Cutting edge: persistently open chromatin at effector gene loci in resting memory CD8+ T cells independent of transcriptional status

Abstract

Memory CD8(+) T cells are characterized by more rapid and robust effector function upon infection compared with naive T cells, but factors governing effector gene responsiveness are incompletely understood. We sought to understand transcriptional control of the effector genes IFN-γ (Ifng), granzyme B (Gzmb), and perforin 1 (Prf1) in murine memory CD8(+) T cells by characterizing their transcriptional profiles and chromatin states during lymphocytic choriomeningitis virus infection. Each effector gene has a distinct transcriptional profile in resting memory cells and following restimulation. Primary infection leads to reduced nucleosomal density near the transcription start sites and reduced H3K27 methylation throughout the Ifng and Gzmb loci, and these chromatin changes persist in the memory phase. Despite similarities in chromatin at the memory stage, PolII recruitment and continuous transcription occur at the Ifng locus but not the Gzmb locus. We propose that these chromatin changes poise effector genes for rapid upregulation, but are insufficient for PolII recruitment and transcription.

Figure 1. Distinct transcriptional profiles of three effector genes in resting and restimulated memory cells

A) Sorted naïve and memory CD8+ T cells were cultured with anti-CD3 and anti-CD28 for the specified time, and analyzed for effector gene transcript abundance by quantitative RT-PCR. The signal for each gene was divided by the Hprt signal, and then relative transcript levels were normalized to the naïve sample at time 0. B and C) Freshly sorted naïve, effector, and memory CD8+ T cells were analyzed for effector gene transcript abundance by quantitative RT-PCR. Effector gene transcript signal was divided by Hprt transcript signal to determine relative quantities.

Figure 2. Depletion of nucleosomes and H3K27me occurs at the Ifng and Gzmb loci in effectors and persist in memory cells

A) Schematics (not to scale) showing the genomic organization of effector genes Ifng, Gzmb, and Prf1, and the inactive Foxp3 gene. Exons are shown as filled rectangles, and the location of PCR amplicons are depicted as dashes marked with lower case letters. Amplicon size averaged 50bp. B) ChIP using antibodies specific for histone H3 was used to detect nucleosomal density at indicated locations across effector genes. Data were normalized by dividing ChIP-PCR signal by input signal. Asterik (*) indicates significant differences between naïve and effector or naïve and memory samples at the same genomic sites, and pound sign (#) indicates significant differences between indicated sites within the Gzmb and Prf1 loci (P<.05). Data are pooled from 9 independent experiments. C) H3K27me3 was detected by ChIP at indicated locations across effector genes. Data were normalized by dividing the H3K27me3 ChIP-PCR signal by the H3 ChIP-PCR signal to normalize the methyl mark to total histone levels. Data are pooled from 4 independent experiments.

Figure 3. PolII recruitment reflects transcriptional status of effector genes

ChIP-PCR on freshly sorted naïve, effector, and memory CD8+ T cells using the antibody 8WG16, which is specific for the RNA polymerase II C-terminal domain. Asterisks (*) denote significant enrichments compared to the same genomic site in naïve cells (P<.05). Data were normalized by dividing ChIP-PCR signal by input signal, and are pooled from 4 independent experiments.