SUMMARY Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obsure. Here, we report the identification of novel N(6)-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer, glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase, ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator, ALKBH1, in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification, N6-mA.

SUMMARY Current genome-editing systems generally rely on the creation of DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. The CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat several mouse models of human diseases. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to observable phenotypic changes, and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.

Transit-amplifying nephron progenitor cells (NPCs) generate all of the nephrons of the mammalian kidney during development. Their limited numbers, poor in vitro expansion, and difficult accessibility in humans have slowed basic and translational research into renal development and diseases. Here, we show that with appropriate 3D culture conditions, it is possible to support long-term expansion of primary mouse and human fetal NPCs as well as NPCs derived from human induced pluripotent stem cells (iPSCs). Expanded NPCs maintain genomic stability, molecular homogeneity, and nephrogenic potential in vitro, ex vivo, and in vivo. Cultured NPCs are amenable to gene targeting and can form nephron organoids that engraft in vivo, functionally couple to the host's circulatory system, and produce urine-like metabolites via filtration. Together, these findings provide a technological platform for studying human nephrogenesis, modeling and diagnosing renal diseases, and drug discovery.

SUMMARY Pancreatic beta-cell mass for appropriate blood glucose control is established during early postnatal life. Beta-cell proliferative capacity declines postnatally but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of beta-cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of beta-cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative beta-cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature beta-cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal beta-cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal beta-cells and paves the way for the identification of novel therapeutic targets to stimulate beta-cell regeneration.

Single nucleus ATAC-seq (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. As pancreatic islets are central to type 2 diabetes (T2D), we profiled 15.3k islet cells using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D GWAS for beta cells as well as enrichment for other endocrine cell types. At T2D signals localized to islet accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS , and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single cell epigenomics for interpreting complex disease genetics.