MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2-7), particularly those in 3Ј untranslated regions (3ЈUTRs), are preferentially conserved. Here, we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3ЈUTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites. The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the "offset 6mer," to be detected. In total, >45,000 miRNA target sites within human 3ЈUTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3Ј end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P CT ) correlating with experimental measurements of repression. Our expanded set of target predictions (including conserved 3Ј-compensatory sites), are available at the TargetScan website, which displays the P CT for each site and each predicted target.[Supplemental material is available online at www.genome.org.]MicroRNAs are ∼22-nucleotide (nt) endogenous RNAs that derive from distinctive hairpin precursors in plants and animals (Bartel 2004). After incorporation into a silencing complex, which contains at its core an Argonaute protein, an miRNA can pair to an mRNA and thereby specify the post-transcriptional repression of that protein-coding message, either by transcript destabilization, translational repression, or both. MicroRNAs constitute one of the more abundant classes of gene-regulatory molecules in animals, with hundreds of distinct miRNAs confidently identified in both human and mouse (Landgraf et al. 2007). A central goal for understanding the functions of all these small regulatory RNAs has been to determine which messages are targeted for repression.The search for biological targets of metazoan miRNAs has benefited greatly from the comparative analysis of orthologous mRNAs. Targets of miRNAs can be predicted above the background of false-positive predictions by requiring conserved Watson-Crick pairing to the 5Ј region of the miRNA, known as the miRNA seed (Lewis et al. 2003). Because so many messages have preferentially preserved their pairing to miRNA seeds, targets can be predicted simply by searching for c...

Posttranscriptional gene regulation frequently occurs through elements in mRNA 3′ untranslated regions (UTRs)1,2. Although crucial roles for 3′UTR-mediated gene regulation have been found in Caenorhabditis elegans3,4,5, most C. elegans genes have lacked annotated 3′UTRs6,7. Here we describe a high-throughput method to reliably identify polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3′UTRs. Compared to standard methods also recently applied to C. elegans UTRs8, 3P-Seq identified 8,581 additional UTRs while excluding thousands of shorter UTR isoforms that do not appear to be authentic. Analysis of this expanded and corrected dataset suggested that the high A/U content of C. elegans 3′UTRs facilitated genome compaction, since the elements specifying cleavage and polyadenylation, which are A/U-rich, can more readily emerge in A/U rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,498 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes utilize palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3′UTRs have median length only one-sixth that of mammalian 3′UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying posttranscriptional gene regulation.

Sequence-specific DNA binding by transcription factors is central to gene expression regulation. While a number of methods for characterizing DNA-protein interactions are currently available1-6, none have demonstrated both quantitative measurement of affinity and high throughput. To address this challenge, we developed HiTS-FLIP, a technique that couples high-throughput sequencing with direct visualization of in vitro binding to provide quantitative protein-DNA binding affinity measurements at unprecedented depth. HiTS-FLIP analysis of GCN4, the master regulator of the yeast amino acid starvation response7, yielded 440 million binding measurements, enabling determination of context-averaged dissociation constants for all 12mer sequences having submicromolar affinity. These data revealed complex interdependency between motif positions, yielded improved discrimination of in vivo GCN4 binding sites and regulatory targets relative to previous models, and identified sets of genes with distinct GCN4 affinity levels which had distinct functions and expression kinetics. This approach promises to deepen understanding of the interactions that drive transcription.

The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host1,2. Within the thousand bacterial species present in the intestine, the symbiont Segmented Filamentous Bacterium (SFB) is unique in its ability to potently stimulate the postnatal maturation of the B and T cell compartments and induce a striking increase in the small intestinal Th17 responses3-5. Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer's patches6,7. This colonization does not result in pathology but rather protects the host from pathogens4. Yet, little is known about the host-SFB interaction that underlies the important immunostimulatory properties of SFB as SFB has resisted in vitro culturing for over 50 years. Here we succeeded in growing mouse SFB outside of its host in an SFB-host cell co-culturing system. Single-celled SFB isolated from monocolonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offsprings or IOs, that can colonize mice to induce signature immune responses. In vitro, IOs can attach to mouse and human host cells and recruit actin. In Author Contributions: NCB, VGR, PSa. and PSc conceived the project and discussed experiments. PSc. designed and performed all in vitro experiments. VGR and MG performed the in vivo challenge experiments, VGR maintained SFB mice and MG analysed the TC7 and RF the mICl2 host response. MMN processed SEM samples and took images with PS. GN assisted in vitro experiments. PSc wrote the paper and PSa, NCB and VGR edited the manuscript.

HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

Antimicrobial peptides (AMP) are defense effectors of the innate immunity playing a crucial role in the intestinal homeostasis with commensals and protection against pathogens. Herein we aimed to investigate AMP gene regulation by deciphering specific characteristics allowing their enhanced expression among innate immune genes, particularly those encoding proinflammatory mediators. Our emphasis was on epigenetic regulation of the gene encoding the AMP β-defensin 2 (HBD2), taken as a model of possibly specific induction, upon challenge with a commensal bacterium, compared with the proinflammatory cytokine IL-8. Using an in vitro model of colonic epithelial cells challenged with Escherichia coli K12, we showed that inhibition of histone deacetylases (HDAC) by trichostatin A dramatically enhanced induction of HBD2 expression, without affecting expression of IL-8. This mechanism was supported by an increased phosphorylation of histone H3 on serine S10, preferentially at the HBD2 promoter. This process occurred through activation of the IκB kinase complex, which also led to activation of NF-κB. Moreover, we demonstrated that NF-κB was modified by acetylation upon HDAC inhibition, partly by the histone acetyltransferase p300, and that both NF-κB and p300 supported enhanced induction of HBD2 expression. Furthermore, we identified additional genes belonging to antimicrobial defense and epithelial restitution pathways that showed a similar pattern of epigenetic control. Finally, we confirmed our finding in human colonic primary cells using an ex vivo organoid model. This work opens the way to use epigenetic pharmacology to achieve induction of epithelial antimicrobial defenses, while limiting the deleterious risk of an inflammatory response.

Transmission distorters (TDs) are genetic elements that favor their own transmission to the detriments of others. Slx/Slxl1 (Sycp3-like-X-linked and Slx-like1) and Sly (Sycp3-like-Y-linked) are TDs which have been co-amplified on the X and Y chromosomes of Mus species. They are involved in an intragenomic conflict in which each favors its own transmission, resulting in sex ratio distortion of the progeny when Slx/Slxl1 vs. Sly copy number is unbalanced. They are specifically expressed in male postmeiotic gametes (spermatids) and have opposite effects on gene expression: Sly knockdown leads to the upregulation of hundreds of spermatid-expressed genes, while Slx/Slxl1-deficiency downregulates them. When both Slx/Slxl1 and Sly are knocked-down, sex ratio distortion and gene deregulation are corrected. Slx/Slxl1 and Sly are, therefore, in competition but the molecular mechanism remains unknown. By comparing their chromatin binding profiles and protein partners, we show that SLX/SLXL1 and SLY proteins compete for interaction with H3K4me3-reader SSTY1 (Spermiogenesis-specific-transcript-on-the-Y1) at the promoter of thousands of genes to drive their expression, and that the opposite effect they have on gene expression is mediated by different abilities to recruit SMRT/N-Cor transcriptional complex. Their target genes are predominantly spermatid-specific multicopy genes encoded by the sex chromosomes and the autosomal Speer/Takusan. Many of them have co-amplified with Slx/Slxl1/Sly but also Ssty during muroid rodent evolution. Overall, we identify Ssty as a key element of the X vs. Y intragenomic conflict, which may have influenced gene content and hybrid sterility beyond Mus lineage since Ssty amplification on the Y pre-dated that of Slx/Slxl1/Sly.

Sperm are haploid, but must be functionally equivalent to distribute alleles equally among progeny. Accordingly, gene products are shared through spermatid cytoplasmic bridges which erase phenotypic differences between individual haploid sperm. Here, we show that a large class of mammalian genes are not completely shared across these bridges. We term these genes “genoinformative markers” (GIMs) and show that a subset can act as selfish genetic elements that spread alleles unevenly through murine, bovine, and human populations. We identify evolutionary pressure to avoid conflict between sperm and somatic function as GIMs are enriched for testis-specific gene expression, paralogs, and isoforms. Therefore, GIMs and sperm-level natural selection may help explain why testis gene expression patterns are an outlier relative to all other tissues.