Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Physics & Astronomy

First Advisor

Arjun Raj

Abstract

Human cells are dynamic: they grow, replicate their genetic information (DNA), and divide. Clonal populations of cells can display marked heterogeneity in size, leading to significant variability in the ratio of DNA to cellular volume. Despite this variability, cells must maintain a constant concentration of RNA and protein, produced from DNA, to ensure proper functionality. How do larger cells produce more output from the same amount of DNA? How do cells that have replicated their DNA prior to cellular division produce the same output as before? Using RNA fluorescence in situ hybridization (RNA FISH), we visualize and count individual RNA molecules in single cells, allowing for precise quantification of transcriptional output of single genes. We also use single-cell RNA sequencing to quantify transcriptional output from all ~20,000 genes encoded in the genome simultaneously. Surprisingly, we discovered that the cell implements two separate transcriptional mechanisms to compensate for changes in cell size and DNA content. Through cell-fusion experiments, we show that a diffusible trans factor, which we believe may be RNA polymerase II, increases transcriptional burst size in larger cells, compensating for changes in volume. Meanwhile, a DNA-linked cis-acting factor reduces the frequency of transcription per gene copy by a factor of two upon DNA replication, allowing the cell to still produce the same amount of RNA after replication, despite having twice the number of DNA copies. We show that transcription depends strongly on volume, and we therefore present a new "noise measure" which provides a measure of gene expression variability that takes volume into account. We perform single-cell RNA sequencing to measure noise genome-wide, and find that cell-type-specific genes tend to exhibit more expression noise than genes that are ubiquitously expressed across cell types. Finally, we have uncovered a fundamental mechanism by which cells are able to functionally compensate for naturally-occurring variability in size and DNA copy number.

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