This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Interferons (IFNs) and other cytokines and growth factors activate the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway. In the IFN signaling, type I IFNs (IFN-a/b) upon binding to their receptor, activate intracellular, receptor-associated, tyrosine kinases JAK1 and Tyk2. These activated JAKs, in turn, phosphorylate specific tyrosine residues on latent cytoplasmic transcription factors which subsequently assemble into a complex called ISGF3 (IFN stimulated gene factor 3). This complex is composed of either STAT1a (91kD) or STAT1b (84kD) and STAT2 (113kD), which together constitute ISGF3-a, and a non-STAT protein called ISGF3-g (48kD) which is a member of the interferon regulatory factor (IRF) family. This complex accumulates in the nucleus, binds to a DNA element, ISRE (IFN-a stimulated response element, a 15 base pair non-dyad symmetrical DNA element), and activates transcription of target genes. ISGF3 is not activated by type II IFN, IFN-g. Instead, IFN-g, upon binding to its receptor and consequent activation of JAK1 and JAK2, induces the phosphorylation of STAT1a (or STAT1b), but not STAT2. The phosphorylated STAT1 then dimerizes and binds to a GAS (IFN-g activated sequence), DNA element that is dyad symmetrical 5?TTN5AA3?. Although both STAT1a and STAT1b can bind to GAS, only STAT1a forms functional GAF (IFN-g activated factor) that induces transcription from the GAS elements. Ultimately, one of the most crucial determinants affecting inherent transactivation potential of induced STATs may be a particular nucleoprotein microenvironment. Our objective is to discern patterns of cooperativity between activated STATs and other transcription factors, coactivator, and corepressor complexes within the context of the native promoter sequences. Such interactions are probably necessary to explain the role of STATs in gene activation at different IFN inducible genes. We have approached this problem by comparative study of the complex binding sites in several STAT-responsive genes. While STAT-binding sites exist and are likely required in chromosomal regulatory regions, single GAS elements give very little or no induction on their own in transient transfection assays. Therefore, we started with known native GAS elements and by adding adjacent native nucleotides determined the minimal size sequence that was IFN-responsive when cloned into a reporter gene. Then we determined whether this reporter inducibility correlated with an appearance of a novel band shift using a corresponding oligonucleotide in electrophoretic mobility shift assays (EMSA). We have detected in EMSA constitutive, low-mobility, protein complex that by mutational analysis is shown to be required for optimal STAT-mediated promoter activation. In particular, GBP promoter sequences that contain intact GAS and ISRE that bind STAT1 homodimer and IRF1, respectively, but that cannot bind constitutive low-mobility complex, when cloned in front of the heterologous reporter gene are inactive. Only sequences that bind STAT1, IRF1 and the constitutive low-mobility complex are able to activate a reporter gene upon IFN-g induction. Studies of the DNA affinity and specificity of this complex revealed that its DNA-binding may be affected by mutations within GAS as well as GAS-like site of the GBP promoter, suggesting a possible physical interaction with STATs or occupation of STAT sites that is relieved after appearance of activated STAT1 dimer. DNA affinity of this complex, observed in EMSA, is completely correlated to the transcriptional activation potential of the corresponding reporter constructs in transfection experiments. Currently we are engaged in obtaining larger quantities of partly purified preparation of constitutive low mobility complex which we will further purify in order to identify constituent subunits.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-38
Application #
8361500
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2011-03-01
Project End
2012-03-31
Budget Start
2011-03-01
Budget End
2012-03-31
Support Year
38
Fiscal Year
2011
Total Cost
$1,304
Indirect Cost
Name
Rockefeller University
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C et al. (2017) Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding. Anal Biochem 519:38-41
Chait, Brian T; Cadene, Martine; Olinares, Paul Dominic et al. (2016) Revealing Higher Order Protein Structure Using Mass Spectrometry. J Am Soc Mass Spectrom 27:952-65
Boice, Michael; Salloum, Darin; Mourcin, Frederic et al. (2016) Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells. Cell 167:405-418.e13
Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Maximizing ion transmission from atmospheric pressure into the vacuum of mass spectrometers with a novel electrospray interface. J Am Soc Mass Spectrom 26:649-58
Mast, Fred D; Rachubinski, Richard A; Aitchison, John D (2015) Signaling dynamics and peroxisomes. Curr Opin Cell Biol 35:131-6
Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Optimizing electrospray interfaces using slowly diverging conical duct (ConDuct) electrodes. J Am Soc Mass Spectrom 26:659-67
Oricchio, Elisa; Papapetrou, Eirini P; Lafaille, Fabien et al. (2014) A cell engineering strategy to enhance the safety of stem cell therapies. Cell Rep 8:1677-1685
Zhong, Yu; Morris, Deanna H; Jin, Lin et al. (2014) Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels. J Biol Chem 289:26021-37
Xue, John Z; Woo, Eileen M; Postow, Lisa et al. (2013) Chromatin-bound Xenopus Dppa2 shapes the nucleus by locally inhibiting microtubule assembly. Dev Cell 27:47-59
Indiani, Chiara; O'Donnell, Mike (2013) A proposal: Source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 18:312-23

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