Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4⁺ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.