Relaxed targeting rules help PIWI proteins silence transposons.

In eukaryotes, small RNA guides, such as small interfering RNAs and microRNAs, direct AGO-clade Argonaute proteins to regulate gene expression and defend the genome against external threats. Only animals make a second clade of Argonaute proteins: PIWI proteins. PIWI proteins use PIWI-interacting RNAs (piRNAs) to repress complementary transposon transcripts1,2. In theory, transposons could evade silencing through target site mutations that reduce piRNA complementarity. Here we report that, unlike AGO proteins, PIWI proteins efficiently cleave transcripts that are only partially paired to their piRNA guides. Examination of target binding and cleavage by mouse and sponge PIWI proteins revealed that PIWI slicing tolerates mismatches to any target nucleotide, including those flanking the scissile phosphate. Even canonical seed pairing is dispensable for PIWI binding or cleavage, unlike plant and animal AGOs, which require uninterrupted target pairing from the seed to the nucleotides past the scissile bond3,4. PIWI proteins are therefore better equipped than AGO proteins to target newly acquired or rapidly diverging endogenous transposons without recourse to new small RNA guides. Conversely, the minimum requirements for PIWI slicing are sufficient to avoid inadvertent silencing of host RNAs. Our results demonstrate the biological advantage of PIWI over AGO proteins in defending the genome against transposons and suggest an explanation for why the piRNA pathway was retained in animal evolution.

Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability.

In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals.

In animals, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased precursor piRNAs (pre-piRNAs). Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals, including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal-the Urmetazoan-used PIWI proteins both to generate piRNA guides and to execute piRNA function.

A-MYB/TCFL5 regulatory architecture ensures the production of pachytene piRNAs in placental mammals.

In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.

The transcription factor TCFL5 responds to A-MYB to elaborate the male meiotic program in mice.

In brief: The testis-specific transcription factor, TCFL5, expressed in pachytene spermatocytes regulates the meiotic gene expression program in collaboration with the transcription factor A-MYB. Abstract: In male mice, the transcription factors STRA8 and MEISON initiate meiosis I. We report that STRA8/MEISON activates the transcription factors A-MYB and TCFL5, which together reprogram gene expression after spermatogonia enter into meiosis. TCFL5 promotes the transcription of genes required for meiosis, mRNA turnover, miR-34/449 production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis. Moreover, Tcfl5+/em1 mutants produce fewer motile sperm.

Conserved, developmentally regulated mechanism couples chromosomal looping and heterochromatin barrier activity at the homeobox gene A locus.

Establishment and segregation of distinct chromatin domains are essential for proper genome function. The insulator protein CCCTC-binding factor (CTCF) is involved in creating boundaries that segregate chromatin and functional domains and in organizing higher-order chromatin structures by promoting chromosomal loops across the vertebrate genome. Here, we investigate the insulation properties of CTCF at the human and mouse homeobox gene A (HOXA) loci. Although cohesin loading at the CTCF binding site is required for looping, we found that cohesin is dispensable for chromatin barrier activity at that site. Using mouse embryonic stem cells in both a pluripotent and differentiated neuronal progenitor state, we determined that embryonic stem cell pluripotency factor OCT4 antagonizes cohesin loading at the CTCF binding site. Loss of OCT4 in the committed and differentiated neuronal progenitor cells results in loading of cohesin and chromosome looping, which contributes to heterochromatin partitioning and selective gene activation across the HOXA locus. Our analysis reveals that chromatin barrier activity of CTCF is evolutionarily conserved and is responsible for the coordinated establishment of chromatin structure, higher-order architecture, and developmental expression of the HOXA locus.

Towards a genome-wide reconstruction of cis-regulatory networks in the human genome.

The vast amount of recent progress made on the sequence of the human genome has allowed an unprecedented examination of cis-regulatory networks. These networks consist of functional elements such as promoters, enhancers, silencers, and insulators, and their coordinated activity is responsible for regulation of gene expression. Recent studies surveyed the entire genome, identifying novel elements and evaluating functional differences in respect to development. These investigations present the first steps towards a global regulatory map for expression in the human genome.

The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility.

Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6-/- fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.

Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans.

In the fetal mouse testis, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons but, after birth, most post-pubertal pachytene piRNAs map to the genome uniquely and are thought to regulate genes required for male fertility. In the human male, the developmental classes, precise genomic origins and transcriptional regulation of postnatal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce postnatal piRNAs in human juvenile and adult testes. As in the mouse, human A-MYB drives transcription of both pachytene piRNA precursor transcripts and messenger RNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that, during mammalian evolution, pachytene piRNA genes are under few selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.

MicroRNA-33-dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis.

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4(+) T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3(+) Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.

Mesenchymal stem cells (MSCs), due to their paracrine, transdifferentiation, and immunosuppressive effects, hold great promise as a therapy for peripheral arterial disease. Diabetes is an important risk factor for peripheral arterial disease; however, little is known of how type II diabetes affects the therapeutic function of MSCs. This review summarizes the current status of preclinical and clinical studies that have been performed to determine the efficacy of MSCs in the treatment of peripheral arterial disease. We also present findings from our laboratory regarding the impact of type II diabetes on the therapeutic efficacy of MSCs neovascularization after the induction of hindlimb ischemia. In our studies, we documented that experimental type II diabetes in db/db mice impaired MSCs' therapeutic function by favoring their differentiation towards adipocytes, while limiting their differentiation towards endothelial cells. Moreover, type II diabetes impaired the capacity of MSCs to promote neovascularization in the ischemic hindlimb. We further showed that these impairments of MSC function and multipotency were secondary to hyperinsulinemia-induced, Nox4-dependent oxidant stress in db/db MSCs. Should human MSCs display similar oxidant stress-induced impairment of function, these findings might permit greater leverage of the potential of MSC transplantation, particularly in the setting of diabetes or other cardiovascular risk factors, as well as provide a therapeutic approach by reversing the oxidant stress of MSCs prior to transplantation.

Transcriptional responses of Mycoplasma gallisepticum strain R in association with eukaryotic cells.

Mycoplasma gallisepticum is an etiologic agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys. Other than proteins important for cytadherence, few M. gallisepticum factors or pathways contributing to host cell interactions have been identified. In this study, an oligonucleotide-based microarray was utilized to investigate transcriptional changes in M. gallisepticum strain R(low) upon exposure to eukaryotic cells. Fifty-eight genes were either up- or downregulated upon exposure to MRC-5 lung fibroblasts grown in vitro, including genes encoding transport-, metabolism-, and translation-associated proteins. Twenty of the 58 regulated genes have no assigned function. These results indicate that M. gallisepticum regulates gene expression upon exposure to eukaryotic cells, revealing genes and pathways likely to be important for host-bacterium interaction.

Chemokine and cytokine gene expression profiles in chickens inoculated with Mycoplasma gallisepticum strains Rlow or GT5.

Mycoplasma gallisepticum infection in chickens leads to tracheitis, airsacculitis, poor feed conversion and reduced egg production, resulting in considerable economic hardship on the poultry industry. The chemokines and cytokines responsible for recruitment, activation and proliferation of leukocytes in affected tissues have not been described. In the current study, chemokine and cytokine gene expression profiles were investigated in tracheas of chickens inoculated with M. gallisepticum strains R(low) (pathogenic) and GT5 (attenuated) at days 1, 4 and 8 post-inoculation. Expression of lymphotactin mRNA was higher in R(low)-inoculated chickens than GT5- or PBS-inoculated chickens, while CXCL13/BCA1 mRNA expression level was higher in both GT5- or R(low)-inoculated chickens than in PBS-inoculated controls on day 1 post-inoculation. However, both R(low) and GT5 strains induced a down-regulation in mRNA expression of CCL20, IL-1beta, IL-8 and IL-12p40 genes, with CCL20 and IL-12 mRNA levels remaining lower on days 4 and 8 post-inoculation. On day 4, R(low)-inoculated chickens exhibited significantly higher tracheal lesion scores and higher levels of lymphotactin, CXCL13, CXCL14, RANTES, MIP-1beta, IL-1beta and IFN-gamma mRNA compared to PBS-inoculated controls. The mRNA levels of these genes were also higher in R(low)-inoculated chickens that had moderate to severe tracheal lesion scores on day 8 post-inoculation. These results reflect the importance of lymphocyte and monocyte chemotactic factors in the development of tracheal lesions in chickens inoculated with M. gallisepticum strain R(low). Our data also suggest that M. gallisepticum may modulate the host response causing dramatic decreases in CCL20, IL-8 and IL-12 mRNA levels in GT5- or R(low)-inoculated chickens as early as one day post-inoculation.

Correlates of immune protection in chickens vaccinated with Mycoplasma gallisepticum strain GT5 following challenge with pathogenic M. gallisepticum strain R(low).

Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain R(low). GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4(+), and CD8(+) cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with R(low). Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4(+) and CD8(+) cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.

Isolation and characterization of Mycoplasma sphenisci sp. nov. from the choana of an aquarium-reared jackass penguin (Spheniscus demersus).

Strain UCMJ was isolated from the choana of a jackass penguin (Spheniscus demersus) with recurrent mucocaseous choanal discharge. Isolation of this mycoplasma expands the known range of species hosting mycoplasmas. The name Mycoplasma sphenisci sp. nov. is proposed for this new species, for which strain UCMJ is the type strain.