Solid Phase Condensates In Vivo Imaging Data
Description
This repository contains raw imaging files and analysis code associated with the following study: Solid phase transitions as a solution to the genome folding paradox Joan Pulupa1,2, *, Natalie G. McArthur3, *, Olga Stathi2, Miao Wang1, 2, Marianna Zazhytska1, 2, Isabella D. Pirozzolo4, Ahana Nayar5, Lawrence Shapiro1, 2, and Stavros Lomvardas1, 2, & 1 Department of Biochemistry and Molecular Biophysics, Vagelos College of Physicians and Surgeons, New York, NY 10032, USA 2 Mortimer B. Zuckerman Mind, Brain, and Behavior Institute, Columbia University, New York, NY 10027, USA 3 Department of Biological Sciences, Columbia University, New York, NY 10027, USA 4 Medical Scientist Training Program, Vagelos College of Physicians and Surgeons, New York, NY 10032, USA 5 Barnard College, New York, NY, 10025, USA * These two authors contributed equally to this work & Corresponding author Abstract Ultra long-range genomic contacts, which emerge as prominent components of neuronal genome architecture1-3, constitute a biochemical enigma. This is because regulatory DNA elements make selective and stable contacts with DNA sequences located hundreds of Kbs away, instead of interacting with proximal sequences occupied by the same exact transcription factors (TFs)1,4. This is exemplified in olfactory sensory neurons (OSNs), where only a fraction of Lhx2/Ebf1/Ldb1-bound sites interact with each other, converging into highly selective multi-chromosomal enhancer hubs5. To obtain biochemical insight to this process, we assembled OR enhancer hubs in vitro with recombinant proteins and enhancer DNA. Cell-free enhancer hub reconstitution revealed that OR enhancers form nucleoprotein condensates with unusual, solid-like characteristics. Assembly of these solid condensates is orchestrated by specific DNA motifs enriched in OR enhancers, which likely impose distinct homotypic properties to their resident Lhx2/Ebf1/Ldb1 complexes. Single molecule tracking and pulse/chase experiments in vivo confirm that Lhx2 and Ebf1 assemble OR transcription-competent condensates with solid properties in OSN nuclei, under physiological protein concentrations. Thus, DNA sequence-influenced homophilic nucleoprotein interactions generate new types of biomolecular condensates, which may provide a generalizable explanation for the stability and specificity of long-range genomic contacts across cell types.
Files
Steps to reproduce
Please refer to the paper for details on how to replicate neuronal culture and imaging conditions. Briefly, primary olfactory neurons were dissociated and plated with astrocytes in order to obtain a primary cell culture. The behavior of endogenously expressed transcription factors was then imaged using a CSU-W1 SoRa Yokogawa spinning disk confocal microscope with Optical Re-Assignment or a a Bruker Vutara VXL. All raw data used for analysis is present.