UC Berkeley

Cis–trans relationships govern gene regulation: it is the interactions between diffusible proteins and chromatinized DNA that dictate nuclear functions. The nature of those interactions must allow for the great regulatory complexity needed for eukaryotic function and particularly of multicellular development. However, many of the current models of cis–trans relationships in the nucleus either lack biophysical rigor or have gone untested. This thesis of three chapters addresses three shortfalls in the current conceptualization of how gene-regulatory proteins function.In the introductory chapter, it is argued that there is a widespread over-reliance on qualitative descriptions of biomolecular behaviors and functions. Because biochemistry is inherently stochastic, the way in which such behaviors and functions are described must eventually be probabilistic and quantitative. Simplistic 1:1 relationships between molecules need to be dropped in favor of probabilistically constituted ensembles of factors with different affinities. Furthermore, because the cell interior is literally fluid and molecular interactions quite transient, the temporal dynamics of the system must always be kept in mind, which largely forbid the use of static structures and monolithic series of events. The second chapter issues a challenge to the notion that an important cis relationship, that between enhancers and promoter, is mediated by a direct interaction between the two elements—a striking instance of the structure–function paradigm questioned in the first chapter. It has been assumed for decades that a complex of proteins forms a bridge connecting enhancer to promoter even over large genomic distances. However, neither evidence nor reason rules this assumption in, and other models have hardly been considered, much less ruled out. Here it is proposed that diffusible biochemical species in the form of modified proteins are generated at the enhancer and can affect the promoter via diffusion. What determines the scale of the gradient of signal emanating from the enhancer is the balance of the local generation rate and the ubiquitous degradation rate. Finally, an imaging study is presented in which the question is asked, how are transcription coactivators (trans factors) distributed among the plethora of cis elements? Through single-molecule tracking of p300 and many mutants thereof, it is shown that direct cis interaction—i.e., by binding modified histones—is not responsible for coactivator targeting. Rather, p300 depends on a combination of transcription factor–interaction domains to associate with chromatin, indicating that atop the cis–trans interactions between transcription factors and chromatin is another layer of trans interactions between transcription factors and their cofactors.