition, steroid-response elements often work through long range regulation of genes located megabases away, making them an appealing model for understanding the role of chromosome structure in gene regulation. We are studying estrogen responsive genes in MCF7 cells, starting with the well-studied paradigm of the TFF1 gene and proceeding to large-scale analysis of many genes. We find that even for TFF1, which shows a 60-fold up-regulation upon induction with estradiol, transcription is a rare event, with long periods of inactivity punctuated by short bursts of RNA synthesis. We show this intermittency is the major driver of non-genetic heterogeneity. The key point is that cellular heterogeneity is a dynamic phenomenon and dynamics must be considered at every step, from experiment through to theory and analysis. Moreover, to understand dynamics is to understand mechanism. We approach this problem in three parts using model systems in addition to the estrogen-responsive human system. First, we consider the role of cis-acting regulatory regions in DNA. Next, we ask how trans-acting factors - both protein and RNA - regulate transcription. Finally, we seek to apply these ideas to large-scale experiments in an attempt to bridge the gap between single-molecule biology and genomics. II. RNA processing in normal and transformed cells. Transcription is carried out by a processive enzyme which can be experimentally reconstituted in vitro from minimal components. The spliceosome is different: it is a single-turnover enzyme which assembles and disassembles dynamically on each and every intron. As such, kinetic studies in living cells have much potential for elucidating the mechanism of splicing. We have also shown that RNA processing is stochastic, with single RNAs generated from the same gene in the same cell following different processing trajectories. Thus, RNA processing is another source of non-genetic heterogeneity. In fact, changes in RNA alternative splicing are one of the hallmarks of cancer. Recently, mutations in the general splicing machinery have emerged as important oncogenes in blood malignancies and solid tumors. T |