Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Initiation of gene transcription is arguably the most influential control point in the development of a multicellular organism. In the simplest version of gene activation, a specific transcription factor binds to a short DNA sequence (6-12 bps) in the control region of a target gene and then somehow recruits the vast biochemical machinery of RNA Polymerase II and its auxiliary proteins, thereby initiating transcription. All of this occurs in an environment of “chromatin”, featuring possible cofactors, differentially modified histones and potentially positioned nucleosomes. We will focus on the all-important early step of gene activation in which a single controlling transcription factor interacts with its binding site in the regulatory region of a target gene. Our goal is to relate precise biophysical measurements made with purified components in vitro to quantitative transcriptional consequences measured in vivo .

For years, we have been analyzing and describing the development of a simple clonal cell lineage, the intestine of the small free-living nematode worm Caenorhabditis elegans . All the major transcription factors are now known but we will focus on ELT-2, the predominant transcription factor driving intestinal differentiation from the early embryo until death. We propose experiments to define three fundamental aspects of the interaction between ELT-2 and its binding sites inside an embryo.

Aim 1: ELT-2 binds preferentially to TGATAA DNA sites but different flanking sequences cause different binding affinities. Using advanced microscopic techniques, we will test if ELT-2 has the same spectrum of affinities binding to DNA in vitro and in vivo, i.e. inside the nucleus of a living embryo. We will also measure the level of free ELT-2 protein inside a living embryo. Both results are necessary to understand gene regulation at a quantitative biophysical level.

Aim 2: We have developed a tightly controlled experimental system in which single base pair variants of the ELT-2 binding site have significant transcriptional consequences inside the animal. We will first define the relation between ELT-2 affinity and transcript levels. and then extend this system to measure influences of developmental stage, chromatin proteins and chromatin modifications. We will ask the key question: do other target genes follow the same rules?

Aim 3: We will combine “low-tech” dissection of C. elegans intestines with state-of-the-art tagging of accessible DNA, thereby defining the position of nucleosomes in regulatory regions of intestine specific genes.

The aim of this research is to understand, at the fundamental level of binding energies, how critical decisions are made in embryonic development. Because ELT-2 is homologous to transcription factors GATA-4/5/6 driving endoderm development in vertebrates, the findings are likely to apply to development of intestines in all animals, including ourselves.