TY - JOUR
T1 - Ehrlichia chaffeensis TRP32 is a nucleomodulin that directly regulates expression of host genes governing differentiation and proliferation
AU - Farris, Tierra R.
AU - Dunphy, Paige S.
AU - Zhu, Bing
AU - Kibler, Clayton E.
AU - McBride, Jere W.
N1 - Funding Information:
We are grateful to all of the lab members both current and past and to Yuriy Fofanov for their insight and advice and for many useful discussions. We also thank Thomas Wood and Steven Widen from the UTMB Next Generation Sequencing Core Facility for their time and expertise. This work, including the efforts of Tierra Farris, Paige Selvy Dunphy, and Jere W. McBride, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI105536). This work, including the efforts of Tierra Farris, Bing Zhu, Clayton E. Kibler, and Jere W. McBride, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI106859). This work, including the efforts of Tierra Farris, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI 007526-15). This work was supported by the National Institute of Allergy and Infectious Diseases grants AI105536 and AI106859 and by funding from the Clayton Foundation for Research (to Jere W. McBride). Tierra Farris was supported by the NIAID T32 AI 007526-15 and by a McLaughlin predoctoral fellowship. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Publisher Copyright:
© 2016, American Society for Microbiology. All Rights Reserved.
PY - 2016
Y1 - 2016
N2 - Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector- host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32- bound genes (n=1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.
AB - Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector- host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32- bound genes (n=1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.
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U2 - 10.1128/IAI.00657-16
DO - 10.1128/IAI.00657-16
M3 - Article
AN - SCOPUS:84994680377
SN - 0019-9567
VL - 84
SP - 3182
EP - 3194
JO - Infection and Immunity
JF - Infection and Immunity
IS - 11
ER -