Ryan A Flynn

All Publications

• Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders NATURE Calo, E., Gu, B., Bowen, M. E., Aryan, F., Zalc, A., Liang, J., Flynn, R. A., Swigut, T., Chang, H. Y., Attardi, L. D., Wysocka, J. 2018; 554 (7690): 112-+

  Abstract

  Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

  View details for DOI 10.1038/nature25449

  View details for Web of Science ID 000424048900041

  View details for PubMedID 29364875

  View details for PubMedCentralID PMC5927778

• The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell Simsek, D., Tiu, G. C., Flynn, R. A., Byeon, G. W., Leppek, K., Xu, A. F., Chang, H. Y., Barna, M. 2017; 169 (6): 1051-1065 e18

  Abstract

  During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.

  View details for DOI 10.1016/j.cell.2017.05.022

  View details for PubMedID 28575669

• Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts BIOCHEMISTRY Chan, D., Feng, C., Zhen, Y., Flynn, R. A., Spitale, R. C. 2017; 56 (13): 1811-1814

  Abstract

  RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2'-hydroxyl acylation can then be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2'-hydroxyl. Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment.

  View details for DOI 10.1021/acs.biochem.7b00128

  View details for Web of Science ID 000398646000002

  View details for PubMedID 28319368

• Comparison of SHAPE reagents for mapping RNA structures inside living cells RNA Lee, B., Flynn, R. A., Kadina, A., Guo, J. K., Kool, E. T., Chang, H. Y. 2017; 23 (2): 169-174

  Abstract

  Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N3 and 1M7 reagents, respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N3 give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.

  View details for DOI 10.1261/rna.058784.116

  View details for Web of Science ID 000392883800006

  View details for PubMedID 27879433

  View details for PubMedCentralID PMC5238792

• Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics Mumbach, M. R., Satpathy, A. T., Boyle, E. A., Dai, C., Gowen, B. G., Cho, S. W., Nguyen, M. L., Rubin, A. J., Granja, J. M., Kazane, K. R., Wei, Y., Nguyen, T., Greenside, P. G., Corces, M. R., Tycko, J., Simeonov, D. R., Suliman, N., Li, R., Xu, J., Flynn, R. A., Kundaje, A., Khavari, P. A., Marson, A., Corn, J. E., Quertermous, T., Greenleaf, W. J., Chang, H. Y. 2017

  Abstract

  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.

  View details for DOI 10.1038/ng.3963

  View details for PubMedID 28945252

• Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing PLOS ONE Xu, J., Spitale, R. C., Guan, L., Flynn, R. A., Torre, E. A., Li, R., Reber, I., Qu, K., Kern, D., Knaggs, H. E., Chang, H. Y., Chang, A. L. 2016; 11 (11)

  View details for DOI 10.1371/journal.pone.0165913

  View details for Web of Science ID 000387724300066

• HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature methods Mumbach, M. R., Rubin, A. J., Flynn, R. A., Dai, C., Khavari, P. A., Greenleaf, W. J., Chang, H. Y. 2016; 13 (11): 919-922

  Abstract

  Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.

  View details for DOI 10.1038/nmeth.3999

  View details for PubMedID 27643841

• An inducible long noncoding RNA amplifies DNA damage signaling. Nature genetics Schmitt, A. M., Garcia, J. T., Hung, T., Flynn, R. A., Shen, Y., Qu, K., Payumo, A. Y., Peres-da-Silva, A., Broz, D. K., Baum, R., Guo, S., Chen, J. K., Attardi, L. D., Chang, H. Y. 2016; 48 (11): 1370-1376

  Abstract

  Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.

  View details for DOI 10.1038/ng.3673

  View details for PubMedID 27668660

  View details for PubMedCentralID PMC5083181

• irCLIP platform for efficient characterization of protein-RNA interactions NATURE METHODS Zarnegar, B. J., Flynn, R. A., Shen, Y., Do, B. T., Chang, H. Y., Khavari, P. A. 2016; 13 (6): 489-?

  Abstract

  The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

  View details for DOI 10.1038/NMETH.3840

  View details for Web of Science ID 000377480100014

  View details for PubMedID 27111506

• RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure CELL Lu, Z., Zhang, Q. C., Lee, B., Flynn, R. A., Smith, M. A., Robinson, J. T., Davidovich, C., Gooding, A. R., Goodrich, K. J., Mattick, J. S., Mesirov, J. P., Cech, T. R., Chang, H. Y. 2016; 165 (5): 1267-1279

  Abstract

  RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2016.04.028

  View details for Web of Science ID 000376478200024

  View details for PubMedID 27180905

• Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress ONCOTARGET Oh, S., Flynn, R. A., Floor, S. N., Purzner, J., Martin, L., Do, B. T., Schubert, S., Vaka, D., Morrissy, S., Li, Y., Kool, M., Hovestadt, V., Jones, D. T., Northcott, P. A., Risch, T., Warnatz, H., Yaspo, M., Adams, C. M., Leib, R. D., Breese, M., Marra, M. A., Malkin, D., Lichter, P., Doudna, J. A., Pfister, S. M., Taylor, M. D., Chang, H. Y., Cho, Y. 2016; 7 (19): 28169-28182

  View details for DOI 10.18632/oncotarget.8612

  View details for Web of Science ID 000377741700097

• Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell Tan, J. L., Fogley, R. D., Flynn, R. A., Ablain, J., Yang, S., Saint-André, V., Fan, Z. P., Do, B. T., Laga, A. C., Fujinaga, K., Santoriello, C., Greer, C. B., Kim, Y. J., Clohessy, J. G., Bothmer, A., Pandell, N., Avagyan, S., Brogie, J. E., van Rooijen, E., Hagedorn, E. J., Shyh-Chang, N., White, R. M., Price, D. H., Pandolfi, P. P., Peterlin, B. M., Zhou, Y., Kim, T. H., Asara, J. M., Chang, H. Y., Young, R. A., Zon, L. I. 2016; 62 (1): 34-46

  Abstract

  Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

  View details for DOI 10.1016/j.molcel.2016.03.013

  View details for PubMedID 27058786

  View details for PubMedCentralID PMC4836061

• 7SK-BAF axis controls pervasive transcription at enhancers. Nature structural & molecular biology Flynn, R. A., Do, B. T., Rubin, A. J., Calo, E., Lee, B., Kuchelmeister, H., Rale, M., Chu, C., Kool, E. T., Wysocka, J., Khavari, P. A., Chang, H. Y. 2016; 23 (3): 231-238

  Abstract

  RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells, and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex, recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers, enhancers and promoters.

  View details for DOI 10.1038/nsmb.3176

  View details for PubMedID 26878240

  View details for PubMedCentralID PMC4982704

• Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature protocols Flynn, R. A., Zhang, Q. C., Spitale, R. C., Lee, B., Mumbach, M. R., Chang, H. Y. 2016; 11 (2): 273-290

  Abstract

  icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

  View details for DOI 10.1038/nprot.2016.011

  View details for PubMedID 26766114

  View details for PubMedCentralID PMC4896316

• Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing. PloS one Xu, J., Spitale, R. C., Guan, L., Flynn, R. A., Torre, E. A., Li, R., Raber, I., Qu, K., Kern, D., Knaggs, H. E., Chang, H. Y., Chang, A. L. 2016; 11 (11)

  Abstract

  While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18-89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3'-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans.

  View details for DOI 10.1371/journal.pone.0165913

  View details for PubMedID 27829007

  View details for PubMedCentralID PMC5102383

• DDX5 and its associated lncRNA Rmrp modulate T(H)17 cell effector functions NATURE Huang, W., Thomas, B., Flynn, R. A., Gavzy, S. J., Wu, L., Kim, S. V., Hall, J. A., Miraldi, E. R., Ng, C. P., Rigo, F. W., Meadows, S., Montoya, N. R., Herrera, N. G., Domingos, A. I., Rastinejad, F., Myers, R. M., Fuller-Pace, F. V., Bonneau, R., Chang, H. Y., Acuto, O., Littman, D. R. 2015; 528 (7583): 517-?

  View details for DOI 10.1038/nature16193

  View details for Web of Science ID 000366991900044

  View details for PubMedID 26675721

• Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation CELL STEM CELL Mazumdar, C., Shen, Y., Xavy, S., Zhao, F., Reinisch, A., Li, R., Corces, M. R., Flynn, R. A., Buenrostro, J. D., Chan, S. M., Thomas, D., Koenig, J. L., Hong, W., Chang, H. Y., Majeti, R. 2015; 17 (6): 1-14

  View details for DOI 10.1016/j.stem.2015.09.017

  View details for Web of Science ID 000366139100010

• Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell stem cell Mazumdar, C., Shen, Y., Xavy, S., Zhao, F., Reinisch, A., Li, R., Corces, M. R., Flynn, R. A., Buenrostro, J. D., Chan, S. M., Thomas, D., Koenig, J. L., Hong, W., Chang, H. Y., Majeti, R. 2015; 17 (6): 675-688

  Abstract

  Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.

  View details for DOI 10.1016/j.stem.2015.09.017

  View details for PubMedID 26607380

  View details for PubMedCentralID PMC4671831

• Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells. Nature Grow, E. J., Flynn, R. A., Chavez, S. L., Bayless, N. L., Wossidlo, M., Wesche, D. J., Martin, L., Ware, C. B., Blish, C. A., Chang, H. Y., Pera, R. A., Wysocka, J. 2015; 522 (7555): 221-225

  Abstract

  Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

  View details for DOI 10.1038/nature14308

  View details for PubMedID 25896322

  View details for PubMedCentralID PMC4503379

• Structural imprints in vivo decode RNA regulatory mechanisms. Nature Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., Chang, H. Y. 2015; 519 (7544): 486-490

  Abstract

  Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  View details for DOI 10.1038/nature14263

  View details for PubMedID 25799993

  View details for PubMedCentralID PMC4376618

• RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature Calo, E., Flynn, R. A., Martin, L., Spitale, R. C., Chang, H. Y., Wysocka, J. 2015; 518 (7538): 249-253

  Abstract

  DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

  View details for DOI 10.1038/nature13923

  View details for PubMedID 25470060

• Dissecting noncoding and pathogen RNA-protein interactomes RNA-A PUBLICATION OF THE RNA SOCIETY Flynn, R. A., Martin, L., Spitale, R. C., Do, B. T., Sagan, S. M., Zarnegar, B., Qu, K., Khavari, P. A., Quake, S. R., Sarnow, P., Chang, H. Y. 2015; 21 (1): 135-143

  Abstract

  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.

  View details for DOI 10.1261/rna.047803.114

  View details for Web of Science ID 000346896200011

  View details for PubMedID 25411354

  View details for PubMedCentralID PMC4274633

• m(6)A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells. Cell stem cell Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., Carter, A. C., Flynn, R. A., Zhou, C., Lim, K., Dedon, P., Wernig, M., Mullen, A. C., Xing, Y., Giallourakis, C. C., Chang, H. Y. 2014; 15 (6): 707-719

  Abstract

  N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

  View details for DOI 10.1016/j.stem.2014.09.019

  View details for PubMedID 25456834

  View details for PubMedCentralID PMC4278749

• RNA structural analysis by evolving SHAPE chemistry. Wiley interdisciplinary reviews. RNA Spitale, R. C., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2014; 5 (6): 867-881

  Abstract

  RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.

  View details for DOI 10.1002/wrna.1253

  View details for PubMedID 25132067

• Long noncoding RNAs in cell-fate programming and reprogramming. Cell stem cell Flynn, R. A., Chang, H. Y. 2014; 14 (6): 752-761

  Abstract

  In recent years, long noncoding RNAs (lncRNAs) have emerged as an important class of regulators of gene expression. lncRNAs exhibit several distinctive features that confer unique regulatory functions, including exquisite cell- and tissue-specific expression and the capacity to transduce higher-order spatial information. Here we review evidence showing that lncRNAs exert critical functions in adult tissue stem cells, including skin, brain, and muscle, as well as in developmental patterning and pluripotency. We highlight new approaches for ascribing lncRNA functions and discuss mammalian dosage compensation as a classic example of an lncRNA network coupled to stem cell differentiation.

  View details for DOI 10.1016/j.stem.2014.05.014

  View details for PubMedID 24905165

• Landscape and variation of RNA secondary structure across the human transcriptome. Nature Wan, Y., Qu, K., Zhang, Q. C., Flynn, R. A., Manor, O., Ouyang, Z., Zhang, J., Spitale, R. C., Snyder, M. P., Segal, E., Chang, H. Y. 2014; 505 (7485): 706-709

  Abstract

  In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

  View details for DOI 10.1038/nature12946

  View details for PubMedID 24476892

• Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency. eLife Yang, Y. W., Flynn, R. A., Chen, Y., Qu, K., Wan, B., Wang, K. C., Lei, M., Chang, H. Y. 2014; 3

  Abstract

  The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001.

  View details for DOI 10.7554/eLife.02046

  View details for PubMedID 24521543

• Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature Kretz, M., Siprashvili, Z., Chu, C., Webster, D. E., Zehnder, A., Qu, K., Lee, C. S., Flockhart, R. J., Groff, A. F., Chow, J., Johnston, D., Kim, G. E., Spitale, R. C., Flynn, R. A., Zheng, G. X., Aiyer, S., Raj, A., Rinn, J. L., Chang, H. Y., Khavari, P. A. 2013; 493 (7431): 231-235

  Abstract

  Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

  View details for DOI 10.1038/nature11661

  View details for PubMedID 23201690

  View details for PubMedCentralID PMC3674581

• RNA SHAPE analysis in living cells. Nature chemical biology Spitale, R. C., Crisalli, P., Flynn, R. A., Torre, E. A., Kool, E. T., Chang, H. Y. 2013; 9 (1): 18-20

  Abstract

  RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

  View details for DOI 10.1038/nchembio.1131

  View details for PubMedID 23178934

• Active chromatin and noncoding RNAs: an intimate relationship CURRENT OPINION IN GENETICS & DEVELOPMENT Flynn, R. A., Chang, H. Y. 2012; 22 (2): 172-178

  Abstract

  Eukaryotic genomes are packaged into chromatin, where diverse histone modifications can demarcate chromatin domains that facilitate or block gene expression. While silent chromatin has been associated with long noncoding RNAs (lncRNAs) for some time, new studies suggest that noncoding RNAs also modulate the active chromatin state. Divergent, antisense, and enhancer-like intergenic noncoding RNAs can either activate or repress gene expression by altering histone H3 lysine 4 methylation. An emerging class of enhancer-like lncRNAs may link chromosome structure to chromatin state and establish active chromatin domains. The confluence of several new technologies promises to rapidly expand this fascinating topic of investigation.

  View details for DOI 10.1016/j.gde.2011.11.002

  View details for Web of Science ID 000304338000016

  View details for PubMedID 22154525

• Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers GENOME BIOLOGY Brunner, A. L., Beck, A. H., Edris, B., Sweeney, R. T., Zhu, S. X., Li, R., Montgomery, K., Varma, S., Gilks, T., Guo, X., Foley, J. W., Witten, D. M., Giacomini, C. P., Flynn, R. A., Pollack, J. R., Tibshirani, R., Chang, H. Y., van de Rijn, M., West, R. B. 2012; 13 (8)

  Abstract

  BACKGROUND: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. RESULTS: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. CONCLUSIONS: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.

  View details for Web of Science ID 000315867500009

• Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Flynn, R. A., Almada, A. E., Zamudio, J. R., Sharp, P. A. 2011; 108 (26): 10460-10465

  Abstract

  Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5' termini and heterogeneous nonpolyadenylated 3' ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1-4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome.

  View details for DOI 10.1073/pnas.1106630108

  View details for Web of Science ID 000292251000024

  View details for PubMedID 21670248

• A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

  Abstract

  The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

  View details for DOI 10.1038/nature09819

  View details for Web of Science ID 000289199400049

  View details for PubMedID 21423168

  View details for PubMedCentralID PMC3670758

• A unique chromatin signature uncovers early developmental enhancers in humans NATURE Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, J. 2011; 470 (7333): 279-?

  Abstract

  Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.

  View details for DOI 10.1038/nature09692

  View details for Web of Science ID 000287144200048

  View details for PubMedID 21160473

• c-Myc Regulates Transcriptional Pause Release CELL Rahl, P. B., Lin, C. Y., Seila, A. C., Flynn, R. A., McCuine, S., Burge, C. B., Sharp, P. A., Young, R. A. 2010; 141 (3): 432-445

  Abstract

  Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.

  View details for DOI 10.1016/j.cell.2010.03.030

  View details for Web of Science ID 000277180800015

  View details for PubMedID 20434984

• Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth MOLECULAR BIOSYSTEMS Huang, P. H., Miraldi, E. R., Xu, A. M., Kundukulam, V. A., Del Rosario, A. M., Flynn, R. A., Cavenee, W. K., Furnari, F. B., White, F. M. 2010; 6 (7): 1227-1237

  Abstract

  To evaluate the role of individual EGFR phosphorylation sites in activating components of the cellular signaling network we have performed a mass spectrometry-based analysis of the phosphotyrosine network downstream of site-specific EGFRvIII mutants, enabling quantification of network-level effects of site-specific point mutations. Mutation at Y845, Y1068 or Y1148 resulted in diminished receptor phosphorylation, while mutation at Y1173 led to increased phosphorylation on multiple EGFRvIII residues. Altered phosphorylation at the receptor was recapitulated in downstream signaling network activation levels, with Y1173F mutation leading to increased phosphorylation throughout the network. Computational modeling of GBM cell growth as a function of network phosphorylation levels highlights the Erk pathway as crucial for regulating EGFRvIII-driven U87MG GBM cell behavior, with the unexpected finding that Erk1/2 is negatively correlated to GBM cell growth. Genetic manipulation of this pathway supports the model, demonstrating that EGFRvIII-expressing U87MG GBM cells are sensitive to Erk activation levels. Additionally, we developed a model describing glioblastoma cell growth based on a reduced set of phosphoproteins, which represent potential candidates for future development as therapeutic targets for EGFRvIII-positive glioblastoma patients.

  View details for DOI 10.1039/c001196g

  View details for Web of Science ID 000278861500012

  View details for PubMedID 20461251

• Divergent Transcription from Active Promoters SCIENCE Seila, A. C., Calabrese, J. M., Levine, S. S., Yeo, G. W., Rahl, P. B., Flynn, R. A., Young, R. A., Sharp, P. A. 2008; 322 (5909): 1849-1851

  Abstract

  Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site-associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.

  View details for DOI 10.1126/science.1162253

  View details for Web of Science ID 000261799400056

  View details for PubMedID 19056940

• Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huang, P. H., Mukasa, A., Bonavia, R., Flynn, R. A., Brewer, Z. E., Cavenee, W. K., Furnari, F. B., White, F. M. 2007; 104 (31): 12867-12872

  Abstract

  Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) commonly found in GBMs that confers enhanced tumorigenic behavior. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have performed a large-scale analysis of EGFRvIII-activated phosphotyrosine-mediated signaling pathways and thereby have identified and quantified 99 phosphorylation sites on 69 proteins. Distinct signaling responses were observed as a function of titrated EGFRvIII receptor levels with the phosphatidylinositol 3-kinase pathway being dominant over the MAPK and STAT3 pathways at a high level of EGFRvIII expression. Within this data set, the activating phosphorylation site on the c-Met receptor was found to be highly responsive to EGFRvIII levels, indicating cross-activation of the c-Met receptor tyrosine kinase by EGFRvIII. To determine the significance of this finding, we devised a combined treatment regimen that used a c-Met kinase inhibitor and either an EGFR kinase inhibitor or cisplatin. This regimen resulted in enhanced cytotoxicity of EGFRvIII-expressing cells compared with treatment with either compound alone. These results suggest that the clinical use of c-Met kinase inhibitors in combination with either EGFR inhibitors or standard chemotherapeutics might represent a previously undescribed therapeutic approach to overcome the observed chemoresistance in patients with GBMs expressing EGFRvIII.

  View details for DOI 10.1073/pnas.0705158104

  View details for Web of Science ID 000248603900050

  View details for PubMedID 17646646

• Diagnosis, treatment, and management of breast cancer in previously augmented women BREAST JOURNAL Tuli, R., Flynn, R. A., Brill, K. L., Sabol, J. L., Usuki, K. Y., Rosenberg, A. L. 2006; 12 (4): 343-348

  Abstract

  Augmentation mammaplasty is rapidly becoming one of the most frequently performed cosmetic surgeries. However, as the augmented patient population ages, major concerns associated with the screening, diagnosis and treatment of breast cancer are being realized. Although current evidence convincingly indicates that breast implants do not play a role in inducing localized or systemic disease, particularly breast cancer, recent studies have shown implants not only reduce the sensitivity of mammography, but interfere with mammographic detection, possibly leading to delayed breast cancer diagnosis. In addition, the risk for local recurrence, as well as unfavorable cosmetic results, breast fibrosis, and capsular contracture following radiation therapy as part of breast-conserving therapy in previously augmented patients are of great concern. Given the overall lack of treatment consensus, paucity of literature, and increasing number of augmented breast cancer patients, we provide a retrospective review of the diagnosis, treatment, and follow-up of 12 augmented patients from 1998 to 2004 who developed breast cancer. Eight of 12 augmented patients presented with a palpable mass on physical examination, which prompted further mammographic evaluation. Abnormalities in the remaining four individuals were detected on routine mammographic screening. Pathology staging results were available for all 12 patients. Breast-conserving therapy was used to treat six patients and adequate negative pathologic margins were obtained in all patients. The remaining six patients were treated with mastectomy due to multifocal disease, inadequate margins, or proximity to the implant capsule. Thus far, one patient has had local recurrence and one patient has had distant recurrence after initial surgery. No evidence of local or systemic recurrence, infection, contracture, poor cosmetic outcome, or other complications has been detected in the remaining 10 patients as of the most recent follow-up. Based on this small cohort of augmented women, the presence of implants led to an increased proportion of palpable tumors, in spite of routine screening mammography. Consistent with other studies, although our results suggest a tendency toward delayed diagnosis in augmented women relative to age-matched controls, this did not appear to influence the overall prognosis.

  View details for DOI 10.1111/j.1075-122X.2006.00273.x

  View details for Web of Science ID 000238656000008

  View details for PubMedID 16848844