UC Berkeley

It has long been appreciated that organization of macromolecules within the cell is paramount to proper cellular function. As the number of tools in the molecular and cell biologist’s toolbox grows, the list of ways cells achieve organization also grows. This is especially true for the process of transcription and its regulation, where the fundamental questions remain a challenge to address, but where a convergence of genetic, genomic, structural and imaging techniques offer the possibility of answers. The proper loading of an RNA polymerase on a gene requires the coordinated action of hundreds of proteins and other macro molecules, as well as cellular genome to be maintained in a topologically permissive state; yet as a system, gene regulation requires interactions to be dynamic enough to respond to the changing needs of the cell in response to internal and external ques. How a cell constructs a system that is, on the one hand robust, and on the other hand flexible remains a challenge to answer.

In Chapter 1 of this thesis, we will introduce the study of transcription regulation as a general topic, and discuss a number of fluorescence imaging techniques that have fundamentally changed the our understanding of transcription regulation. Chapter 2 will focus on one particular aspect of cellular organization which has recently come into vogue—that of membraneless compartment formation through liquid-liquid phase separation. A thorough investigation of the literature, particularly in light of the experiments which we foreshadow and introduce in more depth in Chapter 3, suggests that much more work is needed before phase separation as a means of biological organization can become a general paradigm. Chapter 3 presents a particularly poignant example of the issues raised in Chapter 2. Using Herpes Simplex Virus as a model system because of its ability to form compartments in the nucleus, we demonstrate that recruitment of Pol II and other proteins can be explained through the availability of nonspecific binding sites rather than phase separation. Lastly, Chapter 4 will present technical findings on the optimal fluorescent dyes to use for in vivo labeling of proteins for imaging applications.

In summary, this dissertation underscores the way in which new imaging techniques reveal new biological principles and challenge old models. It will be of great interest to watch these ideas and techniques mature beyond the work presented in this dissertation.