TY - JOUR
T1 - Enhancer associated long non-coding RNA transcription and gene regulation in experimental models of rickettsial infection
AU - Chowdhury, Imran H.
AU - Narra, Hema P.
AU - Sahni, Abha
AU - Khanipov, Kamil
AU - Fofanov, Yuriy
AU - Sahni, Sanjeev K.
N1 - Funding Information:
This work was supported by James W. McLaughlin Postdoctoral Fellowship to IC, Ph.D., at the University of Texas Medical Branch (UTMB), Galveston, TX, USA, exploratory grants R21 AI115231 and R21 AI117483 from the NIAID/NIH, and institutional support funds from the UTMB. We sincerely thank Dr. Thomas Wood, Ph.D. and Dr. Steve Widen, PhD (UTMB Genomic core facility) for their advice and support with the conduct of sequencing and data analysis. We are grateful to Dr. David H. Walker, MD and Dr. Shinji Makino, Ph.D. for generously providing the cell lines and plasmids used in this study.
Funding Information:
This work was supported by James W. McLaughlin Postdoctoral Fellowship to IC, Ph.D., at the University of Texas Medical Branch (UTMB), Galveston, TX, USA, exploratory grants R21 AI115231 and R21 AI117483 from the NIAID/NIH, and institutional support funds from the UTMB.
Publisher Copyright:
© 2007 - 2019 Frontiers Media S.A. All Rights Reserved.
PY - 2019
Y1 - 2019
N2 - Recent discovery that much of the mammalian genome does not encode protein-coding genes (PCGs) has brought widespread attention to long noncoding RNAs (lncRNAs) as a novel layer of biological regulation. Enhancer lnc (elnc) RNAs from the enhancer regions of the genome carry the capacity to regulate PCGs in cis or in trans. Spotted fever rickettsioses represent the consequence of host infection with Gram-negative, obligate intracellular bacteria in the Genus Rickettsia. Despite being implicated in the pathways of infection and inflammation, the roles of lncRNAs in host response to Rickettsia species have remained a mystery. We have profiled the expression of host lncRNAs during infection of susceptible mice with R. conorii as a model closely mimicking the pathogenesis of human spotted fever rickettsioses. RNA sequencing on the lungs of infected hosts yielded reads mapping to 74,964 non-coding RNAs, 206 and 277 of which were determined to be significantly up- and down-regulated, respectively, in comparison to uninfected controls. Following removal of short non-coding RNAs and ambiguous transcripts, remaining transcripts underwent in-depth analysis of mouse lung epigenetic signatures H3K4Me1 and H3K4Me3, active transcript markers (POLR2A, p300, CTCF), and DNaseI hypersensitivity sites to identify two potentially active and highly up-regulated elncRNAs NONMMUT013718 and NONMMUT024103. Using Hi-3C sequencing resource, we further determined that genomic loci of NONMMUT013718 and NONMMUT024103 might interact with and regulate the expression of nearby PCGs, namely Id2 (inhibitor of DNA binding 2) and Apol10b (apolipoprotein 10b), respectively. Heterologous reporter assays confirmed the activity of elncRNAs as the inducers of their predicted PCGs. In the lungs of infected mice, expression of both elncRNAs and their targets was significantly higher than mock-infected controls. Induced expression of NONMMUT013718/Id2 in murine macrophages and NONMMUT024103/Apol10b in endothelial cells was also clearly evident during R. conorii infection in vitro. Finally, shRNA mediated knock-down of NONMMUT013718 and NONMMUT024103 elncRNAs resulted in reduced expression of endogenous Id2 and Apl10b, demonstrating the regulatory roles of these elncRNAs on their target PCGs. Our results provide very first experimental evidence suggesting altered expression of pulmonary lncRNAs and elncRNA-mediated regulation of PCGs involved in immunity and during host interactions with pathogenic rickettsiae.
AB - Recent discovery that much of the mammalian genome does not encode protein-coding genes (PCGs) has brought widespread attention to long noncoding RNAs (lncRNAs) as a novel layer of biological regulation. Enhancer lnc (elnc) RNAs from the enhancer regions of the genome carry the capacity to regulate PCGs in cis or in trans. Spotted fever rickettsioses represent the consequence of host infection with Gram-negative, obligate intracellular bacteria in the Genus Rickettsia. Despite being implicated in the pathways of infection and inflammation, the roles of lncRNAs in host response to Rickettsia species have remained a mystery. We have profiled the expression of host lncRNAs during infection of susceptible mice with R. conorii as a model closely mimicking the pathogenesis of human spotted fever rickettsioses. RNA sequencing on the lungs of infected hosts yielded reads mapping to 74,964 non-coding RNAs, 206 and 277 of which were determined to be significantly up- and down-regulated, respectively, in comparison to uninfected controls. Following removal of short non-coding RNAs and ambiguous transcripts, remaining transcripts underwent in-depth analysis of mouse lung epigenetic signatures H3K4Me1 and H3K4Me3, active transcript markers (POLR2A, p300, CTCF), and DNaseI hypersensitivity sites to identify two potentially active and highly up-regulated elncRNAs NONMMUT013718 and NONMMUT024103. Using Hi-3C sequencing resource, we further determined that genomic loci of NONMMUT013718 and NONMMUT024103 might interact with and regulate the expression of nearby PCGs, namely Id2 (inhibitor of DNA binding 2) and Apol10b (apolipoprotein 10b), respectively. Heterologous reporter assays confirmed the activity of elncRNAs as the inducers of their predicted PCGs. In the lungs of infected mice, expression of both elncRNAs and their targets was significantly higher than mock-infected controls. Induced expression of NONMMUT013718/Id2 in murine macrophages and NONMMUT024103/Apol10b in endothelial cells was also clearly evident during R. conorii infection in vitro. Finally, shRNA mediated knock-down of NONMMUT013718 and NONMMUT024103 elncRNAs resulted in reduced expression of endogenous Id2 and Apl10b, demonstrating the regulatory roles of these elncRNAs on their target PCGs. Our results provide very first experimental evidence suggesting altered expression of pulmonary lncRNAs and elncRNA-mediated regulation of PCGs involved in immunity and during host interactions with pathogenic rickettsiae.
KW - Apolipoprotein L 10b
KW - Enhancer long non-coding (elnc) RNA
KW - Host immune responses
KW - Inhibitor of DNA binding 2 protein
KW - Long non-coding (lnc) RNA
KW - RNA sequencing
KW - Rickettsia
KW - Transcription start site
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U2 - 10.3389/fimmu.2018.03014
DO - 10.3389/fimmu.2018.03014
M3 - Article
C2 - 30687302
AN - SCOPUS:85060649659
SN - 1664-3224
VL - 10
JO - Frontiers in immunology
JF - Frontiers in immunology
IS - JAN
M1 - 3014
ER -