L. Stirling Churchman
Harvard Medical School
Associate Member of the Broad Institute of Harvard and MIT
High-resolution global studies to elucidate the molecular mechanisms that control the expression of both the nuclear and mitochondrial genomes.How do co-transcriptional processes regulate RNA polymerase as it travels along gene bodies? How does this control result in the correct identity and subsequent fate of RNA transcripts? How do the nuclear and mitochondrial genomes coordinate their expression during mitochondrial biogenesis? We study the molecular mechanism and functional consequence of transcription elongation within the context of the cell. We also study the regulation of mitochondrial gene expression to obtain a holistic view of genomic expression in the eukaryotic cell.
To address these questions in human cells and in yeast, we develop high-resolution global strategies to quantitatively observe the density and composition of the RNA polymerase elongation complex. We established a methodology, nascent elongating transcript sequencing (NET-seq), that exploits the extraordinary stability of the DNA-RNA-RNA polymerase ternary complex to capture nascent transcripts directly from live cells (Churchman and Weissman, Nature, 2011). The identity and abundance of the 3’ end of purified transcripts are revealed by deep sequencing thus providing a quantitative, strand-specific measure of RNA polymerase (RNAP) density with single nucleotide precision.
How are cellular factors involved in controlling transcription elongation and in coordinating co-transcriptional activities? We are using NET-seq to investigate how transcription related factors affect transcriptional activity genome wide. In human cells, we are interested in understanding how transcriptional activity changes as cells differentiate in development and in cancer.
Mitochondria have gradually transferred many of the genes from their genome to the nucleus, retaining a small but significant subset on a circular genome tucked within the mitochondrial matrix. Our goal is to directly observe all stages of mitochondrial gene expression and determine their regulatory mechanisms. We are adapting and applying a select set of quantitative genomic approaches to fully dissect the mitochondrial gene expression process, from DNA to protein. In this manner, we plan to map the landscape of mitochondrial gene expression in both budding yeast and human cells using a combination of high-resolution, quantitative approaches that query across the mitochondrial genome and the mitochondrial-encoded proteome.
Nascent transcript sequencing visualizes transcription at nucleotide resolution. L. Stirling Churchman and Jonathan S. Weissman Nature 469: 368-373 (2011)
Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. L. Stirling Churchman, Zeynep Okten, Ronald S. Rock, John F. Dawson and James A. Spudich Proceedings of the National Academy of Sciences 102, 1419-1423 (2005)
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