Search

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

table_S2_housekeeping_genes

All supplementary figures (S1-S17)

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts. In the cancerous state, we observe T cell exhaustion, RUNX1-regulated cancer-associated fibroblasts and increasing accessibility associated with HNF4A motifs in epithelia. DNA methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact “motifs,” whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array (RNA-MaP) to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of “exceptions to the rule” highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.Significance StatementProperly folded RNAs perform essential biological processes. Many RNAs contain tertiary contact “motifs” whose structural and energetic properties are conserved across different RNAs. This study delves into the nature of an RNA motif. We determined the effects of mutations on the energetic properties of a common tertiary contact motif, referred to as the 11-nucleotide receptor. As deep study of the energetic architecture of this and other RNAs requires thermodynamic measurements for many sequence variants, we used RNA-MaP, a quantitative, high-throughput method, to obtain binding free energies for all single and double mutants of the motif. Our results revealed the energetic architecture of this motif and identified rare variants with unexpected properties.

The vast majority of human pancreatic ductal adenocarcinomas (PDACs) harbor TP53 mutations, underscoring p53’s critical role in PDAC suppression. PDAC can arise when pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), giving rise to premalignant pancreatic intraepithelial neoplasias (PanINs), which finally progress to PDAC. The occurrence of TP53 mutations in late-stage PanINs has led to the idea that p53 acts to suppress malignant transformation of PanINs to PDAC. However, the cellular basis for p53 action during PDAC development has not been explored in detail. Here, we leverage a hyperactive p53 variant—p53 53,54 —which we previously showed is a more robust PDAC suppressor than wild-type p53, to elucidate how p53 acts at the cellular level to dampen PDAC development. Using both inflammation-induced and KRAS G12D -driven PDAC models, we find that p53 53,54 both limits ADM accumulation and suppresses PanIN cell proliferation and does so more effectively than wild-type p53. Moreover, p53 53,54 suppresses KRAS signaling in PanINs and limits effects on the extracellular matrix (ECM) remodeling. While p53 53,54 has highlighted these functions, we find that pancreata in wild-type p53 mice similarly show less ADM, as well as reduced PanIN cell proliferation, KRAS signaling, and ECM remodeling relative to Trp53 -null mice. We find further that p53 enhances chromatin accessibility at sites controlled by acinar cell identity transcription factors. These findings reveal that p53 acts at multiple stages to suppress PDAC, both by limiting metaplastic transformation of acini and by dampening KRAS signaling in PanINs, thus providing key new understanding of p53 function in PDAC.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conducted a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identified terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincided with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions showed enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs was inversely related to the SLAM family member CD84. Prospective isolation of CD84loGMPs demonstrated robust lymphoid potentialex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies previously unappreciated lymphoid-primed progenitors, redefining the lympho-myeloid axis in human hematopoiesis.

Genome-wide association studies (GWAS) of hair and skin disease have identified many genetic variants associated with disease phenotypes, but identifying causal variants and interpreting their function requires deciphering gene-regulatory networks in disease-relevant cell types. To this end, we generated matched scRNA- and scATAC-seq profiles of human scalp biopsies, identifying diverse cell types of the hair follicle niche. By interrogating the integrated datasets across multiple levels of cellular resolution, we infer 50-100% more enhancer-gene links than prior approaches, and show that the aggregate accessibility at linked enhancers for highly-regulated genes predicts expression. We use these gene-regulatory maps to prioritize cell types, genes, and causal variants implicated in the pathobiology of androgenetic alopecia (AGA), eczema, and other complex traits. AGA GWAS signals are strongly and specifically enriched in dermal papilla cell open chromatin regions, supporting the role of these cells as key drivers of AGA pathogenesis. Finally, we trained machine-learning models to nominate SNPs that affect gene expression through disruption of specific transcription factor binding motifs, identifying candidate functional SNPs linked to expression of WNT10A in AGA and IL18RAP in eczema. Together, this work reveals principles of gene regulation and identifies gene regulatory consequences of natural genetic variation in complex skin and hair diseases.

Although 3D genome architecture is essential for long-range gene regulation, the significance of distal regulatory chromatin contacts is challenged by recent findings of low correlation between contact propensity and gene expression. To better understand the role of long-range interactions between distal regulatory elements during the early transformation from healthy colon to colorectal cancer, here we performed high resolution chromatin conformation capture for 33 samples including non-neoplastic mucosa, adenomatous polyps and adenocarcinomas, mostly from Familial Adenomatous Polyposis (FAP) patients. We identified hundreds of thousands of chromatin micro-structures, such as architectural stripes and loops, which originated from active cis-regulatory elements. Surprisingly, these structures progressively decayed throughout cancer progression, particularly at promoters. Meta-analyses revealed that this decay was independent of alterations in DNA methylation and chromatin accessibility. Interestingly, the degree of interaction loss was poorly correlated with gene expression changes. Instead, genes whose expression were disproportionately lower and higher than their relative promoter interaction in mucosa shifted their expression in polyps and adenocarcinomas to yield a more direct relationship between strength of interaction and gene expression. Our work provides the first high resolution 3D conformation maps during early cancer formation and progression, and provides novel insights into transcriptional readouts associated with fine-scale chromatin conformation alterations.

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. The genomic targets and regulatory mechanisms of most ncRNAs remain unknown. Using chromatin-associated RNA sequencing (ChAR-seq) we obtained a global map of RNA-chromatin interactions, transcriptome and genome-wide, in human embryonic stem cells and definitive endoderm. We defined the overall architecture of the RNA-chromatin interactome that encompasses all long non-coding RNAs, intronic RNAs, as well as unannotated RNAs derived from regulatory elements, transposable elements, and intergenic regions. We show that the interactome is cell state specific, involves functionally diverse classes of RNAs, and can be accurately predicted by a simple model accounting for RNA expression and distance to their DNA targets. We show that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their transcription locus. We identified a small number of RNAs that deviate from that behavior and interact with many loci across the genome. By relating the changes in the interactome during differentiation to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by the activity of a single ncRNA.

Genomic methods have been valuable for identifying RNA-binding proteins (RBPs) and the genes, pathways, and processes they regulate. Nevertheless, standard motif descriptions cannot be used to predict all RNA targets or test quantitative models for cellular interactions and regulation. We present a complete thermodynamic model for RNA binding to the S. cerevisiae Pumilio protein PUF4 derived from direct binding data for 6180 RNAs measured using the RNA on a massively parallel array (RNA-MaP) platform. The PUF4 model is highly similar to that of the related RBPs, human PUM2 and PUM1, with one marked exception: a single favorable site of base flipping for PUF4, such that PUF4 preferentially binds to a non-contiguous series of residues. These results are foundational for developing and testing cellular models of RNA-RBP interactions and function, for engineering RBPs, for understanding the biophysical nature of RBP binding and the evolutionary landscape of RNAs and RBPs.

Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes. Despite increased interest in archaeal histones in recent years, the properties of archaeal chromatin have been little studied using genomic tools. Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeoteHaloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that whileH. volcaniipromoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection ofH. volcanii, and find thatHaloferaxchromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures. These results provide a foundation for the future functional studies of archaeal chromatin.

Gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed, it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here, we developed an approach for systematic discovery of upstream regulators of critical immune factors-IL2RA, IL-2 and CTLA4-in primary human T cells. Then, we mapped the network of the target genes of these regulators and putative cis-regulatory elements using CRISPR perturbations, RNA-seq and ATAC-seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

Familial adenomatous polyposis (FAP) patients develop hundreds of premalignant polyps that progress to colorectal cancer due to a germline mutation in the APC tumor suppressor. Polyps from FAP patients uniquely facilitate interrogation of the continuum of malignant transformation from histologically normal mucosa to benign and dysplastic polyps and eventual adenocarcinomas. As part of the Human Tumor Atlas Network (HTAN), we performed multi-omic profiling, including whole genome sequencing, on 135 samples from six FAP patients across the pre-malignant continuum spanning all physical regions of the large intestine, and serving as the most comprehensive FAP dataset available. Through a comparative analysis with a published FAP cohort (58 multi-region samples across 5 patients) and sporadic colorectal cancer cohort (n=57), each including benign and malignant samples, we evaluate the timing of driver acquisitions at each stage of malignant progression. Despite being separated by vast regions of histologically normal mucosa, independently evolving polyps show extensive mutation sharing, suggesting FAP polyps are polyclonal in origin. Finally, we leverage a simplified mechanistic model of embryonic colonic development demonstrating that the path to malignant transformation in FAP is consistent with polyclonal origins attributable to early mixing, perhaps as early as in-utero. Taken together, the HTAN FAP Atlas provides a novel window into the earliest stages of cancer formation and may illuminate barriers to malignant transformation and opportunities for earlier intervention. Citation Format: Ryan O. Schenck, Aziz Khan, Aaron Horning, Edward D. Esplin, Emma Monte, Si Wu, Casey Hanson, Nasim Bararpour, Stephanie Neves, Lihua Jiang, Kévin Contrepois, Hayan Lee, Tuhin K. Guha, Zheng Hu, Rozelle Laquindanum, Meredith A. Mills, Hassan Chaib, Roxanne Chiu, Ruiqi Jian, Jeannie Chan, Mathew Ellenberger, Winston R. Becker, Bahareh Bahmani, Basil Michael, Jeanne Shen, Samuel Lancaster, Uri Ladabaum, Anshul Kundaje, Teri A. Longacre, William J. Greenleaf, James M. Ford, Michael P. Snyder, Christina Curtis. The polyclonal path to malignant transformation in familial adenomatous polyposis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3497.

Dinoflagellate chromosomes represent a unique evolutionary experiment, as they exist in a permanently condensed, liquid crystalline state; are not packaged by histones; and contain genes organized into tandem gene arrays, with minimal transcriptional regulation. We analyze the three-dimensional genome of Breviolum minutum, and find large topological domains (dinoflagellate topologically associating domains, which we term ‘dinoTADs’) without chromatin loops, which are demarcated by convergent gene array boundaries. Transcriptional inhibition disrupts dinoTADs, implicating transcription-induced supercoiling as the primary topological force in dinoflagellates., Analysis of the three-dimensional architecture of the dinoflagellate Breviolum minutum genome identifies large topological domains (dinoTADs) demarcated by convergent gene array boundaries. Transcriptional inhibition disrupts dinoTADs.

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and their polymorphisms alter gene expression, yet how they regulate transcription remains unknown. We find that STRs can modulate transcription factor (TF)-DNA affinities and on rates by up to 70-fold by directly binding TF DNA-binding domains, with energetic impacts approaching or exceeding mutations to consensus sites. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density near motifs to speed target search. Confirming that STRs also impact TF binding in cells, neural networks trained only on in vivo occupancies predict identical effects to those observed in vitro. Approximately 90% of TFs preferentially bind STRs that need not resemble known motifs, providing a novel cis-regulatory mechanism to target TFs to cognate sites.

Internet-based scientific communities promise a means to apply distributed, diverse human intelligence toward previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale video game–based crowdsourcing of RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near–thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs. This work suggests a paradigm for widely distributed experimental bioscience.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease. Blum et al. performed single-nucleus RNA sequencing of the adult mouse spinal cord. This analysis revealed heterogeneity in the autonomic and skeletal motor systems and provides a resource to study motor neurons in health and disease.

Genome-wide association studies (GWAS) of neurological diseases have identified thousands of variants associated with disease phenotypes. However, the majority of these variants do not alter coding sequences, making it difficult to assign their function. Here, we present a multi-omic epigenetic atlas of the adult human brain through profiling of single-cell chromatin accessibility landscapes and three-dimensional (3D) chromatin interactions of diverse adult brain regions across a cohort of cognitively healthy individuals. We developed a machine-learning classifier to integrate this multi-omic framework and predict dozens of functional single-nucleotide polymorphisms (SNPs) for Alzheimer’s disease (AD) and Parkinson’s disease (PD), nominating target genes and cell types for previously orphaned GWAS loci. Moreover, we dissected the complex inverted haplotype of the MAPT (encoding tau) PD risk locus, identifying putative ectopic regulatory interactions in neurons that may mediate this disease association. This work expands our understanding of inherited variation and provides a roadmap for the epigenomic dissection of causal regulatory variation in disease.

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq, NOMe-seq and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m(6)A-SMAC-seq and m(6)A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.