a Synthetic fly tad
building transcriptional landscapes from scratch
Transcriptional regulation is essential for almost all biological processes and when it goes awry, can lead to devastating diseases. Although fundamental, the underlying molecular mechanisms are still not completely understood. Since all cells within an organism share the same genome, regulatory elements are needed to tightly control when and where genes are expressed. In particular, enhancers, short (50–1,500 bp) sequences bound by multiple transcription factors (TFs), regulate promoter activity. Yet, many interesting questions remain. What is the relationship between chromatin topology and enhancer function? How do enhancers regulate RNA polymerase activity? This is such a fundamental question one immediately assumes it has been answered, but our understanding is still quite vague. Do enhancers regulate recruitment and transcription initiation or elongation and release from Pol II? Do enhancers regulate the frequency or the amplitude of transcriptional bursts or a combination of both? Enhancers are often located far from the promoters that they regulate. However, the genome is organized into Topologically Associating Domains (TADs) that can facilitate enhancer–promoter interaction. Do TADs act to negate enhancer–promoter distances, as generally assumed? We recently showed that for most genes, shuffling or fusing TADs using genetic inversions did not impact their expression. Similar findings are emerging from a number of TADs in vertebrates and when key proteins are depleted in trans. What then is the role of TADs in gene expression? Do they increase the probability of enhancers finding their correct targets? Given that genes have many enhancers, often with partially overlapping activities, do they work simultaneously or sequentially? If the latter, what is the extent of enhancer competition?
The challenge to address these questions is the inherent complexity and incomplete characterization of endogenous regulatory landscapes. Classic genetic deletion experiments typically perturb other things within the regulatory locus confounding the interpretation for their impact on gene expression. To negate these issues, we will take the opposite approach – to synthesize a TAD de novo, controlling for every element within the regulatory landscape. We will then systematically alter this TAD, in a highly controlled manner, by changing just one parameter at a time. Such a synthetic approach at this scale would not have been possible even 5 years ago, but is highly feasible today thanks to the technology developed by our group. This synthetic technology brings a new era to the study of genome regulation – by systematically changing only one parameter at a time, we can definitively answer long-standing questions in the field.
WORKING ON THIS PROJECT:
Sudarshan Pinglay, PHD, Institute for Systems Genetics at NYU Langone Health
IN COllaboration with:
Eileen Furlong, PHD, EMBL