Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.
2016; 62 (1): 34-46
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
7SK-BAF axis controls pervasive transcription at enhancers
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2016; 23 (3): 231-238
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 Web of Science ID 000371452500010
View details for PubMedID 26878240
Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE
2016; 11 (2): 273-290
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 Web of Science ID 000369084500005
View details for PubMedID 26766114
- Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation CELL STEM CELL 2015; 17 (6): 1-14
Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.
2015; 522 (7555): 221-225
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
Structural imprints in vivo decode RNA regulatory mechanisms.
2015; 519 (7544): 486-490
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
RNA helicase DDX21 coordinates transcription and ribosomal RNA processing.
2015; 518 (7538): 249-253
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
2015; 21 (1): 135-143
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
m(6)A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells
CELL STEM CELL
2014; 15 (6): 707-719
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 Web of Science ID 000347174300010
View details for PubMedID 25456834
RNA structural analysis by evolving SHAPE chemistry.
Wiley interdisciplinary reviews. RNA
2014; 5 (6): 867-881
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
2014; 14 (6): 752-761
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 Web of Science ID 000341248500012
Landscape and variation of RNA secondary structure across the human transcriptome.
2014; 505 (7485): 706-709
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.
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.
2013; 493 (7431): 231-235
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
RNA SHAPE analysis in living cells.
Nature chemical biology
2013; 9 (1): 18-20
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
2012; 22 (2): 172-178
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
2012; 13 (8)
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
2011; 108 (26): 10460-10465
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
2011; 472 (7341): 120-U158
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
A unique chromatin signature uncovers early developmental enhancers in humans
2011; 470 (7333): 279-?
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
2010; 141 (3): 432-445
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
2010; 6 (7): 1227-1237
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
2008; 322 (5909): 1849-1851
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
2007; 104 (31): 12867-12872
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
2006; 12 (4): 343-348
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