Conserved epigenetic hallmarks of T cell aging during immunity and malignancy.

Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such 'epigenetic clocks' appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue 'counting' beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared 'young' regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan.

Efficient activation of hundreds of LTR12C elements reveals cis-regulatory function determined by distinct epigenetic mechanisms.

Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa) system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.