2025

1. High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion Sept, Corriene E, Tak, Y Esther, Goel, Viraat, Bhakta, Mital S, Cerda-Smith, Christian G, Hutchinson, Haley M, Blanchette, Marco, Eyler, Christine E, Johnstone, Sarah E, Joung, J Keith, Hansen, Anders S, and Aryee, Martin J Nat. Commun. 2025 [Abs]

   Cohesin-mediated DNA loop extrusion enables gene regulation by distal enhancers through the establishment of chromosome structure and long-range enhancer-promoter interactions. The best characterized cohesin-related structures, such as topologically associating domains (TADs) anchored at convergent CTCF binding sites, represent static conformations. Consequently, loop extrusion dynamics remain poorly understood. To better characterize static and dynamically extruding chromatin loop structures, we use MNase-based 3D genome assays to simultaneously determine CTCF and cohesin localization as well as the 3D contacts they mediate. Here we present CTCF Analyzer (with) Multinomial Estimation (CAMEL), a tool that identifies CTCF footprints at near base-pair resolution in CTCF MNase HiChiP. We also use Region Capture Micro-C to identify a CTCF-adjacent footprint that is attributed to cohesin occupancy. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF loop) state is rare genome-wide with locus-specific variation from  1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics and find that active regulatory elements impede cohesin extrusion. These findings support a model of topological regulation whereby the transient, partially extruded state facilitates enhancer-promoter contacts that can regulate transcription.

2024

1. Disruption of cellular plasticity by repeat RNAs in human pancreatic cancer You, Eunae, Danaher, Patrick, Lu, Chenyue, Sun, Siyu, Zou, Luli, Phillips, Ildiko E, Rojas, Alexandra S, Ho, Natalie I, Song, Yuhui, Raabe, Michael J, Xu, Katherine H, Richieri, Peter M, Li, Hao, Aston, Natalie, Porter, Rebecca L, Patel, Bidish K, Nieman, Linda T, Schurman, Nathan, Hudson, Briana M, North, Khrystyna, Church, Sarah E, Deshpande, Vikram, Liss, Andrew S, Kim, Tae K, Cui, Yi, Kim, Youngmi, Greenbaum, Benjamin D, Aryee, Martin J, and Ting, David T Cell 2024 [Abs]

   Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single-cell resolution in 46 primary tumors, revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF responses are driven by distinct innate immune signaling through interferon regulatory factor 3 (IRF3). The cell-context-specific viral-like responses to repeat RNAs provide a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment.

2023

1. Enhancer Discovery Reveals a New Model of Enhancer Organization Cerda-Smith, C. G., Hutchinson, H. M., Liu, A., Goel, V. Y., Sept, C., Kim, H., n, S., Burkman, K. G., Bassil, C. F., Hansen, A. S., Aryee, M. J., Johnstone, S. E., Eyler, C. E., and Wood, K. C. bioRxiv 2023
2. Spatial transcriptomics reveals distinct tissue niches linked with steroid responsiveness in acute gastrointestinal GVHD Patel, B. K., Raabe, M. J., Lang, E. R., Song, Y., Lu, C., Deshpande, V., Nieman, L. T., Aryee, M. J., Chen, Y. B., Ting, D. T., and DeFilipp, Z. Blood 2023

2022

1. Epigenetic clocks, aging, and cancer Johnstone, Sarah E, Gladyshev, Vadim N, Aryee, Martin J, and Bernstein, Bradley E Science 2022 [Abs]

   Global methylation changes in aging cells affect cancer risk and tissue homeostasis.

2. Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery Siegenfeld, Allison P, Roseman, Shelby A, Roh, Heejin, Lue, Nicholas Z, Wagen, Corin C, Zhou, Eric, Johnstone, Sarah E, Aryee, Martin J, and Liau, Brian B Nat. Commun. 2022 [Abs]

   The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.

2021

1. Augmenting and directing long-range CRISPR-mediated activation in human cells Tak, Y Esther, Horng, Joy E, Perry, Nicholas T, Schultz, Hayley T, Iyer, Sowmya, Yao, Qiuming, Zou, Luli S, Aryee, Martin J, Pinello, Luca, and Joung, J Keith Nat. Methods 2021 [Abs]

   Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

2020

1. Large-scale topological changes restrain malignant progression in colorectal cancer Johnstone, S. E., Reyes, A., Qi, Y., Adriaens, C., Hegazi, E., Pelka, K., Chen, J. H., Zou, L. S., Drier, Y., Hecht, V., Shoresh, N., Selig, M. K., Lareau, C. A., Iyer, S., Nguyen, S. C., Joyce, E. F., Hacohen, N., Irizarry, R. A., Zhang, B., Aryee, M. J., and Bernstein, B. E. Cell 2020 [Abs]

   Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

2. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing Grünewald, J., Zhou, R., Lareau, C. A., Garcia, S. P., Iyer, S., Miller, B. R., Langner, L. M., Hsu, J. Y., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2020

2019

1. CRISPR DNA base editors with reduced RNA off-target and self-editing activities Grünewald, J., Zhou, R., Iyer, S., Lareau, C. A., Garcia, S. P., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2019
2. Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer Ligorio, M., Sil, S., Malagon-Lopez, J., Nieman, L. T., Misale, S., Di Pilato, M., Ebright, R. Y., Karabacak, M. N., Kulkarni, A. S., Liu, A., Vincent Jordan, N., Franses, J. W., Philipp, J., Kreuzer, J., Desai, N., Arora, K. S., Rajurkar, M., Horwitz, E., Neyaz, A., Tai, E., Magnus, N. K. C., Vo, K. D., Yashaswini, C. N., Marangoni, F., Boukhali, M., Fatherree, J. P., Damon, L. J., Xega, K., Desai, R., Choz, M., Bersani, F., Langenbucher, A., Thapar, V., Morris, R., Wellner, U. F., Schilling, O., Lawrence, M. S., Liss, A. S., Rivera, M. N., Deshpande, V., Benes, C. H., Maheswaran, S., Haber, D. A., Fernandez-Del-Castillo, C., Ferrone, C. R., Haas, W., Aryee, M. J.*, and Ting, D. T.* Cell 2019
3. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors Grünewald, J., Zhou, R., Garcia, S. P., Iyer, S., Lareau, C. A., Aryee, M. J., and Joung, J. K. Nature 2019
4. A (fire)cloud-based DNA methylation data preprocessing and quality control platform Kangeyan, D., Dunford, A., Iyer, S., Stewart, C., Hanna, M., Getz, G., and Aryee, M. J. BMC Bioinformatics 2019
5. Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics Ludwig, L. S., Lareau, C. A., Ulirsch, J. C., Christian, E., Muus, C., Li, L. H., Pelka, K., Ge, W., Oren, Y., Brack, A., Law, T., Rodman, C., Chen, J. H., Boland, G. M., Hacohen, N., Rozenblatt-Rosen, O., Aryee, M. J., Buenrostro, J. D., Regev, A., and Sankaran, V. G. Cell 2019
6. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., Malagon-Lopez, J., Scarf?, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2019

2018

1. In vivo CRISPR editing with no detectable genome-wide off-target mutations Akcakaya, P., Bobbin, M. L., Guo, J. A., Malagon-Lopez, J., Clement, K., Garcia, S. P., Fellows, M. D., Porritt, M. J., Firth, M. A., Carreras, A., Baccega, T., Seeliger, F., Bjursell, M., Tsai, S. Q., Nguyen, N. T., Nitsch, R., Mayr, L. M., Pinello, L., Bohlooly-Y, M., Aryee, M. J., Maresca, M., and Joung, J. K. Nature 2018
2. hichipper: a preprocessing pipeline for calling DNA loops from HiChIP data Lareau, C. A., and Aryee, M. J. Nat. Methods 2018
3. diffloop: a computational framework for identifying and analyzing differential DNA loops from sequencing data Lareau, C. A., and Aryee, M. J. Bioinformatics 2018

2017

1. The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors Nordor, A. V., Nehar-Belaid, D., Richon, S., Klatzmann, D., Bellet, D., Dangles-Marie, V., Fournier, T., and Aryee, M. J. Epigenetics 2017 [Abs]

   The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis-while surviving hypoxia-, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

2. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets Tsai, S. Q., Nguyen, N. T., Malagon-Lopez, J., Topkar, V. V., Aryee, M. J., and Joung, J. K. Nat. Methods 2017
3. Diverse repetitive element RNA expression defines epigenetic and immunologic features of colon cancer Desai, N., Sajed, D., Arora, K. S., Solovyov, A., Rajurkar, M., Bledsoe, J. R., Sil, S., Amri, R., Tai, E., MacKenzie, O. C., Mino-Kenudson, M., Aryee, M. J., Ferrone, C. R., Berger, D. L., Rivera, M. N., Greenbaum, B. D., Deshpande, V., and Ting, D. T. JCI Insight 2017

2016

1. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells Kleinstiver, B. P., Tsai, S. Q., Prew, M. S., Nguyen, N. T., Welch, M. M., Lopez, J. M., McCaw, Z. R., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2016
2. Open-source guideseq software for analysis of GUIDE-seq data Tsai, S. Q., Topkar, V. V., Joung, J. K., and Aryee, M. J. Nat. Biotechnol. 2016

2015

1. Engineered CRISPR-Cas9 nucleases with altered PAM specificities Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P., Li, Z., Peterson, R. T., Yeh, J. R., Aryee, M. J., and Joung, J. K. Nature 2015
2. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2015
3. Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing Ziller, M. J., Hansen, K. D., Meissner, A., and Aryee, M. J. Nat. Methods 2015

2014

1. EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma Riggi, N., Knoechel, B., Gillespie, S. M., Rheinbay, E., Boulay, G., Suv?, M. L., Rossetti, N. E., Boonseng, W. E., Oksuz, O., Cook, E. B., Formey, A., Patel, A., Gymrek, M., Thapar, V., Deshpande, V., Ting, D. T., Hornicek, F. J., Nielsen, G. P., Stamenkovic, I., Aryee, M. J., Bernstein, B. E., and Rivera, M. N. Cancer Cell 2014
2. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing Tsai, S. Q., Wyvekens, N., Khayter, C., Foden, J. A., Thapar, V., Reyon, D., Goodwin, M. J., Aryee, M. J., and Joung, J. K. Nat. Biotechnol. 2014
3. Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays Aryee, M. J., Jaffe, A. E., Corrada-Bravo, H., Ladd-Acosta, C., Feinberg, A. P., Hansen, K. D., and Irizarry, R. A. Bioinformatics 2014