CRISPR/Cas-based screening of long non-coding RNAs (lncRNAs) in macrophages with an NF-κB reporter.

The innate immune system protects against infections by initiating an inducible inflammatory response. NF-κB is one of the critical transcription factors controlling this complex response, but some aspects of its regulation remain unclear. For example, although long non-coding RNAs (lncRNAs) have been shown to critically regulate gene expression, only a fraction of these have been functionally characterized, and the extent to which lncRNAs control NF-κB expression is unknown. Here, we describe the generation of a GFP-based NF-κB reporter system in immortalized murine bone marrow-derived macrophages (iBMDM). Activation of this reporter, using Toll-like receptor ligands, resulted in GFP expression, which could be monitored by flow cytometry. We also established a CRISPR/Cas9 gene deletion system in this NF-κB reporter line, enabling us to screen for genes that regulate NF-κB signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-κB signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting IκBα degradation in the cytoplasm. For lincRNA-AK170409, we provide evidence that this nuclearly-localized lincRNA regulates a number of inflammation-related genes. In conclusion, we have established an NF-κB-GFP iBMDM reporter cell line and a line that stably expresses Cas9. Our approach enabled the identification of lincRNA-Cox2 and lincRNA-AK170409 as NF-κB regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function

Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex.

BackgroundThe genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.ResultsUsing a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.ConclusionsThese results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo

GC-Biased Evolution Near Human Accelerated Regions

Regions of the genome that have been the target of positive selection specifically along the human lineage are of special importance in human biology. We used high throughput sequencing combined with methods to enrich human genomic samples for particular targets to obtain the sequence of 22 chromosomal samples at high depth in 40 kb neighborhoods of 49 previously identified 100–400 bp elements that show evidence for human accelerated evolution. In addition to selection, the pattern of nucleotide substitutions in several of these elements suggested an historical bias favoring the conversion of weak (A or T) alleles into strong (G or C) alleles. Here we found strong evidence in the derived allele frequency spectra of many of these 40 kb regions for ongoing weak-to-strong fixation bias. Comparison of the nucleotide composition at polymorphic loci to the composition at sites of fixed substitutions additionally reveals the signature of historical weak-to-strong fixation bias in a subset of these regions. Most of the regions with evidence for historical bias do not also have signatures of ongoing bias, suggesting that the evolutionary forces generating weak-to-strong bias are not constant over time. To investigate the role of selection in shaping these regions, we analyzed the spatial pattern of polymorphism in our samples. We found no significant evidence for selective sweeps, possibly because the signal of such sweeps has decayed beyond the power of our tests to detect them. Together, these results do not rule out functional roles for the observed changes in these regions—indeed there is good evidence that the first two are functional elements in humans—but they suggest that a fixation process (such as biased gene conversion) that is biased at the nucleotide level, but is otherwise selectively neutral, could be an important evolutionary force at play in them, both historically and at present

Differential Expression of Novel Potential Regulators in Hematopoietic Stem Cells

The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell–cell and cell–matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns). One of the earliest events in this process is recognition of the 3' splice site (3'ss) by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proofreading of 3'ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control and HNRNPA1 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3'ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3'ss

Forces Shaping the Fastest Evolving Regions in the Human Genome

Comparative genomics allow us to search the human genome for segments that were extensively changed in the last ~5 million years since divergence from our common ancestor with chimpanzee, but are highly conserved in other species and thus are likely to be functional. We found 202 genomic elements that are highly conserved in vertebrates but show evidence of significantly accelerated substitution rates in human. These are mostly in non-coding DNA, often near genes associated with transcription and DNA binding. Resequencing confirmed that the five most accelerated elements are dramatically changed in human but not in other primates, with seven times more substitutions in human than in chimp. The accelerated elements, and in particular the top five, show a strong bias for adenine and thymine to guanine and cytosine nucleotide changes and are disproportionately located in high recombination and high guanine and cytosine content environments near telomeres, suggesting either biased gene conversion or isochore selection. In addition, there is some evidence of directional selection in the regions containing the two most accelerated regions. A combination of evolutionary forces has contributed to accelerated evolution of the fastest evolving elements in the human genome

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.No sponso

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta