Thymic epithelia amplify noise in chromatin accessibility via p53 repression to impose immune tolerance [CUT&Run]
Phenotypic plasticity of somatic cells is a principal feature of vertebrate adaptation as well as a hallmark of tumorigenesis. However, the determinants and mechanisms that regulate the stability of somatic cell identities remain unclear. Here, using the somatic plasticity of thymic epithelial cells – which facilitates the selection of a self-discriminating T cell repertoire – as a physiological model system, we show that stochastic fluctuations in background chromatin accessibility at nucleosome-dense regions are amplified by thymic epithelial cells to ectopically express thousands of genes highly restricted to other specialized cell types. We found broad regions of inaccessible chromatin flanking tissue-specific genes become 'destabilized' during thymic epithelial maturation independently of AIRE-induced transcription of these genes, but concurrently with the repression of the tumor suppressor p53. Augmenting p53 activity in thymic epithelial cells reduced noise in chromatin accessibility at nucleosome-dense regions, inhibited ectopic expression of tissue-specific genes and caused multi-organ autoimmunity. Furthermore, we found p53-regulated fluctuations in background chromatin accessibility in lung adenocarcinoma to be associated with high plasticity states that promote tumor progression. Taken together, our findings establish p53-dependent stabilization of nucleosomal barriers to cellular reprogramming as a fulcrum of cell fate integrity that underlies critical components of immune tolerance induction and oncogenic potential. Overall design: Thymic epithelial cells were isolated using a previously published protocol (Kim & Serwold, Methods in Molecular Biology, 2019) with minor modifications. Briefly, thymi from 4-5 week-old mice were harvested and connective tissue was removed. Stromal tissue was perforated using scissors and incubated with rotation in DMEM-F12 (Sigma D6421) at room temperature for for 10min to liberate thymocytes. Remaining stromal tissue was enzymatically digested (0.5mg/mL Collagenase D (Sigma 11088858001), 0.2mg/mL DNaseI (Sigma 10104159001), 0.5mg/mL Papain (Worthington Biochemical LS003126)). Cells were stained with anti-EpCAM antibodies conjugated to APC-Cy7 and EpCAM+ cells were enriched via positive selection using magnetic anti-Cy7 beads (Miltenyi 130-091-652). Enriched fraction was stained with fluorescently conjugated antibodies for FACS or analysis.  For each condition, 500,000 cells were washed three times in wash buffer (20?mM HEPES, pH 7.5, 150?mM NaCl, 0.5?mM spermidine, 1× EDTA-free protease inhibitor cocktail, Roche), then bound to Concanavalin-A beads (Bangs Laboratories) according to the manufacturer's instructions. Cells were incubated with 1:100 dilution of anti-BAF155 antibody (G. Crabtree) for 2?h or overnight at 4?°C in permeabilization buffer (1× permeabilization buffer, eBioscience, 0.5?mM spermidine, 1× EDTA-free protease inhibitor cocktail, 2?mM EDTA). The sample was then incubated with 700?ng?ml-1 protein A-MNase (S. Henikoff) in permeabilization buffer at 4?°C for 1?h. Digestion was performed in 0.5× permeabilization buffer supplemented with 2?mM CaCl2 at 4?°C for 1?h. The reaction was stopped by adding 2× Stop buffer (final concentration 100?mM NaCl, 10?mM EDTA, 2?mM EGTA, 20?µg?ml-1 glycogen, 25?µg?ml-1 RNase A, Thermo Fisher Scientific) and the sample was incubated at 37?°C for 20?min. Protein in the sample was then digested in 0.1% SDS and 250?µg?ml-1 proteinase K (New England Biolabs) for 2?h at 56?°C, shaking gently. CUT&RUN fragments were purified using phenol chloroform extraction. CUT&RUN libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina coupled with NEBNext Multiplex Oligos for Illumina (New England Biolabs) with modifications optimized for small fragments as detailed in https://doi.org/10.17504/protocols.io.wvgfe3w. Paired-end, dual-index sequencing was performed on the Illumina NextSeq 500 system.