Search Talks

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA Polymerase II complex assembly and productive elongation. These studies expose fundamental questions about transcription regulation. Namely, how do transient interactions (~ seconds) between transcription factors and promoters lead to specific responses? We propose an extension of kinetic proofreading to explain transcriptional regulation in eukaryotes. This model suggests active, ATP-consuming processes drive transcription f...

In higher eukaryotic cells, strings of nucleosomes, where long genomic DNA is wrapped around core histones, are irregularly folded into numerous condensed chromatin domains (1,2). Inside these domains, nucleosomes fluctuate and locally behave like a liquid (2,3). While nucleosome behavior is assumed to be highly related to genome functions, it remains unclear how this behavior changes during the cell cycle. During interphase, the nucleus enlarges and genomic DNA doubles. Previous reports have shown that chromatin movements vary during interphase on a minute or longer time-scale. However, using single-nucleosome imaging and tracking (4), we reveal that local nucleosome motion on a second time-scale remains steady throughout the G1, S, and G2 phases in live human cells (4). Combined with Brownian dynamics modeling, our results suggest that this steady-state nucleosome motion is mainly driven by thermal fluctuations. We propose that this observed steady-state nucleosome motion allows cell...

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the het...

The vast majority (~98%) of the mammalian genome is noncoding but has a vital role in gene regulation. Enhancers are noncoding DNA sequences highly enriched in binding sites for tissue-specific transcription factors. Genes with pleiotropic roles in development often have huge regulatory landscapes (~ 1Mb) with multiple enhancers driving precise gene expression.
The human genome is thought to contain 100s of thousands of enhancers with, as yet, uncharacterised activities and a large proportion of disease-causing and disease-predisposing DNA sequence variants map to potential enhancers. To advance understanding of human disease, massive efforts are currently being directed towards establishing genotype to phenotype correlations for sequence variants. A key pre-requisite to these analyses is defining the precise cell- and tissue-specific activities of the enhancers and the transcription factors that bind them.
Our research focuses on building models for understanding cell-type specific ...