Publications by authors named "Elizabeth T Bartom"

29 Publications

  • Page 1 of 1

The transcriptional repressor ID2 supports natural killer cell maturation by controlling TCF1 amplitude.

J Exp Med 2021 Jun;218(6)

Department of Pathology, Committees on Immunology and Cancer Biology, The University of Chicago, Chicago, IL.

Gaining a mechanistic understanding of the expansion and maturation program of natural killer (NK) cells will provide opportunities for harnessing their inflammation-inducing and oncolytic capacity for therapeutic purposes. Here, we demonstrated that ID2, a transcriptional regulatory protein constitutively expressed in NK cells, supports NK cell effector maturation by controlling the amplitude and temporal dynamics of the transcription factor TCF1. TCF1 promotes immature NK cell expansion and restrains differentiation. The increased TCF1 expression in ID2-deficient NK cells arrests their maturation and alters cell surface receptor expression. Moreover, TCF1 limits NK cell functions, such as cytokine-induced IFN-γ production and the ability to clear metastatic melanoma in ID2-deficient NK cells. Our data demonstrate that ID2 sets a threshold for TCF1 during NK cell development, thus controlling the balance of immature and terminally differentiated cells that support future NK cell responses.
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http://dx.doi.org/10.1084/jem.20202032DOI Listing
June 2021

Quantitative and multiplexed chemical-genetic phenotyping in mammalian cells with QMAP-Seq.

Nat Commun 2020 11 12;11(1):5722. Epub 2020 Nov 12.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.

Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types-comprising 12 genetic perturbations in five cell lines-with 1440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.
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http://dx.doi.org/10.1038/s41467-020-19553-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661543PMC
November 2020

Kaposi's Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma.

mBio 2020 08 25;11(4). Epub 2020 Aug 25.

Department of Microbiology-Immunology, Northwestern University, Chicago, Illinois, USA

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas. Kaposi's sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.
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http://dx.doi.org/10.1128/mBio.01457-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448273PMC
August 2020

Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb and DNA methylation.

Nat Genet 2020 06 11;52(6):615-625. Epub 2020 May 11.

Simpson Querrey Center for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.
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http://dx.doi.org/10.1038/s41588-020-0618-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790509PMC
June 2020

Ezh2 Represses Transcription of Innate Lymphoid Genes in B Lymphocyte Progenitors and Maintains the B-2 Cell Fate.

J Immunol 2020 04 24;204(7):1760-1769. Epub 2020 Feb 24.

Committee on Immunology, The University of Chicago, Chicago, IL 60657;

Lymphocyte lineage specification and commitment requires the activation of lineage-specific genes and repression of alternative lineage genes, respectively. The mechanisms governing alternative lineage gene repression and commitment in lymphocytes are largely unknown. In this study, we demonstrate that Ezh2, which represses gene expression through methylation of histone 3 lysine 27, was essential for repression of numerous genes, including genes encoding innate lymphocyte transcription factors, specifically in murine B lymphocyte progenitors, but these cells maintained their B lymphocyte identity. However, adult Ezh2-deficient B lymphocytes expressed , which encodes an RNA-binding protein associated with fetal hematopoietic gene expression programs, and these cells acquired a fetal B-1 lymphocyte phenotype in vitro and in vivo. Therefore, Ezh2 coordinates the repression of multiple gene programs in B lymphocytes and maintains the adult B-2 cell fate.
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http://dx.doi.org/10.4049/jimmunol.1901188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344269PMC
April 2020

6mer Seed Toxicity in Viral microRNAs.

iScience 2020 Feb 11;23(2):100737. Epub 2019 Dec 11.

Division Hematology/Oncology, Department of Medicine, Northwestern University, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL 60611, USA. Electronic address:

MicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.
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http://dx.doi.org/10.1016/j.isci.2019.11.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033618PMC
February 2020

The Oncogenic Kaposi's Sarcoma-Associated Herpesvirus Encodes a Mimic of the Tumor-Suppressive miR-15/16 miRNA Family.

Cell Rep 2019 12;29(10):2961-2969.e6

Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Many tumor viruses encode oncogenes of cellular origin. Here, we report an oncoviral mimic of a cellular tumor suppressor. The Kaposi's sarcoma-associated herpesvirus (KSHV) microRNA (miRNA) miR-K6-5p shares sequence similarity to the tumor-suppressive cellular miR-15/16 miRNA family. We show that miR-K6-5p inhibits cell cycle progression, a hallmark function of miR-16. miR-K6-5p regulates conserved miR-15/16 family miRNA targets, including many cell cycle regulators. Inhibition of miR-K6-5p in KSHV-transformed B cells confers a significant growth advantage. Altogether, our data show that KSHV encodes a functional mimic of miR-15/16 family miRNAs. While it is exceedingly well established that oncogenic viruses encode oncogenes of cellular origin, this is an unusual example of an oncogenic virus that encodes a viral mimic of a cellular tumor suppressor. Encoding a tumor-suppressive miRNA could help KSHV balance viral oncogene expression and thereby avoid severe pathogenesis in the healthy host.
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http://dx.doi.org/10.1016/j.celrep.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939447PMC
December 2019

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity within a loop domain.

J Cell Sci 2019 06 19;132(12). Epub 2019 Jun 19.

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA

Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved β-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plant - but not uncharged yeast or human loop - enhances translation of mRNAs with adenosine-rich 5' untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.
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http://dx.doi.org/10.1242/jcs.228908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602301PMC
June 2019

The Coincidence Between Increasing Age, Immunosuppression, and the Incidence of Patients With Glioblastoma.

Front Pharmacol 2019 27;10:200. Epub 2019 Mar 27.

Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, United States.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and is associated with a median overall survival (mOS) of 16-21 months. Our previous work found a negative association between advanced aging and the survival benefit after treatment with immunotherapy in an experimental brain tumor model. Given the recent phase III clinical success of immunotherapy in patients with many types of cancer, but not for patients with GBM, we hypothesize that aging enhances immunosuppression in the brain and contributes to the lack of efficacy for immunotherapy to improve mOS in patients with malignant glioma. Herein, we compare epidemiological data for the incidence and mortality of patients with central nervous system (CNS) cancers, in addition to immune-related gene expression in the normal human brain, as well as peripheral blood immunological changes across the adult lifespan. Data were extracted from the National Cancer Institute's surveillance, epidemiology, and end results (SEER)-, the Broad Institute's Genotype Tissue Expression project (GTEx)-, and the University of California San Francisco's 10k Immunomes-databases and analyzed for associations with aging. The proportion of elderly individuals, defined as ≥65 years of age, has predominantly increased for more than 100 years in the United States. Over time, the rise in elderly United States citizens has correlated with an increased incidence and mortality rate associated with primary brain and other CNS cancer. With advanced aging, human mRNA expression for factors associated with immunoregulation including immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO) and programmed death-ligand 1 (PD-L1), as well as the dendritic cell surface marker, CD11c, increase in the brain of normal human subjects, coincident with increased circulating immunosuppressive Tregs and decreased cytolytic CD8 T cells in the peripheral blood. Strikingly, these changes are maximally pronounced in the 60-69 year old group; consistent with the median age of a diagnosis for GBM. These data demonstrate a significant association between normal human aging and increased immunosuppression in the circulation and CNS; particularly late in life. Our data raise several hypotheses including that, aging: (i) progressively suppresses normal immunosurveillance and thereby contributes to GBM cell initiation and/or outgrowth; (ii) decreases immunotherapeutic efficacy against malignant glioma.
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http://dx.doi.org/10.3389/fphar.2019.00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446059PMC
March 2019

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Genes Dev 2019 01 20;33(1-2):61-74. Epub 2018 Dec 20.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.

Chromosomal translocations of the Mixed-lineage leukemia 1 () gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.
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http://dx.doi.org/10.1101/gad.319830.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317322PMC
January 2019

6mer seed toxicity in tumor suppressive microRNAs.

Nat Commun 2018 10 29;9(1):4504. Epub 2018 Oct 29.

Department of Medicine, Division Hematology/Oncology, Northwestern University, Chicago, IL, 60611, USA.

Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2-7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3'UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.
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http://dx.doi.org/10.1038/s41467-018-06526-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206098PMC
October 2018

CD95/Fas ligand mRNA is toxic to cells.

Elife 2018 10 16;7. Epub 2018 Oct 16.

Department of Medicine, Division Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, United States.

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported that CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA itself is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) which is required for the toxicity and processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.
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http://dx.doi.org/10.7554/eLife.38621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191286PMC
October 2018

USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia.

Clin Cancer Res 2019 01 17;25(1):222-239. Epub 2018 Sep 17.

Department of Pathology, New York University, New York, New York.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.

Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.

Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth and .

Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320313PMC
January 2019

ZNF598 Plays Distinct Roles in Interferon-Stimulated Gene Expression and Poxvirus Protein Synthesis.

Cell Rep 2018 05;23(5):1249-1258

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address:

Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5' poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.
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http://dx.doi.org/10.1016/j.celrep.2018.03.132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951170PMC
May 2018

A Beginner's Guide to Analysis of RNA Sequencing Data.

Am J Respir Cell Mol Biol 2018 08;59(2):145-157

5 Division of Rheumatology, Department of Medicine, Northwestern University, Chicago, Illinois.

Since the first publications coining the term RNA-seq (RNA sequencing) appeared in 2008, the number of publications containing RNA-seq data has grown exponentially, hitting an all-time high of 2,808 publications in 2016 (PubMed). With this wealth of RNA-seq data being generated, it is a challenge to extract maximal meaning from these datasets, and without the appropriate skills and background, there is risk of misinterpretation of these data. However, a general understanding of the principles underlying each step of RNA-seq data analysis allows investigators without a background in programming and bioinformatics to critically analyze their own datasets as well as published data. Our goals in the present review are to break down the steps of a typical RNA-seq analysis and to highlight the pitfalls and checkpoints along the way that are vital for bench scientists and biomedical researchers performing experiments that use RNA-seq.
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http://dx.doi.org/10.1165/rcmb.2017-0430TRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096346PMC
August 2018

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells.

EMBO Rep 2018 03 12;19(3). Epub 2018 Feb 12.

Division of Hematology/Oncology, Northwestern University, Chicago, IL, USA

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.
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http://dx.doi.org/10.15252/embr.201745336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836092PMC
March 2018

Transcriptional Profiling of Synovial Macrophages Using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis.

Arthritis Rheumatol 2018 06 3;70(6):841-854. Epub 2018 May 3.

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Objective: Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA.

Methods: Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients.

Results: Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy.

Conclusion: Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.
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http://dx.doi.org/10.1002/art.40453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984677PMC
June 2018

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism.

Elife 2017 10 24;6. Epub 2017 Oct 24.

Division of Hematology and Oncology, Department of Medicine, Northwestern University, Chicago, United States.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.
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http://dx.doi.org/10.7554/eLife.29702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655136PMC
October 2017

Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability.

Nat Genet 2017 Nov 2;49(11):1647-1653. Epub 2017 Oct 2.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.
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http://dx.doi.org/10.1038/ng.3965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663216PMC
November 2017

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.

J Exp Med 2017 Aug 10;214(8):2387-2404. Epub 2017 Jul 10.

Division of Thoracic Surgery, Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL.

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.
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http://dx.doi.org/10.1084/jem.20162152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551573PMC
August 2017

EZH2 Regulates the Developmental Timing of Effectors of the Pre-Antigen Receptor Checkpoints.

J Immunol 2017 06 10;198(12):4682-4691. Epub 2017 May 10.

Committee on Immunology, The University of Chicago, Chicago, IL 60637;

The histone methyltransferase EZH2 is required for B and T cell development; however, the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid-specific EZH2 deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of and the consequent stabilization of p53, an effector of the pre-Ag receptor checkpoints. Deletion of in EZH2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-Ag receptor checkpoints.
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http://dx.doi.org/10.4049/jimmunol.1700319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527689PMC
June 2017

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.

Genes Dev 2017 04 9;31(8):787-801. Epub 2017 May 9.

Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two -regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 -regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.
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http://dx.doi.org/10.1101/gad.294744.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435891PMC
April 2017

Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas.

Nat Med 2017 Apr 27;23(4):493-500. Epub 2017 Feb 27.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.
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http://dx.doi.org/10.1038/nm.4296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667640PMC
April 2017

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

Mol Cell 2017 Feb;65(3):460-475.e6

Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, 320 East Superior Street, Chicago, IL 60611, USA; Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, 320 E. Superior St., Chicago, IL 60611, USA. Electronic address:

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.
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http://dx.doi.org/10.1016/j.molcel.2017.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404503PMC
February 2017

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Cell 2017 Jan 5;168(1-2):59-72.e13. Epub 2017 Jan 5.

Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 320 E. Superior St., Chicago, IL 60611, USA; Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, 320 E. Superior St., Chicago, Il 60611, USA. Electronic address:

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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http://dx.doi.org/10.1016/j.cell.2016.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351781PMC
January 2017

Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

Genes Dev 2016 Jan;30(1):92-101

Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA; Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA; Robert H. Lurie National Cancer Institute Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.
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http://dx.doi.org/10.1101/gad.270413.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701981PMC
January 2016

CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia.

Blood 2013 Feb 3;121(6):975-83. Epub 2012 Dec 3.

Institute for Genomics and Systems Biology, Department of Pathology, University of Chicago, IL 60637, USA.

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in hematologic malignancies, including acute myeloid leukemia and therapy-related myeloid neoplasms, and associated with an adverse prognosis. Despite intensive effort by many laboratories, the putative myeloid tumor suppressor(s) on chromosome 7 has not yet been identified.We performed transcriptome sequencing and SNP array analysis on de novo and therapy-related myeloid neoplasms, half with -7/del(7q). We identified a 2.17-Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor. In 1 case, CUX1 was disrupted by a translocation, resulting in a loss-of-function RNA fusion transcript. CUX1 was the most significantly differentially expressed gene within the commonly deleted segment and was expressed at haploinsufficient levels in -7/del(7q) leukemias. Haploinsufficiency of the highly conserved ortholog, cut, led to hemocyte overgrowth and tumor formation in Drosophila melanogaster. Similarly, haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation into immunodeficient mice. Within the RNA-sequencing data, we identified a CUX1-associated cell cycle transcriptional gene signature, suggesting that CUX1 exerts tumor suppressor activity by regulating proliferative genes. These data identify CUX1 as a conserved, haploinsufficient tumor suppressor frequently deleted in myeloid neoplasms.
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http://dx.doi.org/10.1182/blood-2012-04-426965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567344PMC
February 2013

Epigenetic repression of the Igk locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2.

Nat Immunol 2011 Oct 30;12(12):1212-20. Epub 2011 Oct 30.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA.

During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.
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http://dx.doi.org/10.1038/ni.2136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233979PMC
October 2011