Transcriptional regulation is a central component of biology whose importance cannot be overstated. From basal transcription of genes necessary for the physical make-up of a cell, to differential gene expression in response to internal or external signaling, life itself is built upon transcription. Transcription factors (TFs) modulate where and to what extent transcription occurs within the genome, and because of this important role in facilitating life, TFs have been intensely studied since the advent of molecular biology. This dissertation explores the developmental roles and molecular functions of the Arabidopsis thaliana NUCLEAR FACTOR-Y (NF-Y) family of transcription factors, a heterotrimeric TF complex composed of three evolutionarily-diverged subunits, NF-YA, NF-YB, and NF-YC. Interactions between NF-Y subunits have been explored for a number of years, and the interaction of the NF-Y complex with its cognate DNA binding sequence, CCAAT, has been further described through crystallization of a DNA-bound NF-Y complex. Because of this deep understanding, the NF-Y are an excellent candidate for modeling complex TF interactions and their impact on DNA binding.

In Chapter 1, I introduce and describe research into the NF-Y in two parts. In Part 1, I introduce the importance of transcriptional regulation and provide a broad history and description of the NF-Y complex in plants. In Part 2, I provide an exhaustive description of recent plant NF-Y research and future perspectives. Taken together, this chapter aims to establish the importance of studying transcription factors and provide the reader with an up-to-date understanding of what is known regarding the developmental roles and mechanistic functions of the NF-Y.

One of the developmental roles of the NF-Y is in mediating seedling development in response to light, a process known as photomorphogenesis. In Chapter 2, I provide the first description of photomorphogenic roles for NF-YC3, NF-YC4, and NF-YC9. This work describes broad regulation of blue, red, and far red light signaling networks for these NF-YC subunits, where they coordinate light-dependent inhibition of hypocotyl elongation. Further, I identify that these NF-YC subunits function partially independent of the well-described ELONGATED HYPOCOTYL5 (HY5), despite confirming a direct physical interaction between NF-YC9 and HY5 through a novel microscopy-based approach combining Fluorescence Resonance Energy Transfer, Acceptor Photobleaching, and Fluorescence Lifetime Imaging. I further explore the interactions between NF-YC and HY5 in Chapter 4, where I identify a suite of mutually-exclusive phenotypic relationships in seedling primary root elongation in different light conditions.

In Chapter 3 I describe the development and implementation of a yeast synthetic reporter system, the Dynamic Interdependent TF binding Molecular Reporter (DIMR) system. The DIMR system probes oligomeric TF DNA binding in vivo, and has been able to completely recapitulate all tested aspects of NF-Y DNA binding. While my proof-of-concept is limited to the NF-Y, the DIMR system is designed as modularly as possible, and should be readily adaptable to study any TF DNA interaction with minor modifications.