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The genetic mechanisms underlying the expansion in size and complexity of the human brain remains poorly understood. L1 retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution is largely unknown. Using multi-omic profiling we here demonstrate that L1-promoters are dynamically active in the developing and adult human brain. L1s generate hundreds of developmentally regulated and cell-type specific transcripts, many which are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived lncRNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPRi-silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

As guest editors, we are pleased to present this Special Issue on endogenous retroviruses (ERVs) and their impact on mammalian development and disease [...]

The current academic culture facing women in science, technology, engineering, and math (STEM) fields in the United States has sparked the formation of grassroots advocacy groups to empower female scientists in training. However, the impact of these initiatives often goes unmeasured and underappreciated. Our Women in Science and Engineering (WiSE) organization serves postdoctoral researchers, graduate students, and research technicians (trainees) at a private research institute for biological sciences. Here we propose the following guidelines for cultivating a successful women-in-STEM-focused group based upon survey results from our own scientific community as well as the experience of our WiSE group leaders. We hope these recommendations can provide guidance to advocacy groups at other research and academic organizations that wish to strengthen their efforts. Whereas our own group specifically focuses on the underrepresented state of women in science, we hope these guidelines may be adapted and applied to groups that advocate for any minority group within the greater scientific community (i.e., those of gender, race/ethnicity, socioeconomic background, sexual orientation, etc.)., Author summary Women are underrepresented in leadership positions in the fields of science, technology, engineering, and math (STEM) despite obtaining undergraduate and graduate-level degrees at roughly the same rate as men. To take advantage of this resource of highly trained women in STEM fields, there needs to be better career support for female scientists in training. The authors report on their experience running a women-in-STEM-focused group to foster a more supportive, collaborative, and egalitarian scientific community. In particular, the authors use data-driven metrics to evaluate the success of their group in meeting the needs of their community. The results of this evaluation are presented as a set of 10 guidelines for establishing and maintaining advocacy groups that support the careers of women in science.

The current academic culture facing women in science, technology, engineering, and math (STEM) fields in the United States has sparked the formation of grassroots advocacy groups to empower female scientists-in-training. However, the impact of these initiatives often goes unmeasured and underappreciated. Our Women in Science and Engineering (WiSE) organization serves post-doctoral researchers, graduate students, and research technicians (trainees) at a private research institute for biological sciences. Here we propose the following guidelines for cultivating a successful women-in-STEM-focused group based upon survey results from our own scientific community as well as the experience of our WiSE group leaders. We hope these recommendations can provide guidance to advocacy groups at other research and academic organizations that wish to strengthen their efforts. While our own group specifically focuses on the underrepresented state of women in science, we hope these guidelines may be adapted and applied to groups that advocate for any minority group within the greater scientific community (i.e. those of gender, race/ethnicity, socioeconomic background, sexual orientation, etc.).

Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present a single-cell RNA-seq analysis and klustering evaluation (SAKE), a robust method for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range of cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors (BRAFi). Single-cell RNA-seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor-resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. These experimentally validated markers of BRAFi resistance share overlap with previous analyses in different melanoma cell lines, demonstrating the generality of these findings and highlighting the utility of single-cell analysis to elucidate mechanisms of BRAFi resistance.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

Transposable Elements (TEs) are mobile genetic elements whose sequences constitute nearly half of the human genome. Each TE copy can be present in hundreds to thousands of locations within the genome, complicating the genetic and genomic studies of these highly repetitive sequences. The recent development of better tools for evaluating TE derived sequences in genomic studies has enabled an increasing appreciation for the contribution of TEs to human development and disease. While some TEs have contributed novel and beneficial host functions, this review will summarize the evidence for detrimental TE activity in neurodegenerative disorders. Much of the evidence for pathogenicity implicates endogenous retroviruses (ERVs), a subset of TEs that entered the genome by retroviral infections of germline cells in our evolutionary ancestors and have since been passed down as a substantial fraction of the human genome. Human specific ERVs (HERVs) represent some of the youngest ERVs in the genome, and thus are presumed to retain greater function and resultant pathogenic potential.

SummaryAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several inherited pathogenic mutations have been identified as causative, the vast majority of cases are sporadic with no family history of disease. Thus, for the majority of ALS cases, a specific causal abnormality is not known and the disease may be a product of multiple inter-related pathways contributing to varying degrees in different ALS patients. Using unsupervised machine learning algorithms, we stratified the transcriptomes of 148 ALS decedent cortex tissue samples into three distinct and robust molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 20% of the ALS patient samples exhibited high levels of retrotransposon expression and other signatures of TDP-43 dysfunction. Finally, a third group showed predominant signatures of glial activation (19%). Together these results demonstrate that at least three distinct molecular signatures contribute to ALS disease. While multiple dysregulated components and pathways comprising these clusters have previously been implicated in ALS pathogenesis, unbiased analysis of this large survey demonstrated that sporadic ALS patient tissues can be segregated into distinct molecular subsets.

The single cell is considered the basic unit of biology, and the pursuit of understanding how heterogeneous populations of cells can functionally coexist in tissues, organisms, microbial ecosystems, and even cancer, makes them the subject of intense study. Next-generation sequencing (NGS) of RNA and DNA has opened a new frontier of (single)-cell biology. Hundreds to millions of cells now can be assayed in parallel, providing the molecular profile of each cell in its milieu inexpensively and in a manner that can be analyzed mathematically. The goal of this article is to provide a high-level overview of single-cell sequencing for the nonexpert and show how its applications are influencing both basic and applied clinical studies in embryology, developmental genetics, and cancer.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Next-generation sequencing approaches have fundamentally changed the types of questions that can be asked about gene function and regulation. With the goal of approaching truly genome-wide quantifications of all the interaction partners and downstream effects of particular genes, these quantitative assays have allowed for an unprecedented level of detail in exploring biological interactions. However, many challenges remain in our ability to accurately describe and quantify the interactions that take place in those hard to reach and extremely repetitive regions of our genome comprised mostly of transposable elements (TEs). Tools dedicated to TE-derived sequences have lagged behind, making the inclusion of these sequences in genome-wide analyses difficult. Recent improvements, both computational and experimental, allow for the better inclusion of TE sequences in genomic assays and a renewed appreciation for the importance of TE biology. This review will discuss the recent improvements that have been made in the computational analysis of TE-derived sequences as well as the areas where such analysis still proves difficult. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

Chd5 loss links the up-regulation of ribosomal genes to enhanced translation, causing the untimely production of a master transcription factor that unleashes stem cells and alters cell fate., Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

Transposable elements (TE) are mobile genetic elements that can readily change their genomic position. When not properly silenced, TEs can contribute a substantial portion to the cell's transcriptome, but are typically ignored in most RNA-seq data analyses. One reason for leaving TE-derived reads out of RNA-seq analyses is the complexities involved in properly aligning short sequencing reads to these highly repetitive regions. Here we describe a method for including TE-derived reads in RNA-seq differential expression analysis using an open source software package called TEtranscripts. TEtranscripts is designed to assign both uniquely and ambiguously mapped reads to all possible gene and TE-derived transcripts in order to statistically infer the correct gene/TE abundances. Here, we provide a detailed tutorial of TEtranscripts using a published qPCR validated dataset.Barbara McClintock laid the foundation for TE research with her discoveries in maize of mobile genetic elements capable of inserting into novel locations in the genome, altering the expression of nearby genes [1]. Since then, our appreciation of the contribution of repetitive TE-derived sequences to eukaryotic genomes has vastly increased. With the publication of the first human genome draft by the Human Genome Project, it was determined that nearly half of the human genome is derived from TE sequences [2, 3], with varying levels of repetitive DNA present in most plant and animal species. More recent studies looking at distantly related TE-like sequences have estimated that up to two thirds of the human genome might be repeat-derived [4], with the vast majority of these sequences attributed to retrotransposons that require transcription as part of the mobilization process, as discussed below.

Transposable elements (TE) are mobile genetic elements that can readily change their genomic position. When not properly silenced, TEs can contribute a substantial portion to the cell's transcriptome, but are typically ignored in most RNA-seq data analyses. One reason for leaving TE-derived reads out of RNA-seq analyses is the complexities involved in properly aligning short sequencing reads to these highly repetitive regions. Here we describe a method for including TE-derived reads in RNA-seq differential expression analysis using an open source software package called TEtranscripts. TEtranscripts is designed to assign both uniquely and ambiguously mapped reads to all possible gene and TE-derived transcripts in order to statistically infer the correct gene/TE abundances. Here, we provide a detailed tutorial of TEtranscripts using a published qPCR validated dataset.Barbara McClintock laid the foundation for TE research with her discoveries in maize of mobile genetic elements capable of inserting into novel locations in the genome, altering the expression of nearby genes [1]. Since then, our appreciation of the contribution of repetitive TE-derived sequences to eukaryotic genomes has vastly increased. With the publication of the first human genome draft by the Human Genome Project, it was determined that nearly half of the human genome is derived from TE sequences [2, 3], with varying levels of repetitive DNA present in most plant and animal species. More recent studies looking at distantly related TE-like sequences have estimated that up to two thirds of the human genome might be repeat-derived [4], with the vast majority of these sequences attributed to retrotransposons that require transcription as part of the mobilization process, as discussed below.

Single-cell RNA-Seq’s (scRNA-Seq) unprecedented cellular resolution at a genome wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-Seq datasets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present SAKE (Single-cell RNA-Seq Analysis and Klustering Evaluation): a robust method for scRNA-Seq analysis that provides quantitative statistical metrics at each step of the scRNA-Seq analysis pipeline including metrics for: the determination of the number of clusters present, the likelihood that each cell belongs to a given cluster, and the association of each gene marker in determining cluster membership. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors. Single-cell RNA-Seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. In addition, we compare integrated genomic and transcriptomic markers to show that resistance can arise stochastically within multiple distinct clonal populations.

Dicer enzymes function at the core of RNA silencing to defend against exogenous RNA or to regulate endogenous genes. Plant DICER-LIKE4 (DCL4) performs dual functions, acting in antiviral defense and in development via the biogenesis of trans-acting short-interfering RNAs (siRNAs) termed tasiR-ARFs. These small RNAs play an essential role in the grasses, spatially defining the expression domain of AUXIN RESPONSE FACTOR3 (ARF3) transcription factors. However, contrary to tasiR-ARFs' essential function in development, DCL4 proteins exhibit strong evidence of recurrent adaptation typical of host factors involved in antiviral immunity. Here, we address how DCL4 balances its role in development with pressures to diversify in response to viral attack. We show that, in contrast to other tasiR-ARF biogenesis mutants, dcl4 null alleles have an uncharacteristically mild phenotype, correlated with normal expression of select arf3 targets. Loss of DCL4 activity yields a class of 22-nucleotide tasiR-ARF variants associated with the processing of arf3 transcripts into 22-nucleotide secondary siRNAs by DCL1. Our findings reveal a DCL1-dependent siRNA pathway that bypasses the otherwise adverse developmental effects of mutations in DCL4. This pathway is predicted to have important implications for DCL4's role in antiviral defense by reducing the selective constraints on DCL4 and allowing it to diversify in response to viral suppressors.

Biological robustness, the ability of an organism to maintain a steady-state output as genetic or environmental inputs change, is critical for proper development. MicroRNAs have been implicated in biological robustness mechanisms through their post-transcriptional regulation of genes and gene networks. Previous research has illustrated examples of microRNAs promoting robustness as part of feedback loops and genetic switches and by buffering noisy gene expression resulting from environmental and/or internal changes. Here we show that the evolutionarily conserved microRNAs mir-34 and mir-83 (homolog of mammalian mir-29) contribute to the robust migration pattern of the distal tip cells in Caenorhabditis elegans by specifically protecting against stress from temperature changes. Furthermore, our results indicate that mir-34 and mir-83 may modulate the integrin signaling involved in distal tip cell migration by potentially targeting the GTPase cdc-42 and the beta-integrin pat-3. Our findings suggest a role for mir-34 and mir-83 in integrin-controlled cell migrations that may be conserved through higher organisms. They also provide yet another example of microRNA-based developmental robustness in response to a specific environmental stress, rapid temperature fluctuations.

The mechanisms by which TGF-β promotes lung adenocarcinoma (ADC) metastasis are largely unknown. Here, we report that in lung ADC cells, TGF-β potently induces expression of DOCK4, but not other DOCK family members, via the Smad pathway and that DOCK4 induction mediates TGF-β’s prometastatic effects by enhancing tumor cell extravasation. TGF-β-induced DOCK4 stimulates lung ADC cell protrusion, motility, and invasion without affecting epithelial-to-mesenchymal transition. These processes, which are fundamental to tumor cell extravasation, are driven by DOCK4-mediated Rac1 activation, unveiling a novel link between TGF-β and Rac1. Thus, our findings uncover the atypical Rac1 activator DOCK4 as a key component of the TGF-β/Smad pathway that promotes lung ADC cell extravasation and metastasis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

SummaryPancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer in women and lacks an effective targeted therapy. Therefore, finding common vulnerabilities in these tumors represents an opportunity for more effective treatment. Despite the growing appreciation of G-protein-coupled receptor (GPCR)-mediated signaling in cancer pathogenesis, very little is known about the role GPCRs play in TNBC. Using genomic information of human breast cancer, we have discovered that the orphan GPCR, G-protein-coupled receptor 161 (GPR161) is overexpressed specifically in TNBC and correlates with poor prognosis. Knockdown of GPR161 impairs proliferation of human basal breast cancer cell lines. Overexpression of GPR161 in human mammary epithelial cells increases cell proliferation, migration, intracellular accumulation of E-cadherin, and formation of multiacinar structures in 3D culture. GPR161 forms a signaling complex with the scaffold proteins β-arrestin 2 and Ile Gln motif containing GTPase Activating Protein 1, a regulator of mammalian target of rapamycin complex 1 and E-cadherin. Consistently, GPR161 amplified breast tumors and cells overexpressing GPR161 activate mammalian target of rapamycin signaling and decrease Ile Gln motif containing GTPase Activating Protein 1 phosphorylation. Thus, we identify the orphan GPCR, GPR161, as an important regulator and a potential drug target for TNBC.

SummaryChromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD., Author summary Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.

Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs after the second larval stage in response to environmental stimuli encountered during the first two larval stages. Dauer larvae are stress resistant and long lived, permitting survival in harsh conditions. When environmental conditions improve, dauer larvae re-enter development, and progress through two post-dauer larval stages to adulthood. Strikingly, all of these life history options (whether continuous or interrupted) involve an identical pattern and sequence of cell division and cell fates. To identify microRNAs with potential functions in buffering development in the context of C. elegans life history options, we used multiplex real-time PCR to assess the expression of 107 microRNAs throughout development in both continuous and interrupted life histories. We identified 17 microRNAs whose developmental profile of expression is affected by dauer life history and/or L1 diapause, compared to continuous development. Hence these microRNAs could function to regulate gene expression programs appropriate for different life history options in the developing worm.

MicroRNAs (miRNAs) are short RNA molecules that regulate the post-transcriptional expression of their target genes. This regulation may take the form of stable translational repression or degradation of the target transcript, although the mechanisms governing the outcome of miRNA-mediated regulation remain largely unknown. While it is becoming clear that miRNAs are core components of gene regulatory networks, elucidating precise roles for each miRNA within these networks will require an accurate means of identifying target genes and assessing the impact of miRNAs on individual targets. Numerous computational methods for predicting targets are currently available. These methods vary widely in their emphasis, accuracy, and ease of use for researchers. This review will focus on a comparison of the available computational methods in animals, with an emphasis on approaches that are informed by experimental analysis of microRNA:target complexes.

Hubble Space Telescope images of the core-collapse supernova remnant Cassiopeia A are used to identify high-velocity knots of ejecta located outside the remnant's main emission shell of expanding debris. These ejecta fragments are found near or ahead of the remnant's forward shock front and mostly lie from 12000 to 30000 in radial distance from the remnant's center of expansion. Filter flux ratios when correlated with published spectra show that these knots can be divided into three emission classes: (1) knots dominated by [N II] lambda lambda 6548, 6583 emissions, ( 2) knots dominated by [O II] kk7319, 7330 emissions, and (3) knots displaying filter flux ratios suggestive of [ S II], [ O II], and [Ar III] lambda 7135 emission line strengths similar to the "fast-moving knots'' (FMKs) found in the remnant's bright main shell. Of 1825 knots identified, 444 are strong [N II] emission knots, 192 are strong [O II] emission knots, and 1189 are FMK-like knots. In terms of location around the remnant, 972, 207, and 646 knots are found in the remnant's northeast jet, southwest jet, and non-jet regions, respectively. Assuming a distance of 3.4 kpc, derived knot transverse velocities based on proper motion measurements spanning a 9 month interval indicate maximum transverse expansion velocities for these three knot classes of 14,500, 13,500, and 11,500 km s(-1), respectively. We present a catalog of these outlying ejecta clumps comprising finding charts, epoch 2004.2 knot positions, proper motions, photometric filter fluxes, and estimated knot emission type, along with cross-references to previous knot identifications and data. This compilation represents a nearly tenfold increase in the number of outlying, high-velocity ejecta knots identified around the Cassiopeia A remnant.

Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5' seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.

We present HST images of the remnant of SN 1885 seen in absorption against M31's bulge via resonance lines of Ca I, Ca II, Fe I, and Fe II. Viewed in CaII H & K line absorption, the remnant appears as a nearly black circular spot with an outermost angular radius of 0.40" +/- 0.025" implying r = 1.52 pc and a 120 yr average expansion velocity of 12400 +/-1400 km/s. The strongest Ca II absorption is organized in a broken ring structure with a radius of 0.20" (=6000 km/s) with several apparent absorption `clumps' of an angular size near the pixel scale of 0.05" (= 1500 km/s). The detection of Ca II clumps is the first direct evidence for some instabilities and the existence of a deflagration phase in SNe Ia or, alternatively, mixing induced by radioactive decay of 56^Ni over time scales of seconds or days. However, the degree of mixing allowed by the observed images is much smaller than current 3D calculations for Rayleigh-Taylor dominated deflagration fronts. The images also require a central region of no or little Ca but iron group elements indicative of burning under sufficiently high densities for electron capture taking place, i.e., burning prior to a significant pre-expansion of the WD.

PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer., The p53-inducible antiproliferative gene BTG2 is suppressed in many cancers, in the absence of inactivating gene mutations. Here the authors show that the histone lysine methyltransferase SETD1A suppresses the expression of several p53 target genes including BTG2 by inducing a network of microRNAs.

Motivation: Most RNA-seq data analysis software packages are not designed to handle the complexities involved in properly apportioning short sequencing reads to highly repetitive regions of the genome. These regions are often occupied by transposable elements (TEs), which make up between 20 and 80% of eukaryotic genomes. They can contribute a substantial portion of transcriptomic and genomic sequence reads, but are typically ignored in most analyses. Results: Here, we present a method and software package for including both gene- and TE-associated ambiguously mapped reads in differential expression analysis. Our method shows improved recovery of TE transcripts over other published expression analysis methods, in both synthetic data and qPCR/NanoString-validated published datasets. Availability and implementation: The source code, associated GTF files for TE annotation, and testing data are freely available at http://hammelllab.labsites.cshl.edu/software. Contact: mhammell@cshl.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well., Author Summary Mutations in maize leafbladeless1 (lbl1) that disrupt ta-siRNA biogenesis give rise to plants with thread-like leaves that have lost top/bottom polarity. We used genomic approaches to identify lbl1-dependent small RNAs and their targets to determine the basis for these polarity defects. This revealed substantial diversity in small RNA pathways across plant species and identified unexpected roles for LBL1 in the regulation of repetitive elements within the maize genome. We further show that only ta-siRNA loci belonging to the TAS3 family function in the maize vegetative apex. The TAS3-derived tasiR-ARFs are the main ta-siRNA active in the apex, and misregulation of their ARF3 targets emerges as the basis for the lbl1 leaf polarity defects. Supporting this, we show that plants expressing arf3a transcripts insensitive to tasiR-ARF-directed cleavage recapitulate the phenotypes observed in lbl1. The TAS3 ta-siRNA pathway, including the regulation of ARF3 genes, is conserved throughout land plant evolution, yet the phenotypes of plants defective for ta-siRNA biogenesis are strikingly different. Our data leads us to propose that divergence in the processes regulated by the ARF3 transcription factors or the spatiotemporal pattern during development in which these proteins act, underlies the diverse developmental contributions of this small RNA pathway across plants.

Pancreatic Ductal Adenocarcinoma (PDA) is the 4th leading cause of cancer death in the USA. Lethality of PDA is largely ascribed to poor drug delivery and augmented cell survival pathways. We previously found that oncogenic Kras expression induced an important regulator of redox control, the transcription factor Nuclear factor erythroid-derived 2-like 2, Nfe2l2/Nrf2. Expression of Nrf2-dependent proteins is critical for neutralizing or eliminating toxicants and to maintain cellular redox homeostasis. The NRF2 transcriptional program is believed to protect neoplastic cells from oxidative stress, and may confer the resistance of these cells to chemotherapy. Thus, antagonizing NRF2 effector functions represents an attractive therapeutic strategy. Activation of Nrf2 leads to alterations in cellular redox levels, to which cysteine residues are particularly reactive. Redox modifications on reactive cysteines may regulate the activity of their corresponding protein, rendering these proteins as candidate redox-sensitive effectors of NRF2. To decipher changes in the cysteine proteome, we devised a highly sensitive proteomic method that combines a selectively cleavable cysteine-reactive affinity tag to enrich for and identify reduced cysteines, with amine-reactive isobaric tags for relative and absolute quantification of the total proteome. Using this approach, we identified cysteines on translational regulatory proteins to be explicitly oxidized in Nrf2-deficient, Kras mutant cells. Both cap-dependent and -independent mRNA translation was impaired in Nrf2-deficient pancreatic cancer cells, and can be rescued upon supplementation with antioxidants. In addition to stimulating translation through maintaining the reduced state of specific cysteine residues, redox regulation by Nrf2 also promotes EGFR autocrine signaling through AKT in KRAS mutant cells to fuel cap-dependent translation initiation. These functions converge to promote global protein synthesis in PDA. As a consequence, combined inhibition of AKT signaling and glutathione synthesis hampered the survival of PDA cells in vitro and in vivo, presenting a new opportunity for therapeutic intervention. This abstract is also being presented as Poster B31. Citation Format: Iok In Christine Chio, Seyed Mehdi Jafarnejad, Mariano Ponz-Sarvise, Youngkyu Park, Keith Rivera, Wilhelm Palm, Daniel Ohlund, Molly Hammell, Howard Crawford, Edward Schmidt, Craig Thompson, Darryl Pappin, Nahum Sonenberg, David Tuveson. NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR03.

International audience; The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.

Pancreatic Ductal Adenocarcinoma (PDA) is the 4th leading cause of cancer death in the USA. Lethality of PDA is largely ascribed to poor drug delivery and augmented cell survival pathways. We previously found that oncogenic Kras expression induced an important regulator of redox control, the transcription factor Nuclear factor erythroid-derived 2-like 2, Nfe2l2/Nrf2. Expression of Nrf2-dependent proteins is critical for neutralizing or eliminating toxicants and to maintain cellular redox homeostasis. The NRF2 transcriptional program is believed to protect neoplastic cells from oxidative stress, and may confer the resistance of these cells to chemotherapy. Thus, antagonizing NRF2 effector functions represents an attractive therapeutic strategy. Activation of Nrf2 leads to alterations in cellular redox levels, to which cysteine residues are particularly reactive. Redox modifications on reactive cysteines may regulate the activity of their corresponding protein, rendering these proteins as candidate redox-sensitive effectors of NRF2. To decipher changes in the cysteine proteome, we devised a highly sensitive proteomic method that combines a selectively cleavable cysteine-reactive affinity tag to enrich for and identify reduced cysteines, with amine-reactive isobaric tags for relative and absolute quantification of the total proteome. Using this approach, we identified cysteines on translational regulatory proteins to be explicitly oxidized in Nrf2-deficient, Kras mutant cells. Both cap- dependent and -independent mRNA translation was impaired in Nrf2-deficient pancreatic cancer cells, and can be rescued upon supplementation with antioxidants. In addition to stimulating translation through maintaining the reduced state of specific cysteine residues, redox regulation by Nrf2 also promotes EGFR autocrine signaling through AKT in KRAS mutant cells to fuel cap-dependent translation initiation. These functions converge to promote global protein synthesis in PDA. As a consequence, combined inhibition of AKT signaling and glutathione synthesis hampered the survival of PDA cells in vitro and in vivo, presenting a new opportunity for therapeutic intervention. This abstract is also being presented as Poster B02 Citation Format: Iok In Christine Chio, Seyed Mehdi Jafarnejad, Mariano Ponz-Sarvise, Keith Rivera, Daniel Öhlund, Vineet Sangar, Kevin Wright, Dea Fillippi, Yuan Hao, John Erby Wilkinson, Herve Tiriac, Molly Hammell, Edward Schmidt, Youngkyu Park, Darryl Pappin, Nahum Sonenberg, David Tuveson.{Authors}. Nrf2 promotes mRNA translation in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR04.

Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins—Piwi, Aubergine (Aub), and Ago3—acting in concert with their small RNA guides, the Piwi-interacting RNAs (piRNAs). Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell type-specific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable model systems to interrogate pathways involved in pancreatic tumorigenesis and to probe individual responses to novel therapies are urgently needed. To that end, we established methods to culture normal and neoplastic pancreatic duct cells as three-dimensional organoid cultures. Pancreatic organoids can be rapidly generated from resected tumors or fine needle biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Following orthotopic transplant, neoplastic organoids recapitulated the full spectrum of tumor development by forming early-grade neoplasms that progressed to locally invasive and metastatic carcinomas, demonstrating the utility of organoids to model the stages of PDA tumorigenesis. Monolayer cell lines were generated from organoid cultures with high efficiency, creating a diverse collection of new PDA cell lines. To better understand pathways involved in PDA progression, we performed transcriptomic and proteomic analyses of murine organoids derived from normal pancreatic ducts, pancreatic intraepithelial neoplasias (PanINs), and PDAs. These datasets revealed expression changes associated with early and late pancreatic tumorigenesis. To identify genes dysregulated during pancreatic tumorigenesis whose depletion impaired human PDA cells, a CRISPR-Cas competition assay was employed. Taken together, pancreatic organoids offer a novel model system for studying pancreatic cancer biology and can be used to screen for genetic dependencies in PDA. Citation Format: Lindsey A. Baker, Hervé Tiriac, Vincenzo Corbo, Sylvia F. Boj, Chang-il Hwang, Iok In Christine Chio, Danielle D. Engle, Myrthe Jager, Mariano Ponz-Sarvise, Mona S. Spector, Ana Gracanin, Tobiloba Oni, Kenneth H. Yu, Ruben van Boxtel, Meritxell Huch, Keith D. Rivera, John P. Wilson, Michael E. Feigin, Daniel Öhlund, Abram Handly-Santana, Christine M. Ardito-Abraham, Michael Ludwig, Ela Elyada, Brinda Alagesan, Giulia Biffi, Georgi N. Yordanov, Bethany Delcuze, Brianna Creighton, Kevin Wright, Youngkyu Park, Folkert H.M. Morsink, I. Quintus Molenaar, Inne H. Borel Rinkes, Edwin Cuppen, Yuan Hao, Ying Jin, Isaac J. Nijman, Christine Iacobuzio-Donahue, Steven D. Leach, Darryl J. Pappin, Molly Hammell, David S. Klimstra, Olca Basturk, Ralph H. Hruban, George Johan Offerhaus, Robert G.J. Vries, Hans Clevers, David A. Tuveson. Using human patient-derived organoids to identify genetic dependencies in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B16.

microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNA-target duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely nonoverlapping results and experimental assessment of their impact has been limited. Here, we compare two large sets of miRNA targets: targets identified using an improved Ago1 immunopurification method and targets identified among transcripts up-regulated after Ago1 depletion. We found surprisingly limited overlap between these sets. The two sets showed enrichment for target sites with different molecular, structural and functional properties. Intriguingly, we found a strong correlation between UTR length and other contextual features that distinguish the two groups. This finding was extended to all predicted microRNA targets. Distinct repression mechanisms could have evolved to regulate targets with different contextual features. This study reveals a complex relationship among different features in miRNA-target recognition and poses a new challenge for computational prediction.

MicroRNAs (miRNAs) and transcription factors (TFs) are primary metazoan gene regulators. Whereas much attention has focused on finding the targets of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. Here, we present the first genome-scale Caenorhabditis elegans miRNA regulatory network that contains experimentally mapped transcriptional TF → miRNA interactions, as well as computationally predicted post-transcriptional miRNA → TF interactions. We find that this integrated miRNA network contains 23 miRNA ↔ TF composite feedback loops in which a TF that controls a miRNA is itself regulated by that same miRNA. By rigorous network randomizations, we show that such loops occur more frequently than expected by chance and, hence, constitute a genuine network motif. Interestingly, miRNAs and TFs in such loops are heavily regulated and regulate many targets. This “high flux capacity” suggests that loops provide a mechanism of high information flow for the coordinate and adaptable control of miRNA and TF target regulons.

HST ACS images of the young SN remnant Cas A are used to explore the expansion and spatial distribution of its highest velocity debris. Proper motions of over 1800 outlying ejecta knots are reported. The distribution of transverse expansion velocities for these knots shows a striking bipolar asymmetry with the highest velocity knots confined to nearly opposing northeast and southwest `jets'. The jets appear kinematically and chemically distinct with respect to the remnant's highest velocity debris seen in other directions. Significant gaps in the spatial distribution of outlying ejecta lie in directions which are approximately perpendicular to the jets. Extrapolations of 9 month proper motions for all outer ejecta knots and a subsample of 72 bright and compact knots suggest explosion dates (assuming no knot deceleration) of 1662 +/- 27 and 1672 +/- 18, respectively. We find some evidence for non-uniform deceleration in different directions with knots located along the northwestern limb among the least decelerated ejecta suggesting a convergence date of 1681 +/-19. The remnant's central X-ray point source lies some $7''$ to the southeast of the estimated expansion center (PA = 169 deg) indicating a projected motion of ~350 km/s toward the middle of the broad southern outer ejecta knot gap., Comment: 13 pages, 5 figures, ApJ, in press

Hubble Space Telescope images of the young Galactic supernova remnant Cassiopeia A reveal a far larger population of outlying, high-velocity knots of ejecta with a broader range of chemical properties than previously suspected. We identify three main classes of outer ejecta: 1) Knots dominated by [N II] 6548,6583 emission; 2) Knots dominated by oxygen emission lines especially [O II] 7319,7330; and 3) Knots with emission line strengths similar to the [S II] strong FMK ejecta commonly seen in the main emission shell. The discovery of a significant population of O-rich ejecta situated in between the suspected N-rich outer photospheric layer and S-rich FMK-like ejecta suggests that the Cas A progenitor's chemical layers were not completely disrupted by the supernova explosion outside of the remnant's NE and SW high velocity `jet' regions. In addition, we find the majority of O-rich outer ejecta at projected locations out beyond (v = 6500 - 9000 km/s) the remnant's fastest moving Fe-rich X-ray emission material (6000 km/s) seen in Chandra and XMM data along the eastern limb. This suggests that penetration of Fe-rich material up through the S and Si-rich mantle did not extend past the progenitor's N or O-rich outer layers for at least this section of the remnant., 17 pages including 3 tables and 7 figures. To appear in ApJ

We present results from observations of SN 1979C with XMM-Newton in X-rays and in the UV, archival X-ray and HST data, and follow-up ground-based optical imaging. The XMM-Newton MOS spectrum shows two-temperature thermal plasma emission characteristics of both the forward (4.1 keV) and reverse shock (0.78 keV) with no intrinsic absorption. The long-term X-ray lightcurve of SN 1979C shows no sign of a decline over 16-23 yrs after its outburst. The high X-ray luminosity (8 x 10^38 ergs/s) is caused by the interaction of the shock with dense circumstellar matter, likely deposited by a strong stellar wind from the progenitor with a high mass-loss rate of 1.5 x 10^-4 M_sun/yr. The X-ray data support a strongly decelerated shock and show a mass-loss rate history which is consistent with a constant progenitor mass-loss rate and wind velocity over the past >16,000 yrs in the evolution of the progenitor. We find a CSM density profile of rho_CSM \propto r^-s with index s10^4 cm^-3) out to large radii from the site of the explosion (> 4 x 10^17 cm). Using XMM-Newton Optical Monitor data we further detect optical/UV emission from SN 1979C, with B, U, and UVW1-band lum. of 5,7, and 9 x 10^36 ergs/s. The young stellar cluster in the vicinity of the SN, as imaged by the HST and follow-up ground-based optical imaging, can only provide a fraction of the total observed flux, so that a significant contribution to the output likely arises from the strong interaction of SN 1979C with dense CSM., 13 pages, 9 Postscript figures, submitted to ApJ, uses emulateapj5.sty. A high-resolution version is available at http://lheawww.gsfc.nasa.gov/users/immler/Immler_SN1979C.ps.gz