In this work, dRNA-seq (differential RNA sequencing) and RNAtag-seq were applied to first define the global transcriptome architecture of C. difficile, followed by Hfq RIP-seq (RNA immunoprecipitation followed by RNA-seq) and RIL-seq (RNA interaction by ligation and sequencing) to characterize the Hfq-mediated sRNA interactome on a transcriptome-wide scale. These approaches resulted in the annotation of > 60 novel sRNAs. Notably, it not only revealed 50 Hfq-bound sRNAs, but also > 1000 mRNA-sRNA interactions, confirming Hfq as a global RNA matchmaker in C. difficile. Similar to its function in Gram-negative species, deletion of Hfq resulted in decreased sRNA half-lives, providing evidence that Hfq affects sRNA stability in C. difficile. Finally, several sRNAs and their function in various infection relevant conditions were characterized. The sRNA nc085 directly interacts with the two-component response regulator eutV, resulting in regulation of ethanolamine utilization, an abundant intestinal carbon and nitrogen source known to impact C. difficile pathogenicity. Meanwhile, SpoY and SpoX regulate translation of the master regulator of sporulation spo0A in vivo, thereby affecting sporulation initiation. Furthermore, SpoY and SpoX deletion significantly impacts C. difficile gut colonization and spore burden in a mouse model of C. difficile infection. / Der anaerobe Gram-positive humanpathogene Erreger Clostridioides difficile (C. difficile) gilt als Hauptursache für nosokomiale Antibiotika-assoziierte Diarrhöe. Verschiedene Virulenzfaktoren und -eigenschaften beeinflussen das Fortschreiten und den Schweregrad der Krankheit, darunter Toxinexpression und Sporenbildung. Kleine regulatorische RNAs (sRNAs) sind bekannte post- transkriptionelle Regulatoren von Virulenz- und Stress-assoziierten Stoffwechselwegen in vielen pathogenen Bakterien. In Gram-negativen Arten wird sRNA-abhängige post-transkriptionelle Regulierung häufig durch das RNA-Chaperon Hfq vermittelt, welches die sRNA-mRNA- Basenpaarung erleichtert. Trotz ihrer Bedeutung in Gram-negativen Bakterien ist vergleichsweise wenig über die verschiedenen Aspekte der post-transkriptionellen Regulation in Gram-positiven Arten bekannt. Erste Daten deuten auf eine wichtige Funktion von Hfq bei der Regulierung verschiedener infektionsassoziierter Signalwege in C. difficile hin, sowie auf die Existenz eines umfangreichen post-transkriptionellen Netzwerks. Eine globale Identifizierung von Hfq- assoziierten RNAs und deren Einfluss auf die Virulenz von und Kolonisierung durch C. difficile ist jedoch bisher noch nicht erfolgt. In dieser Arbeit wurde dRNA-seq (differentielle RNA-Sequenzierung) und RNAtag-seq angewandt, um zunächst die globale Transkriptom-Architektur von C. difficile zu definieren. Anschließend wurde Hfq RIP-seq (RNA-Immunpräzipitation gefolgt von RNA-seq) und RIL-seq (RNA-Interaktion durch Ligation und Sequenzierung) durchgeführt, um das Hfq-vermittelte sRNA-Interaktom auf globaler Ebene zu charakterisieren. Diese Ansätze führten zur Annotation von > 60 neuen sRNAs. Darüber hinaus wurden 50 Hfq-gebundene sRNAs, sowie > 1000 mRNA- sRNA-Interaktionen identifiziert, wodurch Hfq als globaler RNA-Matchmaker in C. difficile bestätigt wurde. Analog zu seiner Funktion in Gram-negativen Arten, führte die Deletion von Hfq zu verringerten sRNA-Halbwertszeiten, was darauf hindeutet, dass Hfq die sRNA-Stabilität in C. difficile beeinflusst. Schließlich wurden mehrere sRNAs und ihre Funktion unter verschiedenen infektionsrelevanten Bedingungen charakterisiert. Die sRNA nc085 interagiert direkt mit dem Zweikomponenten-Regulator eutV, was zu einer Regulierung der Ethanolaminverwertung führt. Als häufig vorkommenden Kohlenstoff- und Stickstoffquelle im Darm, kann Ethanolamin die Pathogenität von C. difficile beeinflussen. SpoY und SpoX regulieren dagegen die Translation des Hauptregulators der Sporulation spo0A in vivo und damit die Sporulationsinitiation. Darüber hinaus hat die Deletion von SpoY und SpoX signifikante Auswirkungen auf die Besiedlung des Darms mit C. difficile sowie die Sporenbelastung in einem Mausmodell der C. difficile-Infektion. Insgesamt liefert diese Arbeit Beweise für eine umfassende Hfq-abhängige post-transkriptionelle Regulierung, die die Physiologie und Virulenz eines Gram-positiven Erregers beeinflusst. Auch wenn mit dieser Arbeit die Charakterisierung der sRNA-vermittelten Regulation in C. difficile gerade erst begonnen hat, können die RIL-seq-Daten als Grundlage für zukünftige mechanistische Studien der RNA-basierten Genregulation in C. difficile herangezogen werden.

Receptor-interacting protein kinase 1 (RIPK1) is a major upstream mediator of inflammation and cell death. These processes are key to common inflammatory diseases such as atherosclerosis, where macrophages play an important role in their progression. Closely linked to the expression of downstream genes, long non-coding RNAs (lncRNAs) are critical to controlling cellular processes in health and disease. As post-transcriptional regulatory mechanisms for RIPK1 are largely unknown, this project seeks to study the stability of Ripk1 mRNA and RIPK1 protein, along with Ripk1 mRNA interactions with relevant lncRNAs under various conditions. Using transcription and translation inhibitors, we determined that both Ripk1 mRNA and RIPK1 protein are relatively unstable with half-lives of approximately 3 h. Their turnover in macrophages is further influenced by the timing and duration of inflammation. We also implemented a novel RNA pull-down procedure to capture Ripk1 mRNA and attached lncRNAs for next-generation sequencing. Through differential expression analysis, we discovered significant upregulation of known lncRNA AC125611 and novel lncRNA MSTRG.5894.1 in Ripk1-targeted samples subject to inflammation. MSTRG.7477.1 was upregulated during necroptosis, while MSTRG.5684.5 was upregulated during both inflammation and necroptosis. GapmeR-mediated knockdowns of AC125611 and MSTRG.5684.5 under inflammatory conditions resulted in decreased Ripk1 mRNA expression and RIPK1 protein expression, respectively. Meanwhile, MSTRG.7477.1 knockdowns were connected to decreased RIPK1 at both the mRNA and protein levels. Our research ultimately advances the current understanding of RIPK1 regulation by focusing on Ripk1 mRNA-lncRNA associations and turnover of its mRNA and protein in macrophages, paving the way for future investigations into their capacity to act as therapeutic targets.

RNAs play essential roles in various biological processes. Mounting evidence has demonstrated that RNA subcellular localization and intercellular trafficking govern their functions in coordinating plant growth at the organismal level. Beyond that, plants constantly encounter foreign RNAs (i.e., RNAs from pathogens including viruses and viroids). The subcellular localizations of RNAs are crucial for their function. While numerous types of RNAs (i.e., mRNAs, small RNAs, rRNAs, tRNAs, and long noncoding RNAs) have been found to traffic in a non-cell-autonomous fashion within plants, the underlying regulatory mechanism remains unclear. Viroids are single-stranded circular noncoding RNAs, which entirely rely on their RNA motifs to exploit cellular machinery for organelle entry and exit, cell-to-cell movement through plasmodesmata, and systemic trafficking. Viroids represent an excellent model to dissect the role of RNA 3-dimensional (3D) structural motifs in regulating RNA movement. Using nuclear-replicating viroids as a model, we showed that cellular Importin alpha-4 is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (Virp1) that binds both Importin alpha-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs Virp1 binding, viroid nuclear accumulation and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on Virp1 for nuclear import. On the other hand, no viroid can systemically infect the model plant Arabidopsis thaliana, suggesting the existence of non-host resistance yet to be understood. Here, we attempted to test whether a gene involved in RNA silencing, RNA-dependent RNA polymerase 6 (RDR6), plays a role in non-host resistance in Arabidopsis. I will discuss the data below in detail.

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No description available.

<p>Bacterial small RNAs have emerged as a class of molecules having important regulatory roles. Accumulating numbers of <em>cis</em>-encoded sRNAs (antisense RNAs) have been recently discovered to be transcribed from the chromosomal DNA of many bacterial species, including the streptomycetes. Here, we investigate potential regulatory roles for two <em>S. coelicolor</em> antisense RNAs, scr4677 and α-abeA.</p> <p>The scr4677 antisense RNA is transcribed from the intergenic region between <em>SCO4676</em> (a gene encoding a conserved protein of unknown function) and <em>SCO4677</em>, encoding a regulatory protein with proposed anti-sigma factor activity. Transcription profiling revealed that scr4677 may not only interact with <em>SCO4676</em> mRNA but also with <em>SCO4677-4676</em> read-through transcripts. Our study suggested that scr4677 functioned to destabilize <em>SCO4676</em> mRNA, at the same time that it stabilized the <em>SCO4677-4676</em> read-through transcript. The potential role for scr4677 in destabilizing <em>SCO4676</em> mRNA was not mediated by the double stranded ribonuclease RNase III. Genetic analysis showed <em>scr4677</em> transcription was affected by SCO4677, and the transcription was apparently dependent on an unknown protein binding to the <em>SCO4676 </em>coding sequence.</p> <p>A second independent study focused on investigating the regulation of a previously uncharacterized genetic region, <em>SCO3287-3290</em>, since renamed <em>abeABCD</em>. This region contains an antisense RNA (α-abeA)-encoding gene, and is adjacent to the downstream <em>SCO3291</em> (<em>abeR</em>) gene, which encodes a putative regulatory protein. Genetic analysis revealed that overexpression of <em>abeR </em>or <em>abeABCD</em> stimulated the production of the blue-pigmented antibiotic actinorhodin, and deletion of <em>abeR</em> impaired actinorhodin production. Transcription analysis revealed the <em>abe</em> genes (including α-<em>abeA</em>) to be subject to multiple levels of regulation. We found an internal promoter within the <em>abeA</em> coding sequence and that required AbeR for expression. Furthermore, biochemical experiments demonstrated that AbeR regulated <em>abeBCD</em> directly, by binding to four heptameric repeats in its promoter region. The expression of α-<em>abeA</em> and other <em>abe</em> genes were differentially affected by RNase III.</p> / Doctor of Science (PhD)

Epigenetics was a term first coined to understand how cells with the same genetic make up can differentiate into various cell types. Elegant research over the past 30 years has shown that these mechanisms include heritable marks such as DNA methylation and histone modifications along with stable expression of non- coding RNAs. Within the realm of epigenetics is a phenomenon known as genomic imprinting. Imprints are marks that distinguish the maternal from the paternal chromosomes in the form of methylation. Methylation marks can influence transcript expression, resulting in only one allele being expressed. One imprinted domain is the Kcnq1 domain located on chromosome 11p15.5 in humans and chromosome 7 in the mouse. This domain is thought to be under the control of a paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1. The Kcnq1ot1 ncRNA is expressed on the paternal chromosome due to a differentially methylation region located within its promoter. The promoter is methylated on the maternal allele thus inhibiting ncRNA expression, whereas the promoter is unmethylated on the paternal allele. In the placenta, a most of the genes located within a one mega-basepair region are exclusively expressed from the maternal chromosome, whereas the transcripts on the paternal chromosome are silenced by the ncRNA. The placenta seems to follow the classic idea of an imprinted domain. However, in the embryo and more specifically, in the embryonic heart, this is not the case. In the embryonic heart, only a 400kb region is restricted to maternal expression. In addition, one the genes, Kcnq1, starts out expressed exclusively from the maternal allele in early development but switches to biallelic expression during mid-gestation. The purpose of my research is to determine the underlying complexities that are involved in the regulation of transcripts within the Kcnq1 domain. This involves the Kcnq1 gene itself, which has been shown to transition from mono- to biallelic expression during mid-gestation and the Kcnq1ot1 ncRNA per se. I hypothesize that regulation by the Kcnq1ot1 ncRNA is not occurring in a uniform manner in the embryo; rather, the amount of regulation by the ncRNA is dependent on the developmental stage and specific tissue. In addition, this regulation involves complex interactions between enhancers, insulators and other regulatory elements to control the amount of silencing by the Kcnq1ot1 ncRNA. First, through a series of experiments looking at the Kcnq1 promoter, the mechanism of Kcnq1 paternal expression was determined. It was confirmed that Kcnq1 becomes biallelic during mid-gestation in the heart. Bisulfite mutagenesis and methylation sensitive chromatin immunoprecipitation were used to test the hypothesis that the Kcnq1 promoter was methylated in early development and then lost its methylation mark. However, a lack of methylation disproved this mechanism of paternal Kcnq1 activation. Rather, chromosome conformation capture (3C) determined that the Kcnq1 promoter interacts in a tissue-specific manner with regions within the domain that have enhancer activity. The role of the ncRNA within our system was also investigated. Interestingly, when Kcnq1ot1 allelic expression was profiled throughout development in heart, it transitioned to biallelic expression during heart development but remained monoallelic in the liver and brain. Several possibilities could account for this phenomenon, including loss of promoter methylation and/or an alternative transcript start site. Both of these options were explored using bisulfite mutagenesis and 5' RACE. However, the Kcnq1ot1 promoter region retained its methylation mark even after the maternal transcript was turned on, disproving this idea. Rather, a maternal specific transcript was found in the heart to start downstream of the CpG islands. Lastly, to gain a better understand of the Kcnq1ot1 ncRNA, experiments were carried out on a mutant mouse in which a truncated form of the ncRNA was transmitted paternally; this is dubbed the "Kterm" mouse. Unexpectedly, Kcnq1 still followed the same mono- to biallelic transition as seen in the wild-type, whereas the head and body counterparts from the same stage embryos were biallelic for Kcnq1. Also, the immediate upstream genes, Cdk1nc and Slc22a18, lost their mono-allelic expression in neonatal heart, liver and brain when the Kterm mutation was transmitted. This suggested that Kcnq1ot1 did not function as a silencer for Kcnq1 paternal expression in the heart, but rather had an alternative and previously unknown function. From qRT-PCR, 3C and ChIP assays, it was determined that the Kcnq1ot1 ncRNA plays a role in regulating Kcnq1 gene expression in the heart by limiting its interaction to specific cis-acting enhancers. When the ncRNA was absent, the Kcnq1 promoter interacted with non-native sites along the domain, possibly causing the increase in transcript expression. This phenomenon was specific to the heart and was not seen in other tissues. These findings showed that Kcnq1 paternal expression is the result of strong developmental and tissue specific enhancers. Chromatin interactions in cis put a strong enhancer in contact with the Kcnq1 promoter to increase its expression in later development. In addition, a truncation mutation model identified a key role for the Kcnq1ot1 ncRNA in regulating Kcnq1 expression. Instead of regulating the imprinting status of Kcnq1, the ncRNA regulates the amount of Kcnq1 transcript being produced in the heart by regulating chromatin interactions. Finally, these studies identified a maternally expressed Kcnq1ot1 transcript whose role in heart development is still not fully understood. Taken together, these findings support a model where an inhibitory factor(s) silence the paternal Kcnq1 transcript and maternal Kcnq1ot1 transcript and in later development, this factor is released allowing for expression and chromatin interactions to occur. / Molecular Biology and Genetics

Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Introduction: Myocardial infarction is the most common form of acute cardiac injury attributed to heart failure. Despite advancements in prognosis and treatment, acute MI (AMI) still bears a considerable mortality rate within the initial year, with a significant portion of patients succumbing within the initial 30 days post-MI. The overall prognosis hinges on factors such as the extent of heart muscle damage, duration of the inflammatory response, and the efficacy of administered treatments in mitigating myocardial cell death and injury. This underscores the need for deeper mechanistic understanding and development of targeted therapies for cardiovascular diseases. In response to cardiac injury, macrophages are initially recruited to the infarcted myocardium and undergo phenotypic change from pro-inflammatory (M1) macrophages in the early stage to an anti-inflammatory (M2) macrophages phenotype in the later stage, orchestrating the initiation, maintenance, and eventual resolution of the inflammatory response. However, in chronic ischemia or severe infarction, continuous cardiomyocyte death prolongs pro-inflammatory macrophage activation resulting in robust secretion of pro-inflammatory cytokines perpetuating the inflammatory response and resulting in increased myocardial damage. Despite some understanding, further research is needed on the mechanisms and factors influencing macrophage function during injury. Circular RNAs are newly discovered non-coding RNA generated from protein-coding genes ubiquitously expressed in mammalian tissue, highly conserved among species, and recently implicated in the possible regulation of macrophage activation. However, their role in immunomodulation during cardiovascular injury remains unknown. Objective: This study focused on determining the specific role of circ-cdr1as in phenotypic switching between pro-, and anti-inflammatory macrophages and to determine whether functional regulation of circ-cdr1as modulates macrophage function post-cardiac injury. Methods and Results: We performed circular RNA microarray analyses to assess circular RNA transcriptome changes using RNA isolated from bone marrow derived macrophages (BMDM) polarized to a M1 phenotype by INFγ and TNFα or a M2 phenotype by IL-10, IL-4, and TGF-β. Following RNA isolation, samples are treated with RNaseR for enrichment of circular RNA and removal of linear RNA. We identified circRNAs differentially expressed in pro-and anti-inflammatory macrophages, including circRNA cdr1as (circ-cdr1as). RT-qPCR analysis revealed circ-cdr1as was one of the most downregulated in pro-inflammatory macrophages and significantly upregulated in anti-inflammatory macrophages in vitro. We established a circ-cdr1as overexpression system by generating a circ-cdr1as plasmid using pc3.1 plasmid with flanking regions that allows circularization of specified sequence for in vitro studies. For knockdown of circ-cdr1as, we used small hairpin RNA targeting the splicing junction found only in circular RNA. RT-qPCR and fluorescence activated cell sorting (FACS) analyses showed that overexpression of circ-Cdr1as increased transcription of anti-inflammatory markers and percentage of CD206+ (M2 macrophage marker) cells in naïve and pro-inflammatory macrophages in vitro. Meanwhile, knockdown decreased transcription of anti-inflammatory markers and increased the percentage of CD86+ (M1 macrophage marker) cells in naïve and anti-inflammatory macrophages in vitro. Disease enrichment analysis based on IPA system of the diseases associated with circular RNA involved in macrophage polarization indicated that cardiac fibrosis and cardiac enlargement as the top diseases. Therefore, we investigated the expression levels of circ-cdr1as in the heart after myocardial infarction (MI) injury in a mouse model. RT-qPCR analysis revealed downregulation of circ-cdr1as in the heart 3 days post MI, suggesting a possible physiological role for circ-cdr1as in MI pathophysiology. We isolated fibroblast, cardiomyocytes, CD31+ endothelial cells, and F4/80+ macrophages and investigated the transcriptional changes of circ-cdr1as to determine if it is cell-type specific. RT-qPCR analysis showed no significant difference in transcription of circ-cdr1as in fibroblast and endothelial cells. However, in cardiomyocytes and macrophages there was a significant downregulation of circ-cdr1as. To understand the role of circ-cdr1asmodulated macrophages in post-MI cardiac repair in vivo, we overexpressed circ-cdr1as in fluorescently labeled BMDMs and directly injected them into the ischemic myocardium immediately following MI surgery. FACS and immunohistochemistry analyses showed that these macrophages retained their anti-inflammatory phenotype during the initial stages of cardiac injury, and in the later stages improved cardiac left ventricular (LV) functions and reduced infarct size. Since circ-cdr1as was also decreased in cardiomyocytes post-MI, we additionally generated circ-cdr1as adeno associated virus 9 (circ-cdr1as-AAV-9) vectors to overexpress circ-cdr1as in mouse hearts. We performed tail vein injections of circ-cdr1as-AAV9 vectors 14 days prior to MI and conducted physiological and histological studies. Administration of circ-cdr1as-AAV9 significantly improved post-MI LV functions including ejection fraction (%EF) and fractional shortening (%FS) at 21-28D post MI, decreased infarct size, and improved angiogenesis. Interestingly, in the initial stages of cardiac injury, overexpression of circ-cdr1as reduced cardiomyocyte apoptosis and increased percentage of anti-inflammatory macrophages at injury site. Lastly, emerging evidence suggests that some circular RNAs function as microRNA (miR) sponges. Therefore, we investigated this mechanism to assess whether circular cdr1as invokes phenotypic changes in macrophages in both the steady-state and injured heart by acting as a sponge for miRNA to inhibit its function. Circ-cdr1as contains over 70 binding sites for miR-7, a microRNA known to exacerbate inflammation in lung related diseases through inhibition of KLF4. Pull-down assay indicated that circ-cdr1as directly interacts with miR-7. We found a reciprocal relationship between circ-cdr1as and miR-7 in macrophages and in the heart 3 days post-MI. Overexpression of miR-7 by miR-7-5p mimic increased the percentage of pro-inflammatory marker CD86 in naïve, pro-, and anti-inflammatory macrophages and upregulated transcription of pro-inflammatory markers. Meanwhile, inhibition of miR-7 had the opposite effect. We also found that expression of miR-7 target gene, KLF4, was reduced when macrophages were treated with miR-7 and increased when miR-7 was inhibited. Conclusions: In summary, this study suggests that circ-cdr1as plays a key role in regulating anti-inflammatory phenotype of macrophages through the modulation of miR-7 and its targets and exogenous delivery of circ-cdr1as may improve LV function over time. Therefore, circ-cdr1as may potentially be developed as an anti-inflammatory regulator in tissue inflammation after cardiac injury. / Biomedical Sciences

Yes / The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), an unrestricted research grant from the Dow Chemical Company, and the Johan Throne Holst Foundation to J.M. G.G. was supported by European Union Seventh Framework Program (FP7-PEOPLE2013-COFUND) under the Grant Agreement n609020 - Scientia Fellows