(F-226) Visualizing and Manipulating a Stochastic Epigenetic Switch Controlling T cell Lineage Commitment at Single-locus Resolution

Introduction:: Epigenetic switches, acting at single genomic loci, enable stem cells to turn on lineage-specifying genes, and commit to different cell fates during development. These switches often activate in a stochastic, all-or-none manner, with signals and transcription factors modulating probabilities of gene activation and lineage decision making. This stochastic control critically enables for multiple differentiated cell types to emerge in defined proportions at a population level. However, the molecular mechanisms that underlie such stochastic control have remained unclear. Here, we study a stochastic epigenetic switch controlling the T cell lineage-specifying gene Bcl11b, whose activation in thymic progenitors results in irreversible expression of this gene and commitment to the T cell fate. We previously found that the repressive histone modification H3K27me3 regulates Bcl11b activation probabilities (Pease et al. 2021), suggesting that heterogeneity in H3K27me3 levels between loci may be a critical factor in generating heterogeneity in activation responses. To determine whether this heterogeneity exists, we are quantifying H3K27me3 levels at single Bcl11b loci in single cells using a technique we have developed named SCEPTRE, that uses expansion microscopy to both visualize and quantify histone modification states and physical conformations of single genomic loci (Woodworth et al. 2021). Using SCEPTRE, we are assessing H3K27me3 modification levels at the Bcl11b locus in both its OFF and ON states, prior and after lineage commitment. Further, we are altering H3K27me3 levels at the Bcl11b locus using dCas9 epigenome modulation tools, and reading out resultant effects on histone modification and gene expression states at the Bcl11b locus.


Materials and Methods:: To visualize and quantify histone modifications at Bcl11b, we will utilize an in vitro differentiation system to generate progenitor cells at different stages of T cell development, using a Bcl11b-YFP fluorescent reporter allele in conjunction with fluorescence activated cell sorting to purify cells before and after lineage commitment. Using SCEPTRE (Single Cell Evaluation of Post-TRanslational Epigenetic Encoding), we will then label the Bcl11b promoter and enhancer using DNA-FISH, concurrently stain for H3K27me3 modifications using immunofluorescence staining, then perform expansion microscopy to enhance the resolution of this locus to ~75 nm for resolution of histone marks. To modulate the activity of Bcl11b, we will transduce T cell progenitors with dCas9 fused to both epigenetic repressors and activators, including H3K27me3 modifiers, then performing these experiments.


Results, Conclusions, and Discussions:: SCEPTRE reveals heterogeneity in H3K27me3 levels at the Bcl11b promoter and enhancer at different stages of activation. In preliminary evidence, we found that cells that are silent for Bcl11b expression have H3K27me3 modifications at a timing enhancer located 850 kb downstream of Bcl11b, but lower or baseline levels of these modifications at the Bcl11b promoter. A similar absence of H3K27me3 modifications was seen in cells that had turned on the Bcl11b locus. These findings suggest that epigenetic histone modifications of key regulatory elements may play a role in the mechanism of irreversible epigenetic switching at Bcl11b. Additionally, by using dCas9 to recruit epigenetic modifiers to the Bcl11b locus, we are working to further clarify the mechanism of epigenetic switching through modulating activation states and cell lineage commitment. Thus, in our work we aim to elucidate the fundamental mechanisms of epigenetic timers to optimize the production of immune cells for regenerative medicine.


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References (Optional): : Pease NA, Nguyen PHB, Woodworth MA, et al. Tunable, division-independent control of gene activation timing by a polycomb switch. Cell Rep. 2021;34(12):108888. doi:10.1016/j.celrep.2021.108888

Woodworth MA, Ng KKH, Halpern AR, et al. Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy. Nucleic Acids Res. 2021;49(14):e82. doi:10.1093/nar/gkab423