Quantitative assessment of gene dosage effects on cellular phenotypes [electronic resource]
Keywords: Chemical Engineering.

In biological systems, the level of gene expression and activity has a powerful effect on a cell's behavior. When a gene's expression level is above or below the range of "normal" expression, disease can occur. In order to study these deviations in gene expression, researchers typically knock out gene expression entirely or overexpress the gene at a high level. For some applications, these methods do not resemble physiological levels. Using tools and methods developed in this lab and others, we investigated methods of controlling gene dosage and measuring its effect in several applications related to cancer, a disease whose etiology can include perturbations in gene expression: I. To study the effect of increasing the expression of an oncogene, we developed a constitutive synthetic gene promoter library and a method to analyze cell phenotypes in a high-throughput manner using flow cytometry. Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. We have demonstrated that quantitative trends can be fit to single-cell flow cytometry data generated from a heterogeneous population. We engineered transformed pre-B cells to express a broad range of wild-type and oncogenic Ras levels by using retroviral vectors equipped with different strength promoters. Cells were "shotgun" transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a nearly continuous range of expression levels. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression led to even greater MAPK signal transduction, this increased expression had minimal or even decreasing effects on the proliferation rate. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. In addition, this study introduces a general method and a software application that performs LOWESS on two-parameter total population flow cytometry data to quantify genetic dose-response relationships. II. Sometimes, the dosage effects of a gene's activity have a profound effect on a cell phenotype. In cancers caused by activating mutations, tumors are often initially responsive to chemotherapy but relapse after a period of treatment, often due to secondary mutations. Activation-induced cytidine deaminase (AID) is a genomic mutator that produces much of the diversity in antibodies via class-switch recombination and somatic hypermutation activity. Although AID is tightly regulated, aberrant expression of (AID) has been observed in a range of cancers. Overexpression of AID has been linked to the generation of somatic mutations outside of the normally-targeted immunoglobulin gene context. We show that induction of AID activity has a negative effect on cell viability and demonstrates mutational activity on reporter constructs in a dose-dependent manner. III. Using a gene repression platform recently adapted for use in mammalian cells, we investigated the effect on gene expression of targeting various regions in a commonly-studied tumor suppressor gene TP53. This platform, CRISPR (clustered regularly interspaced short palindromic repeat), has been developed from a native prokaryotic adaptive immune system to a method of directing proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves a DNA target, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, coding sequence, and regions flanking the endogenous human TP53 gene. p53, the gene product of TP53, is a transcription factor whose activation by DNA damage or other cellular stresses results in responses that promote cell cycle arrest, apoptosis, or other tumor suppressing processes and has, previously and presently, served as a well-studied model gene for the purpose of studying gene repression. We showed that repression up to 7-fold can be achieved with dCas9 alone by targeting the complex to sites near the TP53 transcriptional start site. Additionally, we found that the addition of the KRAB repressor domain to dCas9 did not increase overall repression of TP53 expression nor did it have long-distance repressive effects when targeted to regions flanking the TP53 gene locus. Finally, we examined if a similar repression could be achieved by targeting sites surrounding the transcriptional start site in other genes. Of five additional genes, we achieved significant, though modest, repression in four by targeting the TSS. This work demonstrates that efficient transcriptional repression of endogenous genes is possible using dCas9-enabled CRISPRi in human cells, but efficiency can vary depending on the choice of gene and the position of the target site.