Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumor suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small cell lung cancer (NSCLC). Concurrent KRAS mutation and LKB1 loss (KL) specifies aggressive oncological behavior1,2. We show that KL cells and tumors share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate (CP) in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. CPS1 transcription is suppressed by LKB1 via AMPK, and CPS1 expression anticorrelates with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumor growth. Surprisingly, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine/purine ratio, compromises S-phase progression, and induces DNA polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncogenotype imposes a novel metabolic vulnerability related to exquisite dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.

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Recent studies have highlighted super-enhancers (SEs) as important regulatory elements for gene expression, but their intrinsic properties remain incompletely characterized. Through an integrative analysis of Hi-C and ChIP-seq data, here we find that a significant fraction of SEs are hierarchically organized, containing both hub and non-hub enhancers. Hub enhancers share similar histone marks with non-hub enhancers, but are distinctly associated with cohesin and CTCF binding sites and disease-associated genetic variants. Genetic ablation of hub enhancers results in profound defects in gene activation and local chromatin landscape. As such, hub enhancers are the major constituents responsible for SE functional and structural organization.

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Small cell lung cancer (SCLC) is a rapidly lethal disease with few therapeutic options. We studied metabolic heterogeneity in SCLC to identify subtype-selective vulnerabilities. Metabolomics in SCLC cell lines identified two groups correlating with high or low expression of the Achaete-scute homolog-1 (ASCL1) transcription factor (ASCL1 and ASCL1), a lineage oncogene. Guanosine nucleotides were elevated in ASCL1 cells and tumors from genetically engineered mice. ASCL1 tumors abundantly express the guanosine biosynthetic enzymes inosine monophosphate dehydrogenase-1 and -2 (IMPDH1 and IMPDH2). These enzymes are transcriptional targets of MYC, which is selectively overexpressed in ASCL1 SCLC. IMPDH inhibition reduced RNA polymerase I-dependent expression of pre-ribosomal RNA and potently suppressed ASCL1 cell growth in culture, selectively reduced growth of ASCL1 xenografts, and combined with chemotherapy to improve survival in genetic mouse models of ASCL1/MYC SCLC. The data define an SCLC subtype-selective vulnerability related to dependence on de novo guanosine nucleotide synthesis.

The apoptotic or necrotic death of renal tubule epithelial cells is the main pathogenesis of renal ischemia-reperfusion-induced acute kidney injury (AKI). Pyroptosis is a programmed cell death pathway that depends on the activation of the caspase cascade and IL-1 cytokine family members. However, the role of pyroptosis in AKI induced by ischemia-reperfusion remains unclear. In this study, we found that the levels of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1β, were significantly increased after 6 h of renal ischemia-reperfusion injury (IRI) and peaked at 12 h after IRI. Enhanced pyroptosis was accompanied by elevated renal structural and functional injury. Similarly, hypoxia-reoxygenation injury (HRI) also induced pyroptosis in renal tubule epithelial NRK-52E cells, which was characterized by increased pore formation and elevated lactate dehydrogenase release. In addition, obvious upregulation of the endoplasmic reticulum (ER) stress biomarkers glucose-regulated protein 78 and C/EBP homologous protein (CHOP) preceded the incidence of pyroptosis in cells treated with IRI or HRI. Pretreatment with a low dose of tunicamycin, an inducer of ER stress, relieved IRI-induced pyroptosis and renal tissue injury. Silencing of CHOP by small interfering RNA significantly decreased HRI-induced pyroptosis of NRK-52E cells, as evidenced by reduced caspase-11 activity and IL-1β generation. Therefore, we conclude that pyroptosis of renal tubule epithelial cells is a key event during IRI and that CHOP-caspase-11 triggered by overactivated ER stress may be an essential pathway involved in pyroptosis.

Tissue-specific gene expression requires coordinated control of gene-proximal and-distal cisregulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of rewriting enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.