Margaret Goodell, Wei Li and colleagues use double-knockout mice for Dnmt3a and Tet2 to model leukemia development. Through epigenetic and transcriptional analyses, they show that loss of DNMT3A and TET2 upregulates lineage-specific transcription factors such as KLF1 in hematopoietic stem cells and accelerates malignancy. Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation–hydroxymethylation pathway. Using Dnmt3a and Tet2 double-knockout mice in which the development of malignancy is accelerated, we show that the double-knockout methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulators Klf1 and Epor, is upregulated in double-knockout hematopoietic stem cells (HSCs). DNMT3A and TET2 both repress Klf1, suggesting a model of cooperative inhibition by epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, the loss of which, along with DNMT3A, obstructs differentiation, leading to transformation.

Neoadjuvant immune checkpoint blockade (ICB) demonstrates promise in operable esophageal squamous cell carcinoma (ESCC), but lacks available efficacy biomarkers. Here, we perform single-cell RNA-sequencing of tumors from patients with ESCC undergoing neoadjuvant ICB, revealing a subset of exhausted CD8⁺ T cells expressing SPRY1 (CD8⁺ Tex-SPRY1) that displays a progenitor exhausted T cell (Tpex) phenotype and correlates with complete response to ICB. We validate CD8⁺ Tex-SPRY1 cells as an ICB-specific predictor of improved response and survival using independent ICB-/non-ICB cohorts and demonstrate that expression of SPRY1 in CD8⁺ T cells enforces Tpex phenotype and enhances ICB efficacy. Additionally, CD8⁺ Tex-SPRY1 cells contribute to proinflammatory phenotype of macrophages and functional state of B cells, which thereby promotes antitumor immunity by enhancing CD8⁺ T cell effector functions. Overall, our findings unravel progenitor-like CD8⁺ Tex-SPRY1 cells' role in effective responses to ICB for ESCC and inform mechanistic biomarkers for future individualized immunotherapy.

DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. Here, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest. We demonstrate that dCas9-SunTag-DNMT3A dramatically increases CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. Furthermore, using a single guide RNA, dCas9-SunTag-DNMT3A is able to methylate a 4.5-kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing and RNA-seq show that dCas9-SunTag-DNMT3A methylates regions of interest with minimal impact on the global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship between DNA methylation and gene expression.

Comprehensive studies have shown that DNA methylation plays vital roles in both loss of pluripotency and governance of the transcriptome during embryogenesis and subsequent developmental processes. Aberrant DNA methylation patterns have been widely observed in tumorigenesis, ageing and neurodegenerative diseases, highlighting the importance of a systematic understanding of DNA methylation and the dynamic changes of methylomes during disease onset and progression. Here we describe a facile and convenient approach for efficient targeted DNA methylation by fusing inactive Cas9 (dCas9) with an engineered prokaryotic DNA methyltransferase MQ1. Our study presents a rapid and efficient strategy to achieve locus-specific cytosine modifications in the genome without obvious impact on global methylation in 24 h. Finally, we demonstrate our tool can induce targeted CpG methylation in mice by zygote microinjection, thereby demonstrating its potential utility in early development.