Repress Target Gene Expression Research Articles

Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

On a stake-out: Mycobacterial small RNA identification and regulation.

Persistence of mycobacteria in the hostile environment of human macrophage is pivotal for its successful pathogenesis. Rapid adaptation to diverse stresses is the key aspect for their survival in the host cells. A range of heterogeneous mechanisms operate in bacteria to retaliate stress conditions. Small RNAs (sRNA) have been implicated in many of those mechanisms in either a single or multiple regulatory networks to post-transcriptionally modulate bacterial gene expression. Although small RNA profiling in mycobacteria by advanced technologies like deep sequencing, tilling microarray etc. have identified hundreds of sRNA, however, a handful of those small RNAs have been unearthed with precise regulatory mechanism. Extensive investigations on sRNA-mediated gene regulations in eubacteria like Escherichia coli revealed the existence of a plethora of distinctive sRNA mechanisms e.g. base pairing, protein sequestration, RNA decoy etc. Increasing studies on mycobacterial sRNA also discovered several eccentric mechanisms where sRNAs act at the posttranscriptional stage to either activate or repress target gene expression that lead to promote mycobacterial survival in stresses. Several intrinsic features like high GC content, absence of any homologue of abundant RNA chaperones, Hfq and ProQ, isolate sRNA mechanisms of mycobacteria from that of other bacteria. An insightful approach has been taken in this review to describe sRNA identification and its regulations in mycobacterial species especially in Mycobacterium tuberculosis.

Default Activation and Nuclear Translocation of the Plant Cellular Energy Sensor SnRK1 Regulate Metabolic Stress Responses and Development.

Energy homeostasis is vital to all living organisms. In eukaryotes, this process is controlled by fuel gauging protein kinases: AMP-activated kinase in mammals, Sucrose Non-Fermenting1 (SNF1) in yeast (Saccharomyces cerevisiae), and SNF1-related kinase1 (SnRK1) in plants. These kinases are highly conserved in structure and function and (according to this paradigm) operate as heterotrimeric complexes of catalytic-α and regulatory β- and γ-subunits, responding to low cellular nucleotide charge. Here, we determined that the Arabidopsis (Arabidopsis thaliana) SnRK1 catalytic α-subunit has regulatory subunit-independent activity, which is consistent with default activation (and thus controlled repression), a strategy more generally used by plants. Low energy stress (caused by darkness, inhibited photosynthesis, or hypoxia) also triggers SnRK1α nuclear translocation, thereby controlling induced but not repressed target gene expression to replenish cellular energy for plant survival. The myristoylated and membrane-associated regulatory β-subunits restrict nuclear localization and inhibit target gene induction. Transgenic plants with forced SnRK1α-subunit localization consistently were affected in metabolic stress responses, but their analysis also revealed key roles for nuclear SnRK1 in leaf and root growth and development. Our findings suggest that plants have modified the ancient, highly conserved eukaryotic energy sensor to better fit their unique lifestyle and to more effectively cope with changing environmental conditions.

MicroRNAs in Microglia: How do MicroRNAs Affect Activation, Inflammation, Polarization of Microglia and Mediate the Interaction Between Microglia and Glioma?

The essential roles of microglia in maintaining homeostasis in the healthy brain and contributing to neuropathology are well documented. Emerging evidence suggests that epigenetic modulation regulates microglial behavior in both physiological and pathological conditions. MicroRNAs (miRNAs) are short, non-coding epigenetic regulators that repress target gene expression mostly via binding to 3′-untranslated region (3′-UTR) of mRNA in a Dicer-dependent manner. Dysregulation of certain miRNAs can contribute to microglial hyper-activation, persistent neuroinflammation, and abnormal macrophage polarization in the brain. These abnormal conditions can support the pathogenesis of neurological disorders such as glioma, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), stroke, ischemia, and spinal cord injury (SCI). However, the roles of miRNAs in microglia in health and neurological disease have not been systematically summarized. This review will first report the role of Dicer, a key endoribonulease that is responsible for most miRNA biogenesis in microglia. Second, we will focus on recent research about the function of miRNAs in activation, inflammation and polarization of microglia, respectively. In addition, potential crosstalk between microglia and glioma cells via miRNAs will be discussed in this part. Finally, the role of two essential miRNAs, miR-124, and miR-155, in microglia will be highlighted.

Structural and Dynamics of Glucocorticoid Receptor Ligand Binding Domain in Complex with PGC1α and Synthetic Glucocorticoids

Synthetic glucocorticoids are important pharmaceutical drugs that are known to bind to the ligand binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. These binding will induce conformational changes within GR for the subsequent DNA binding and different co‐regulators recruitments, ultimately lead to activate or repress target gene expression. One of the crucial co‐regulators is peroxisome proliferator‐activated gamma coactivator 1‐α (PGC1α), known to regulates energy metabolism by direct interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α changes GR conformation to drive transcription are unknown. Here, an ancestral variant of GR (GR2) was utilized as a tool to produce stable protein for biochemical and structural studies. We report the first high resolution X‐ray crystal structures of GR2 LBD in complexes with PGC1α and dexamethasone and hydrocortisone. These structural analyses reveal how distinct steroid drugs bind to GR with different affinities via unique hydrogen bonding and hydrophobic interactions. Hydrogen‐deuterium exchange mass spectrometry showed different effects on GR2 LBD allosteric communication after glucocorticoids binding. Molecular dynamics simulations and network analysis identified the key residues involved in the intramolecular communication between ligand binding site and activation function surface, which is the PGC1α binding site.Support or Funding InformationThis work was supported by W.M. Keck Foundation Medical Research Grant to E. A. O. X. L. was supported by an American Heart Association postdoctoral fellowship [17POST33660110].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aberrant Activation of β-Catenin Signaling Drives Glioma Tumorigenesis via USP1-Mediated Stabilization of EZH2.

Aberrant activation of β-catenin signaling is a critical driver for tumorigenesis, but the mechanism underlying this activation is not completely understood. In this study, we demonstrate a critical role of β-catenin signaling in stabilization of enhancer of zeste homolog 2 (EZH2) and control of EZH2-mediated gene repression in oncogenesis. β-Catenin/TCF4 activated the transcription of the deubiquitinase USP1, which then interacted with and deubiquitinated EZH2 directly. USP1-mediated stabilization of EZH2 promoted its recruitment to the promoters of CDKN1B, RUNX3, and HOXA5, resulting in enhanced enrichment of histone H3K27me3 and repression of target gene expression. In human glioma specimens, expression levels of nuclear β-catenin, USP1, and EZH2 correlated with one another. Depletion of β-catenin/USP1/EZH2 repressed glioma cell proliferation in vitro and tumor formation in vivo. Our findings indicate that a β-catenin-USP1-EZH2 axis orchestrates the interplay between dysregulated β-catenin signaling and EZH2-mediated gene epigenetic silencing during glioma tumorigenesis. SIGNIFICANCE: These findings identify the β-catenin-USP1-EZH2 signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma.

Detection of MicroRNA-Mediated Target mRNA Cleavage and 3'-Uridylation in Human Cells by a SLA-RT-PCR Analysis.

MicroRNA (miRNA) plays an important role in posttranscriptional regulation of gene expression by dominantly binding to the 3'-UTR regions of target mRNAs in the miRNA-induced silencing complex (miRISC), triggering off their sequential cleavage and 3'-uridylation, facilitating their degradation, repressing target gene expression, and leading to a reduced protein output. The miRNA-mediated target mRNA cleavage activity generates cleaved mRNA fragments with varied termini, which creates major technical challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs and the resulting mRNA fragments in transition. Here we described a sensitive stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the miRNA-mediated target mRNA cleavage sites by determining precise sequences at the 3'- termini of cleaved mRNA fragments and their 3'-uridylation in human cells under physiological conditions. The SLA-RT-PCR methods have been demonstrated as a sensitive, cost-efficient, and high-throughput tool to systematically detect miRNA-targeted mRNA cleavage sites and fragments with 3'-uridylation in human cells.

KLF1 Acts As a Pioneer Transcription Factor to Open Chromatin and Facilitate Recruitment of GATA1

Only a small subset of transcription factors (TFs) can act as pioneer factors; i.e. those that can ‘open’ otherwise ‘closed’ chromatin to facilitate assembly of TF complexes and co-factors to enable transcription. The KLF/SP family of TFs bind to a 9-10 bp consensus motif in DNA to activate or repress target gene expression. We have studied the potential for KLF1, which is essential for erythropoiesis, to provide a pioneering function in erythroid progentior cells. Previous ChIP-seq studies have shown KLF1 binds a few thousand enhancers and promoters to activate erythroid cell gene expression 1. It often binds near to other key erythroid TFs such as GATA1 and SCL/TAL1, so is likely to work in concert with them in some contexts. We have employed an inducible stable KLF1-ERTM construct to rescue gene expression and differentiation of Klf1-/- erythroid cell lines 2. We employed ChIP-seq, ATAC-seq and DNAse1 HS to show KLF1 can bind to closed sites in chromatin and induce an open state. We show this is essential for recruitment of the settler transcription, GATA1, at certain co-bound sites but not others. This pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such the one at -26kb in the a-globin LCR and one within the body of the E2f2 gene 3 but rarely at promoters. We further show that two different neomorphic mutations in the KLF1 DNA-binding domain lead to ectopic pioneering (opening of closed chromatin) and aberrant gene activation 4. We generated a series of N-terminal deletions in KLF1 and employed ATAC-seq to map the domain/s within KLF1 responsible for the pioneering activity and show it is distinct from DNA-binding activity. The domain is responsible for bromodomain protein recruitment, the likely effector of chromatin remodelling. We have also examined whether KLF3, which acts as a transcription repressor via recruitment of the co-repressor, CtBP2, can force the closure of otherwise open chromatin 5. We find it cannot. Rather, KLF3 (and likely other members of this subclade) works via active recruitment of co-repressors rather than rendering chromatin inaccessible. This likely enables rapid reactivation of pioneered enhancers without the need to reprogram chromatin. This work has broad implications for how the KLF/SP family of TFs work in vivo to reprogram cells and direct differentiation. We will present data for such activity in non-erythroid cell systems.

The polycomb group protein Yaf2 regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner

The polycomb group (PcG) proteins are key epigenetic regulators in stem cell maintenance. PcG proteins have been thought to act through one of two polycomb repressive complexes (PRCs), but more recent biochemical analyses have challenged this model in the identification of noncanonical PRC1 (nc-PRC1) complexes characterized by the presence of Rybp or Yaf2 in place of the canonical Chromobox proteins. However, the biological significance of these nc-PRC1s and the potential mechanisms by which they mediate gene repression are largely unknown. Here, we explore the functional consequences of Yaf2 disruption on stem cell regulation. We show that deletion of Yaf2 results in compromised proliferation and abnormal differentiation of mouse embryonic stem cells (mESCs). Genome-wide profiling indicates Yaf2 functions primarily as a transcriptional repressor, particularly impacting genes associated with ectoderm cell fate in a manner distinct from Rybp. We confirm that Yaf2 assembles into a noncanonical PRC complex, with deletion analysis identifying the region encompassing amino acid residues 102-150 as required for this assembly. Furthermore, we identified serine 166 as a Yaf2 phosphorylation site, and we demonstrate that mutation of this site to alanine (S166A) compromises Ring1B-mediated H2A monoubiquitination and in turn its ability to repress target gene expression. We therefore propose that Yaf2 and its phosphorylation status serve as dual regulators to maintain the pluripotent state in mESCs.

RYBP modulates stability and function of Ring1B through targeting UBE3A.

Ring1 and yin yang 1-binding protein (RYBP) are central components of noncanonical polycomb-repressive complex 1 (nc-PRC1), which represses target gene expression and is required for normal organismal development. However, the molecular function of RYBP in this complex is obscure. In this study, we showed that RYBP inhibits the polyubiquitination-mediated proteasomal degradation of Ring1B independently of its ubiquitin (Ub)-protein isopeptide ligase (E3) ligase activity, leading to its stabilization and increased catalytic activity toward monoubiquitination of histone H2A at lysine 119. Mechanistic dissection further disclosed that RYBP directly binds to ubiquitin protein ligase E3A (UBE3A) to promote its ubiquitination and proteasomal degradation in an autoubiquitination-independent manner. The resultant reduction of UBE3A protein level alleviates its effect on ubiquitination-mediated degradation of Ring1B, therefore resulting in increased stability and enhanced transcriptional repressor activity on its target genes. Thus, our current findings lay a foundation for understanding how RYBP functions in nc-PRC1 complexes, which is involved in development, stem cell maintenance, and carcinogenesis.-Li, M., Zhang, S., Zhao, W., Hou, C., Ma, X., Li, X., Huang, B., Chen, H., Chen, D. RYBP modulates stability and function of Ring1B through targeting UBE3A.

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase

Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

The 'how' and 'where' of plant microRNAs.

Contents 1002 I. 1002 II. 1007 III. 1010 IV. 1013 1013 References 1013 SUMMARY: MicroRNAs (miRNAs) are small non-coding RNAs, of typically 20-24nt, that regulate gene expression post-transcriptionally through sequence complementarity. Since the identification of the first miRNA, lin-4, in the nematode Caenorhabditis elegans in 1993, thousands of miRNAs have been discovered in animals and plants, and their regulatory roles in numerous biological processes have been uncovered. In plants, research efforts have established the major molecular framework of miRNA biogenesis and modes of action, and are beginning to elucidate the mechanisms of miRNA degradation. Studies have implicated restricted and surprising subcellular locations in which miRNA biogenesis or activity takes place. In this article, we summarize the current knowledge on how plant miRNAs are made and degraded, and how they repress target gene expression. We discuss not only the players involved in these processes, but also the subcellular sites in which these processes are known or implicated to take place. We hope to raise awareness that the cell biology of miRNAs holds the key to a full understanding of these enigmatic molecules.

Role of small RNAs in abiotic stress responses in plants

Abiotic stresses in plants are responsible for considerable yield losses globally. Scientific efforts have been made in understanding the complex stress-response mechanisms. Abiotic stress responses in plants have been known to be associated with the regulation of specific gene expression at the transcriptional and translational level. However, various studies over the years have demonstrated the role of endogenous small RNAs (sRNAs) like micro RNAs (miRNAs), short interfering RNAs (siRNAs), trans acting siRNAs (ta-siRNAs), natural antisense transcript derived siRNAs (nat-siRNAs) and piwi interacting RNAs (piRNAs) in plant abiotic stress response. sRNAs are involved in abiotic stresses like drought and salinity stress, oxidative and mineral nutrient deficiency, thermal (cold and heat) stress etc. These sRNAs are differentially expressed during various abiotic stress responses and repression of target gene expression appears to be the major mechanism of action to combat abiotic stresses (Fig. 1). High throughput techniques have been instrumental in identification and characterization of various stress associated sRNAs in plants. Utilization of innovative bioinformatical and computational tools has also contributed immensely towards genome wide identification of sRNAs and their target genes. In the future, RNA interference approach mediated by various sRNAs can be used for development of transgenic plants aimed for combating abiotic stress tolerance in plants. This review focuses on (i) interpretation and understanding of the roles of various sRNAs involved in abiotic stress tolerance (ii) identification of sRNAs involved in abiotic stress tolerance and study of sRNA-target gene interaction and validation.

Overexpression of gma-miR1510a/b suppresses the expression of a NB-LRR domain gene and reduces resistance to Phytophthora sojae

MicroRNAs (miRNAs) are universal regulators that repress target gene expression in eukaryotes and play essential roles in plant immune responses. miRNAs were recently found to be involved in soybean and Phytophthora sojae interactions. Here, we screened miR1510, which was repressed in soybean during infection with P. sojae, indicating that it might be involved in soybean response to pathogens. To further uncover the roles of miRNAs in soybean, gma-miR1510a/b was overexpressed in the hairy roots of soybean using an Arabidopsis miR319a precursor as the backbone. The gma-miR1510a/b-overexpressing hairy roots showed enhanced susceptibility to P. sojae, and the results showed that miR1510 guides the cleavage of the Glyma.16G135500 gene, which encodes a classic type of plant disease resistance-associated gene that harbors the Toll-interleukin-like receptor (TIR) domain and nucleotide-binding site-leucine-rich repeat (NB-LRR) domain. Noticeably, several biotic stresses and hormone-responsive cis-regulatory elements were found to be present in the promoters of gma-MIR1510a and the target gene. Collectively, the results obtained in the current study reveal that gma-miR1510 regulates the target NB-LRR immune receptor gene Glyma.16G135500 and thus plays a crucial role in regulating the resistance of soybean to P. sojae.

Heterogeneous nuclear ribonucleoprotein A1 regulates rhythmic synthesis of mouse Nfil3 protein via IRES-mediated translation

Nuclear factor, interleukin 3, regulated (Nfil3, also known as E4 Promoter-Binding Protein 4 (E4BP4)) protein is a transcription factor that binds to DNA and generally represses target gene expression. In the circadian clock system, Nfil3 binds to a D-box element residing in the promoter of clock genes and contributes to their robust oscillation. Here, we show that the 5′-untranslated region (5′-UTR) of Nfil3 mRNA contains an internal ribosome entry site (IRES) and that IRES-mediated translation occurs in a phase-dependent manner. We demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds to a specific region of Nfil3 mRNA and regulates IRES-mediated translation. Knockdown of hnRNP A1 almost completely abolishes protein oscillation without affecting mRNA oscillation. Moreover, we observe that intracellular calcium levels, which are closely related to bone formation, depend on Nfil3 levels in osteoblast cell lines. We suggest that the 5′-UTR mediated cap-independent translation of Nfil3 mRNA contributes to the rhythmic expression of Nfil3 by interacting with the RNA binding protein hnRNP A1. These data provide new evidence that the posttranscriptional regulation of clock gene expression is important during bone metabolism.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

Photoclickable MicroRNA for the Intracellular Target Identification of MicroRNAs.

MicroRNAs (miRNAs) are important gene regulators that bind with target genes and repress target gene expression at the post-transcriptional level. The identification of target genes associated with miRNAs inside different cells is a major challenge in miRNA chemical biology due to the lack of functional miRNAs bearing appropriate tags. Here we report photoclickable miRNAs as appropriately pretagged miRNAs that keep the intracellular function of miRNAs and allow the addition of molecular handles through photoclick reaction. The photoclickable miRNAs upon transfection inside cells were able to form functional complexes with target genes and repress target gene expression. Target genes associated with the photoclickable miRNAs in the complexes were then tagged with the molecular handle through photoclick reaction for pull-down and identification. Using photoclickable miR-106a, miR-27, and miR-122, we first verified that their intracellular function was comparable to that of intact miRNAs, which showed obvious advantage over corresponding biotinylated miRNAs. After attaching the biotin handle to the associated complexes containing the photoclickable miRNAs through the tetrazole-ene photoclick reaction, target genes previously bound with these miRNAs inside cells were successfully pulled town and analyzed. The application of this strategy was demonstrated by the identification of several new target genes of miR-122, followed by revealing a novel regulatory pathway in HepG2 cells with regard to the role of PEG10 in miR-122-promoted cell apoptosis.

MiR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts.

Substantial variability exists in the presentation of complex neurological disorders, and the study of single nucleotide polymorphisms (SNPs) has shed light on disease mechanisms and pathophysiological variability in some cases. However, the vast majority of disease-linked SNPs have unidentified pathophysiological relevance. Here, we tested the hypothesis that SNPs within the miRNA recognition element (MRE; the region of the target transcript to which the miRNA binds) can impart changes in the expression of those genes, either by enhancing or reducing transcript and protein levels. To test this, we cross-referenced 7,153 miRNA-MRE brain interactions with the SNP database (dbSNP) to identify candidates, and functionally assessed 24 SNPs located in the 3’UTR or the coding sequence (CDS) of targets. For over half of the candidates tested, SNPs either enhanced (4 genes) or disrupted (10 genes) miRNA binding and target regulation. Additionally, SNPs causing a shift from a common to rare codon within the CDS facilitated miRNA binding downstream of the SNP, dramatically repressing target gene expression. The biological activity of the SNPs on miRNA regulation was also confirmed in induced pluripotent stem cell (iPSC) lines. These studies strongly support the notion that SNPs in the 3’UTR or the coding sequence of disease-relevant genes may be important in disease pathogenesis and should be reconsidered as candidate modifiers.

Stuxnet Facilitates the Degradation of Polycomb Protein during Development

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity.