Publications by authors named "Matthew D Galbraith"

23 Publications

  • Page 1 of 1

Multi-omics analysis reveals contextual tumor suppressive and oncogenic gene modules within the acute hypoxic response.

Nat Commun 2021 03 2;12(1):1375. Epub 2021 Mar 2.

Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Cellular adaptation to hypoxia is a hallmark of cancer, but the relative contribution of hypoxia-inducible factors (HIFs) versus other oxygen sensors to tumorigenesis is unclear. We employ a multi-omics pipeline including measurements of nascent RNA to characterize transcriptional changes upon acute hypoxia. We identify an immediate early transcriptional response that is strongly dependent on HIF1A and the kinase activity of its cofactor CDK8, includes indirect repression of MYC targets, and is highly conserved across cancer types. HIF1A drives this acute response via conserved high-occupancy enhancers. Genetic screen data indicates that, in normoxia, HIF1A displays strong cell-autonomous tumor suppressive effects through a gene module mediating mTOR inhibition. Conversely, in advanced malignancies, expression of a module of HIF1A targets involved in collagen remodeling is associated with poor prognosis across diverse cancer types. In this work, we provide a valuable resource for investigating context-dependent roles of HIF1A and its targets in cancer biology.
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http://dx.doi.org/10.1038/s41467-021-21687-2DOI Listing
March 2021

Seroconversion stages COVID19 into distinct pathophysiological states.

medRxiv 2020 Dec 7. Epub 2020 Dec 7.

COVID19 is a heterogeneous medical condition involving a suite of underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Seronegative COVID19 patients harbor hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, neutropenia, lymphopenia and thrombocytopenia. In seropositive patients, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased markers of platelet activation, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.
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http://dx.doi.org/10.1101/2020.12.05.20244442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743101PMC
December 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

Nutlin-Induced Apoptosis Is Specified by a Translation Program Regulated by PCBP2 and DHX30.

Cell Rep 2020 03;30(13):4355-4369.e6

Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, via Sommarive 9, 38123 Trento, Italy. Electronic address:

Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits. In this study, we report a translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to associate with the enhanced translation of mRNAs carrying multiple copies of an identified 3' UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identify PCBP2 and DHX30 as CGPD-motif interactors. We find that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs increases, and the response to Nutlin shifts toward apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells leads to decreased translation of CGPD-motif mRNAs.
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http://dx.doi.org/10.1016/j.celrep.2020.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182397PMC
March 2020

Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome.

Cell Rep 2019 11;29(7):1893-1908.e4

Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO 80302, USA. Electronic address:

People with Down syndrome (DS; trisomy 21) display a different disease spectrum relative to the general population, including lower rates of solid malignancies and higher incidence of neurological and autoimmune conditions. However, the mechanisms driving this unique clinical profile await elucidation. We completed a deep mapping of the immune system in adults with DS using mass cytometry to evaluate 100 immune cell types, which revealed global immune dysregulation consistent with chronic inflammation, including key changes in the myeloid and lymphoid cell compartments. Furthermore, measurement of interferon-inducible phosphorylation events revealed widespread hypersensitivity to interferon-α in DS, with cell-type-specific variations in downstream intracellular signaling. Mechanistically, this could be explained by overexpression of the interferon receptors encoded on chromosome 21, as demonstrated by increased IFNAR1 surface expression in all immune lineages tested. These results point to interferon-driven immune dysregulation as a likely contributor to the developmental and clinical hallmarks of DS.
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http://dx.doi.org/10.1016/j.celrep.2019.10.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871766PMC
November 2019

Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions.

Mol Cell 2019 11 5;76(3):485-499.e8. Epub 2019 Sep 5.

Max Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9, Vienna, Austria. Electronic address:

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
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http://dx.doi.org/10.1016/j.molcel.2019.07.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842433PMC
November 2019

SIX2 Mediates Late-Stage Metastasis via Direct Regulation of and Induction of a Cancer Stem Cell Program.

Cancer Res 2019 02 3;79(4):720-734. Epub 2019 Jan 3.

Integrated Physiology Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Srr2 enhancer, promoting expression and downstream expression of , which are both key pluripotency factors. Regulation of by Six2 enhanced cancer stem cell properties and increased metastatic colonization. and expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites. SIGNIFICANCE: These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells. http://cancerres.aacrjournals.org/content/canres/79/4/720/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586234PMC
February 2019

Therapeutic targeting of transcriptional cyclin-dependent kinases.

Transcription 2019 04 9;10(2):118-136. Epub 2018 Nov 9.

a Linda Crnic Institute for Down Syndrome, School of Medicine , University of Colorado Anschutz Medical Campus , Aurora , CO , USA.

The fact that many cancer types display transcriptional addiction driven by dysregulation of oncogenic enhancers and transcription factors has led to increased interest in a group of protein kinases, known as transcriptional cyclin dependent kinases (tCDKs), as potential therapeutic targets. Despite early reservations about targeting a process that is essential to healthy cell types, there is now evidence that targeting tCDKs could provide enough therapeutic window to be effective in the clinic. Here, we discuss recent developments in this field, with an emphasis on highly-selective inhibitors and the challenges to be addressed before these inhibitors could be used for therapeutic purposes. Abbreviations: CAK: CDK-activating kinase;CDK: cyclin-dependent kinase;CMGC group: CDK-, MAPK-, GSK3-, and CLK-like;CTD: C-terminal repeat domain of the RPB1 subunit of RNA polymerase II;DRB: 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole;mCRPC: metastatic castration-resistant prostate cancer;NSCLC: non-small cell lung cancer;P-TEFb: positive elongation factor b;RNAPII: RNA polymerase II;S2: serine-2 of CTD repeats;S5: serine-5 of CTD repeats;S7: serine-7 of CTD repeats;SEC: super elongation complex;tCDK: transcriptional cyclin-dependent kinase;TNBC: triple-negative breast cancer.
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http://dx.doi.org/10.1080/21541264.2018.1539615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602565PMC
April 2019

ΔNp63α Suppresses TGFB2 Expression and RHOA Activity to Drive Cell Proliferation in Squamous Cell Carcinomas.

Cell Rep 2018 09;24(12):3224-3236

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA; Functional Genomics Facility, University of Colorado School of Medicine, Aurora, CO 80045, USA; Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, CO 80045, USA; Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO 80203, USA. Electronic address:

The transcriptional repressor ΔNp63α is a potent oncogene widely overexpressed in squamous cell carcinomas (SCCs) of diverse tissue origins, where it promotes malignant cell proliferation and survival. We report here the results of a genome-wide CRISPR screen to identify pathways controlling ΔNp63α-dependent cell proliferation, which revealed that the small GTPase RHOA blocks cell division upon ΔNp63α knockdown. After ΔNp63α depletion, RHOA activity is increased, and cells undergo RHOA-dependent proliferation arrest along with transcriptome changes indicative of increased TGF-β signaling. Mechanistically, ΔNp63α represses transcription of TGFB2, which induces a cell cycle arrest that is partially dependent on RHOA. Ectopic TGFB2 activates RHOA and impairs SCC proliferation, and TGFB2 neutralization restores cell proliferation during ΔNp63α depletion. Genomic data from tumors demonstrate inactivation of RHOA and the TGFBR2 receptor and ΔNp63α overexpression in more than 80% of lung SCCs. These results reveal a signaling pathway controlling SCC proliferation that is potentially amenable to pharmacological intervention.
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http://dx.doi.org/10.1016/j.celrep.2018.08.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219633PMC
September 2018

Mechanisms of transcriptional regulation by p53.

Cell Death Differ 2018 01 10;25(1):133-143. Epub 2017 Nov 10.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

p53 is a transcription factor that suppresses tumor growth through regulation of dozens of target genes with diverse biological functions. The activity of this master transcription factor is inactivated in nearly all tumors, either by mutations in the TP53 locus or by oncogenic events that decrease the activity of the wild-type protein, such as overexpression of the p53 repressor MDM2. However, despite decades of intensive research, our collective understanding of the p53 signaling cascade remains incomplete. In this review, we focus on recent advances in our understanding of mechanisms of p53-dependent transcriptional control as they relate to five key areas: (1) the functionally distinct N-terminal transactivation domains, (2) the diverse regulatory roles of its C-terminal domain, (3) evidence that p53 is solely a direct transcriptional activator, not a direct repressor, (4) the ability of p53 to recognize many of its enhancers across diverse chromatin environments, and (5) mechanisms that modify the p53-dependent transcriptional program in a context-dependent manner.
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http://dx.doi.org/10.1038/cdd.2017.174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729533PMC
January 2018

CDK8 Kinase Activity Promotes Glycolysis.

Cell Rep 2017 Nov;21(6):1495-1506

Linda Crnic Institute for Down Syndrome, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309, USA. Electronic address:

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancerous tissues. Despite its importance in cancer development, our understanding of mechanisms driving this form of metabolic reprogramming is incomplete. We report here an analysis of colorectal cancer cells engineered to carry a single point mutation in the active site of the Mediator-associated kinase CDK8, creating hypomorphic alleles sensitive to bulky ATP analogs. Transcriptome analysis revealed that CDK8 kinase activity is required for the expression of many components of the glycolytic cascade. CDK8 inhibition impairs glucose transporter expression, glucose uptake, glycolytic capacity and reserve, as well as cell proliferation and anchorage-independent growth, both in normoxia and hypoxia. Importantly, CDK8 impairment sensitizes cells to pharmacological glycolysis inhibition, a result reproduced with Senexin A, a dual inhibitor of CDK8/CDK19. Altogether, these results contribute to our understanding of CDK8 as an oncogene, and they justify investigations to target CDK8 in highly glycolytic tumors.
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http://dx.doi.org/10.1016/j.celrep.2017.10.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726794PMC
November 2017

Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.

Genome Res 2017 10 13;27(10):1645-1657. Epub 2017 Sep 13.

Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.

The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.
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http://dx.doi.org/10.1101/gr.220533.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630028PMC
October 2017

A Kinase-Independent Role for Cyclin-Dependent Kinase 19 in p53 Response.

Mol Cell Biol 2017 07 15;37(13). Epub 2017 Jun 15.

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA

The human Mediator complex regulates RNA polymerase II transcription genome-wide. A general factor that regulates Mediator function is the four-subunit kinase module, which contains either cyclin-dependent kinase 8 (CDK8) or CDK19. Whereas CDK8 is linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. We discovered that osteosarcoma cells (SJSA) are naturally depleted of CDK8 protein. Whereas stable CDK19 knockdown was tolerated in SJSA cells, proliferation was reduced. Notably, proliferation defects were rescued upon the reexpression of wild-type or kinase-dead CDK19. Comparative RNA sequencing analyses showed reduced expression of mitotic genes and activation of genes associated with cholesterol metabolism and the p53 pathway in CDK19 knockdown cells. SJSA cells treated with 5-fluorouracil, which induces metabolic and genotoxic stress and activates p53, further implicated CDK19 in p53 target gene expression. To better probe the p53 response, SJSA cells (shCDK19 versus shCTRL) were treated with the p53 activator nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state after nutlin-3 treatment, and this effect was kinase independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a role for CDK19 in cellular resistance to nutlin-3.
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http://dx.doi.org/10.1128/MCB.00626-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472832PMC
July 2017

Trisomy 21 consistently activates the interferon response.

Elife 2016 07 29;5. Epub 2016 Jul 29.

Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, United States.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.
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http://dx.doi.org/10.7554/eLife.16220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012864PMC
July 2016

The TIP60 Complex Is a Conserved Coactivator of HIF1A.

Cell Rep 2016 06 16;16(1):37-47. Epub 2016 Jun 16.

Linda Crnic Institute for Down Syndrome, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO 80309, USA. Electronic address:

Hypoxia-inducible factors (HIFs) are critical regulators of the cellular response to hypoxia. Despite their established roles in normal physiology and numerous pathologies, the molecular mechanisms by which they control gene expression remain poorly understood. We report here a conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells. TIP60 (KAT5) is required for HIF1-dependent gene expression in fly cells and embryos and colorectal cancer cells. HIF1A interacts with and recruits TIP60 to chromatin. TIP60 is dispensable for HIF1A association with its target genes but is required for HIF1A-dependent chromatin modification and RNA polymerase II activation in hypoxia. In human cells, global analysis of HIF1A-dependent gene activity reveals that most HIF1A targets require either TIP60, the CDK8-Mediator complex, or both as coactivators for full expression in hypoxia. Thus, HIF1A employs functionally diverse cofactors to regulate different subsets of genes within its transcriptional program.
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http://dx.doi.org/10.1016/j.celrep.2016.05.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957981PMC
June 2016

NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia.

Cell Rep 2016 Mar 17;14(12):2938-49. Epub 2016 Mar 17.

Division of Experimental Medicine, Department of Oncology, Segal Cancer Centre and Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, QC H3T 1E2, Canada. Electronic address:

Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.
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http://dx.doi.org/10.1016/j.celrep.2016.02.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814328PMC
March 2016

Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms.

Elife 2014 May 27;3:e02200. Epub 2014 May 27.

Howard Hughes Medical Institute, University of Colorado, Boulder, Boulder, United States Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, United States

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033189PMC
http://dx.doi.org/10.7554/eLife.02200DOI Listing
May 2014

ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1.

Nucleic Acids Res 2013 Dec 17;41(22):10241-53. Epub 2013 Sep 17.

School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK, Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, CO 80309, USA, Department of Neurology, Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 90089, USA and Department of Pharmacology, Ningxia Medical University, Yinchuan 750004, China.

The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.
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http://dx.doi.org/10.1093/nar/gkt837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905876PMC
December 2013

HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia.

Cell 2013 Jun;153(6):1327-39

Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, CO 80309, USA.

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.
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http://dx.doi.org/10.1016/j.cell.2013.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681429PMC
June 2013

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX.

EMBO J 2012 Mar 13;31(5):1266-78. Epub 2012 Jan 13.

Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, The University of Colorado, Boulder, CO 80309-0347, USA.

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.
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http://dx.doi.org/10.1038/emboj.2011.498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298004PMC
March 2012

CDK8: a positive regulator of transcription.

Transcription 2010 Jul-Aug;1(1):4-12

University of Colorado, Boulder, USA.

CDK8 belongs to a group of cyclin-dependent kinases involved in transcriptional regulation from yeast to mammals. CDK8 associates with the Mediator complex, but functions outside of Mediator are also likely. Historically, CDK8 has been described mostly as a transcriptional repressor, but a growing body of research provides unequivocal evidence for various roles of CDK8 in gene activation. Several transcriptional programs of biomedical importance employ CDK8 as a co-activator, including the p53 network, the Wnt/β-catenin pathway, the serum response network, and those governed by SMADs and the thyroid hormone receptor, thus highlighting the importance of further investigation into this enigmatic transcriptional regulator.
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http://dx.doi.org/10.4161/trns.1.1.12373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035184PMC
March 2012

Lessons on transcriptional control from the serum response network.

Curr Opin Genet Dev 2011 Apr;21(2):160-6

Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, CO, United States.

Response to environmental stimuli is critical for cell survival and function and requires high fidelity signal transduction into the nucleus to facilitate the coordinated transcriptional regulation of appropriate gene networks. The cellular response to mitogenic stimuli provides an excellent paradigm to decipher the mechanisms mediating precise gene expression control at the transcriptional level. Here we review recent advances in our understanding of this so-called serum response network, which illuminate novel aspects of nuclear signaling mechanisms, combinatorial control by DNA binding proteins and regulation of RNA polymerase II (RNAPII) elongation.
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http://dx.doi.org/10.1016/j.gde.2011.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070842PMC
April 2011

Role of glutamine synthetase in phenazine antibiotic production by Pantoea agglomerans Eh1087.

Can J Microbiol 2004 Oct;50(10):877-81

School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic D-alanylgriseoluteic acid. A glutamine auxotroph harboring an insertion in a putative glnA gene was obtained by transposon-mutagenesis of Eh1087 that produced less D-alanylgriseoluteic acid than the parental strain (strain Eh7.1). Cosmids encoding the Eh1087 glnA were isolated by their ability to complement the mutant for prototrophy. The role of the Eh1087 glnA locus was functionally confirmed by complementation of an Escherichia coli glnA mutant. Analysis of the nucleotide and deduced amino acid sequences of the Eh1087 glnA gene indicated a high degree of similarity to the glnA genes and glutamine synthetase enzymes of other Enterobacteriaceae. Isotopic labelling experiments with 15N-labelled ammonium sulfate demonstrated that wild-type Eh1087 incorporated 15N into griseoluteic acid more readily than the glnA mutant Eh7.1. We conclude that the 2 nitrogens in the phenazine nucleus originate from glutamine and the intracellular glutamine synthesized by Eh1087 is a source of the phenazine nucleus nitrogens even in glutamine-rich environments.
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http://dx.doi.org/10.1139/w04-076DOI Listing
October 2004