Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection

Abstract

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.

Global Transcriptomic Profiling of Pulmonary Gene Expression in an Experimental Murine Model of Rickettsia conorii Infection

“…The quantity and quality of enriched total RNA was ascertained, respectively, on a Multiskan TM Go Spectrophotometer (Thermo Fisher Scientific) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and only RNA samples with an RNA integrity number (RIN) of >9 were included in our experiments. The enriched RNA was subjected to reverse transcription, followed by preparation of barcoded and strand-specific complementary DNA (cDNA) libraries using TruSeq RNA Sample Prep Kit (Illumina), as described earlier [30]. The cDNA libraries were then processed for sequencing as 50-base-long paired-end reads on an Illumina HiSeq 1500 at the Next Generation Sequencing core facility of the UTMB.…”

Emerging roles of non-coding RNAs in vector-borne infections

“…Chowdhury and colleagues provided the very first experimental evidence suggesting altered expression of pulmonary host cell ncRNAs and their involvement in the host defense response (Chowdhury et al, 2019). The profiling of the expression of host lung cell lncRNAs during infection of mice with R. conorii, simulating the pathogenesis of human spotted fever rickettsioses (Fig.…”

“…Additional in-depth analyses concluded that the genomic loci of NONMMUT013718 and NONMMUT024103 might regulate the expression of nearby protein-coding genes (PCGs), namely inhibitor of DNA binding 2 (Id2) and apolipoprotein 10b (Apol10b), respectively (Chowdhury et al, 2019). The regulatory role of these elncRNAs was confirmed during R. conorii infection in vitro, with induced expression of NONMMUT013718 (Id2) in murine macrophages and NONMMUT024103 (Apol10b) in endothelial cells clearly visible (Chowdhury et al, 2019). As expected, shRNAmediated knockdown of both elncRNAs reduced the expression of their endogenous target PCGs.…”

“…Symptoms are usually mild and nonspecific during the acute phase; during the chronic phase, most patients remain asymptomatic but are infected for life. However, 30% of people suffer from cardiac disorders and up to 10% suffer The very first experimental evidence suggesting altered expression of pulmonary host cell ncRNAs and their involvement in the host defense response has only been recently obtained (Chowdhury et al, 2019). Here, mice were infected with high-dose R. conorii via tail vein injection.…”

“…Long non-coding RNA transcription and gene regulation in experimental models of rickettsial infection. The very first experimental evidence suggesting altered expression of pulmonary host cell ncRNAs and their involvement in the host defense response has only been recently obtained(Chowdhury et al, 2019). Here, mice were infected with high-dose R. conorii via tail vein injection.…”