Multiplexed, Single-Cell Profiling of Chromatin States with Expansion Microscopy

Abstract

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications can act in combination to specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one modification at a time. I developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at in situ labeled genomic regions in single cells. Using SCEPTRE, I distinguished multiple histone modifications at different alleles of the same gene, quantified histone modification levels at multiple developmentally-regulated genes and identified a relationship between histone H3K4me3 and the loading of paused RNA polymerase II at individual loci. I observed extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements, which emphasizes the need for single cell methods when defining the chromatin state of genes. Lastly, I further developed SCEPTRE for use with live-cell tracking to recognize how cell cycle and potential state inheritance impact H3K27me3 mark heterogeneity at a developmental gene. I therefore have demonstrated the potential SCEPTRE may have for uncovering the contribution epigenetic states have to cell fate decision making.