Investigating the Central Dogma of Molecular Biology in the Context of Nuclear Architecture and Cell Cycle

Abstract

Number of figures: 4 Number of characters in the title: 81 Number of words in abstract: 177 Number of words excluding Abstract, Materials and Methods and References: 2498 Number of references: 39 Number of supplementary movies: 1 Number of supplementary Figures: 1 Number of supplementary tables: 1 Abstract Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position-which usually lies close to nuclear lamina-nor on the expression from the other copies. Keywords Cell cycle, 3D nuclear architecture, central dogma, DNA FISH, smFISH, Cyclin A2, nuclear lamina, immunofluorescence AM, SD conceived and designed the study. SD performed the experiments and the analysis.AM, SD interpreted the data and wrote the manuscript. AM monitored the project and associated grants. Competing interestsNone.