Benjamin Soibam

Proinflammatory role for let-7 microRNAS in experimental asthma.

MicroRNAs (miRNAs) are short, non-coding RNAs that target and silence protein coding genes through 3′-UTR elements. Evidence increasingly assigns an immunosuppressive role for miRNAs in immunity, but relatively few miRNAs have been studied, and an overall understanding of the importance of these regulatory transcripts in complex in vivo systems is lacking. Here we have applied multiple technologies to globally analyze miRNA expression and function in allergic lung disease, an experimental model of asthma. Deep sequencing and microarray analyses of the mouse lung short RNAome revealed numerous extant and novel miRNAs and other transcript classes. Similar to mRNAs, lung miRNA expression changed dynamically during the transition from the naive to the allergic state, suggesting numerous functional relationships. A possible role for miRNA editing in altering the lung mRNA target repertoire was also identified. Multiple members of the highly conserved let-7 miRNA family were the most abundant lung miRNAs, and we confirmed in vitro that interleukin 13 (IL-13), a cytokine essential for expression for allergic lung disease, is regulated by mmu-let-7a. However, inhibition of let-7 miRNAs in vivo using a locked nucleic acid profoundly inhibited production of allergic cytokines and the disease phenotype. Our findings thus reveal unexpected complexity in the miRNAome underlying allergic lung disease and demonstrate a proinflammatory role for let-7 miRNAs.

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Open‐field arena boundary is a primary object of exploration for Drosophila

Drosophila adults, when placed into a novel open‐field arena, initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity and spend a majority of time near the arena edge, executing motions along the walls. In order to determine the environmental features that are responsible for the initial high activity and wall‐following behavior exhibited during exploration, we examined wild‐type and visually impaired mutants in arenas with different vertical surfaces. These experiments support the conclusion that the wall‐following behavior of Drosophila is best characterized by a preference for the arena boundary, and not thigmotaxis or centrophobicity. In circular arenas, Drosophila mostly move in trajectories with low turn angles. Since the boundary preference could derive from highly linear trajectories, we further developed a simulation program to model the effects of turn angle on the boundary preference. In an hourglass‐shaped arena with convex‐angled walls that forced a straight versus wall‐following choice, the simulation with constrained turn angles predicted general movement across a central gap, whereas Drosophila tend to follow the wall. Hence, low turn angled movement does not drive the boundary preference. Lastly, visually impaired Drosophila demonstrate a defect in attenuation of the elevated initial activity. Interestingly, the visually impaired w1118 activity decay defect can be rescued by increasing the contrast of the arenas edge, suggesting that the activity decay relies on visual detection of the boundary. The arena boundary is, therefore, a primary object of exploration for Drosophila.

miR-322/-503 cluster is expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification.

Significance Compared with microRNAs (miRNAs) enriched in cardiac and skeletal muscles, little is known about miRNAs expressed in early cardiac progenitors. Here, we show that mesoderm posterior 1 (Mesp1) transactivates a large number of miRNAs that may promote cardiomyocyte formation. The miR-322/-503 cluster has the highest enrichment in the Mesp1 lineage of cardiac progenitor cells, is specifically expressed in the developing heart tube, and drives precocious cardiomyocyte formation by targeting an RNA-binding factor, CUG-binding protein Elav-like family member 1 (Celf1). This study fills a gap in our knowledge about miRNAs acting early in the cardiac program and identifies previously unreported candidates in promoting cardiac regeneration. Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.

Genome‐Wide Identification of MESP1 Targets Demonstrates Primary Regulation Over Mesendoderm Gene Activity

MESP1 is considered the first sign of the nascent cardiac mesoderm and plays a critical role in the appearance of cardiac progenitors, while exhibiting a transient expression in the developing embryo. We profiled the transcriptome of a pure population of differentiating MESP1‐marked cells and found that they chiefly contribute to the mesendoderm lineage. High‐throughput sequencing of endogenous MESP1‐bound DNA revealed that MESP1 preferentially binds to two variants of E‐box sequences and activates critical mesendoderm modulators, including Eomes, Gata4, Wnt5a, Wnt5b, Mixl1, T, Gsc, and Wnt3. These mesendoderm markers were enriched in the MESP1 marked population before the appearance of cardiac progenitors and myocytes. Further, MESP1‐binding is globally associated with H3K27 acetylation, supporting a novel pivotal role of it in regulating target gene epigenetics. Therefore, MESP1, the pioneer cardiac factor, primarily directs the appearance of mesendoderm, the intermediary of the earliest progenitors of mesoderm and endoderm organogenesis. Stem Cells 2015;33:3254–3265

Modeling Drosophila Positional Preferences in Open Field Arenas with Directional Persistence and Wall Attraction

In open field arenas, Drosophila adults exhibit a preference for arena boundaries over internal walls and open regions. Herein, we investigate the nature of this preference using phenomenological modeling of locomotion to determine whether local arena features and constraints on movement alone are sufficient to drive positional preferences within open field arenas of different shapes and with different internal features. Our model has two components: directional persistence and local wall force. In regions far away from walls, the trajectory is entirely characterized by a directional persistence probability, , for each movement defined by the step size, , and the turn angle, . In close proximity to walls, motion is computed from and a local attractive force which depends on the distance between the fly and points on the walls. The directional persistence probability was obtained experimentally from trajectories of wild type Drosophila in a circular open field arena and the wall force was computed to minimize the difference between the radial distributions from the model and Drosophila in the same circular arena. The two-component model for fly movement was challenged by comparing the positional preferences from the two-component model to wild type Drosophila in a variety of open field arenas. In most arenas there was a strong concordance between the two-component model and Drosophila. In more complex arenas, the model exhibits similar trends, but some significant differences were found. These differences suggest that there are emergent features within these complex arenas that have significance for the fly, such as potential shelter. Hence, the two-component model is an important step in defining how Drosophila interact with their environment.

Mouse myofibers lacking the SMYD1 methyltransferase are susceptible to atrophy, internalization of nuclei and myofibrillar disarray

ABSTRACT The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity. Summary: Elimination of the lysine methyltransferase SMYD1 from mouse skeletal muscle caused myopathy with excessive internal nuclei, atrophy, myofibrillar disorganization and broad upregulation of muscle gene expression.

Mesp1 Marked Cardiac Progenitor Cells Repair Infarcted Mouse Hearts

Mesp1 directs multipotential cardiovascular cell fates, even though it’s transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1’s transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1Cre/+; Rosa26EYFP/+ ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.

Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis

ABSTRACT HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. Summary: Deletion of HIRA, a gene encoding a histone chaperone that lies within the critical region of 22q11.2 deletion syndrome, alters cardiac gene expression and leads to cardiomyopathy.

Novel MicroRNAs regulating proliferation and apoptosis in uterine papillary serous carcinomas.

MicroRNAs (miRNAs) are endogenous, non-coding RNA transcripts that regulate gene expression. Here, we report 175 putative novel miRNAs identified in uterine cancers profiled by Next Generation Sequencing. Our data indicate that one of these putative miRNAs (BCM-173) is conserved across multiple species and is expressed at levels similar to known human miRNAs. Functionally, this miRNA promotes the growth and migration of uterine cancer cell lines by targeting vinculin and altering the distribution of focal adhesions. These results expand our insight into the repertoire of human miRNAs and identify novel pathways by which dysregulated miRNA expression promotes uterine cancer growth.