A Phase Separation Model for Transcriptional Control.

Phase-separated multi-molecular assemblies provide a general regulatory mechanism to compartmentalize biochemical reactions within cells. We propose that a phase separation model explains established and recently described features of transcriptional control. These features include the formation of super-enhancers, the sensitivity of super-enhancers to perturbation, the transcriptional bursting patterns of enhancers, and the ability of an enhancer to produce simultaneous activation at multiple genes. This model provides a conceptual framework to further explore principles of gene control in mammals.

Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control.

Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). ED neural plasticity is driven by histone acetylation (HA) mediated epigenetic mechanisms that regulate dynamic activity-dependent gene transcription profiles in response to neuronal stimulation. Yet, how histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. We previously demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. Here, we focus on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. We used immunohistochemistry (IHC) to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, we found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in our well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that we identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. Our results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

A second riboswitch class for the enzyme cofactor NAD.

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found "pnuC motif" RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.

Exploring and engineering PAM-diverse Streptococci Cas9 for PAM-directed bifunctional and titratable gene control in bacteria.

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria.

A rare bacterial RNA motif is implicated in the regulation of the purF gene whose encoded enzyme synthesizes phosphoribosylamine.

The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by using comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound 5-phospho-D-ribosylamine (PRA), which is the first committed step in purine biosynthesis. The consensus model for Fibro-purF motif RNAs includes a predicted three-stem junction that carries numerous conserved nucleotide positions within the regions joining the stems. This architecture appears to be of sufficient size and complexity for the formation of the ligand-binding aptamer portion of a riboswitch. In this study, we conducted biochemical analyses of a representative Fibro-purF motif RNA to confirm that the RNA generally folds according to the predicted consensus model. However, due to the instability of PRA, binding of this ligand candidate by the RNA could not be directly assessed. Genetic analyses were used to demonstrate that Fibro-purF motif RNAs regulate gene expression in accordance with predicted PRA concentrations. These findings indicate that Fibro-purF motif RNAs are genetic regulation elements that likely suppress PRA biosynthesis when sufficient levels of this purine precursor are present.

Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD.

The nadA motif is a riboswitch candidate present in various Acidobacteria species that was previously identified by bioinformatic analysis of bacterial DNA data sets. More than 100 unique representatives have been identified exclusively upstream of nadA genes, which code for an enzyme in the biosynthetic pathway of the ubiquitous coenzyme NAD+ The architecture of nadA motif RNAs suggests they use structurally similar tandem ligand-binding aptamer domains to control translation initiation. Biochemical analyses reveal that the first domain selectively binds ligands carrying an adenosine 5'-diphosphate (5' ADP) moiety, including NAD+ and its reduced form, NADH. Genetic analyses indicate that a tandem nadA motif RNA suppresses gene expression when NAD+ is abundant, and that both aptamer domains are required for maximal gene regulation. However, we have not observed selective binding of the nicotinamide moiety of NAD+ or binding by the second putative aptamer in vitro, despite sequence and structural similarities between the tandem domains.

Singlet glycine riboswitches bind ligand as well as tandem riboswitches.

The glycine riboswitch often occurs in a tandem architecture, with two ligand-binding domains (aptamers) followed by a single expression platform. Based on previous observations, we hypothesized that "singlet" versions of the glycine riboswitch, which contain only one aptamer domain, are able to bind glycine if appropriate structural contacts are maintained. An initial alignment of 17 putative singlet riboswitches indicated that the single consensus aptamer domain is flanked by a conserved peripheral stem-loop structure. These singlets were sorted into two subtypes based on whether the active aptamer domain precedes or follows the peripheral stem-loop, and an example of each subtype of singlet riboswitch was characterized biochemically. The singlets possess glycine-binding affinities comparable to those of previously published tandem examples, and the conserved peripheral domains form A-minor interactions with the single aptamer domain that are necessary for ligand-binding activity. Analysis of sequenced genomes identified a significant number of singlet glycine riboswitches. Based on these observations, we propose an expanded model for glycine riboswitch gene control that includes singlet and tandem architectures.

Epigenetics: What does it mean for paediatric practice?

'Epigenetics' involves the study of gene expression and the environmental exposures that influence expression. In paediatrics, it is recognized that different physiological and developmental stages of the young individual are affected by both genetic control and environmental influence. It appears that changes in gene expression - not changes in the DNA itself - can be passed on from one generation to another. The importance for paediatricians is recognizing disorders involving epigenetics, recording events during childhood that could affect epigenetic control of gene expression, and being aware of new therapies as they become available. Paediatricians need to be able to recognize the relevant risk factors.

L'épigénétique désigne l'étude de l'expression des gènes et de l'exposition environnementale qui influe sur cette expression. En pédiatrie, il est établi que le contrôle génétique et l'influence environnementale ont tous deux une incidence sur diverses phases physiologiques et développementales du jeune. Il semble que des modifications à l'expression des gènes, et non à l'ADN même, peuvent être transmises d'une génération à l'autre. Pour les pédiatres, il est important de dépister les troubles liés à l'épigénétique, de prendre note des événements de l'enfance qui pourraient influer sur le contrôle épigénétique de l'expression des gènes et d'être informé des nouveaux traitements à mesure qu'ils sont mis en marché. Les pédiatres doivent savoir reconnaître les facteurs de risque pertinents.

Synthetic Gene Circuits for Regulation of Next-Generation Cell-Based Therapeutics.

Arming human cells with synthetic gene circuits enables to expand their capacity to execute superior sensing and response actions, offering tremendous potential for innovative cellular therapeutics. This can be achieved by assembling components from an ever-expanding molecular toolkit, incorporating switches based on transcriptional, translational, or post-translational control mechanisms. This review provides examples from the three classes of switches, and discusses their advantages and limitations to regulate the activity of therapeutic cells in vivo. Genetic switches designed to recognize internal disease-associated signals often encode intricate actuation programs that orchestrate a reduction in the sensed signal, establishing a closed-loop architecture. Conversely, switches engineered to detect external molecular or physical cues operate in an open-loop fashion, switching on or off upon signal exposure. The integration of such synthetic gene circuits into the next generation of chimeric antigen receptor T-cells is already enabling precise calibration of immune responses in terms of magnitude and timing, thereby improving the potency and safety of therapeutic cells. Furthermore, pre-clinical engineered cells targeting other chronic diseases are gathering increasing attention, and this review discusses the path forward for achieving clinical success. With synthetic biology at the forefront, cellular therapeutics holds great promise for groundbreaking treatments.

A spermidine riboswitch class in bacteria exploits a close variant of an aptamer for the enzyme cofactor S-adenosylmethionine.

Natural polyamines such as spermidine and spermine cations have characteristics that make them highly likely to be sensed by riboswitches, such as their general affinity to polyanionic RNA and their broad contributions to cell physiology. Despite previous claims that polyamine riboswitches exist, evidence of their biological functions has remained unconvincing. Here, we report that rare variants of bacterial S-adenosylmethionine-I (SAM-I) riboswitches reject SAM and have adapted to selectively sense spermidine. These spermidine-sensing riboswitch variants are associated with genes whose protein products are directly involved in the production of spermidine and other polyamines. Biochemical and genetic assays demonstrate that representatives of this riboswitch class robustly function as genetic "off" switches, wherein spermidine binding causes premature transcription termination to suppress the expression of polyamine biosynthetic genes. These findings confirm the existence of natural spermidine-sensing riboswitches in bacteria and expand the list of variant riboswitch classes that have adapted to bind different ligands.

A GRA2 minimal promoter improves the efficiency of TATi / Tet-Off conditional regulation of gene expression in Toxoplasma gondii.

A tetracycline-responsive transcription system (Tet-Off) adapted for use in Toxoplasma gondii (nicknamed TATi) is useful for molecular biological studies of this organism. Previous studies using TATi incorporated minimal promoters derived from the gene promoters for TgSAG1 or TgSAG4. The present study achieves improved activation and suppression of an integrated reporter gene in the absence and presence of anhydrotetracycline, respectively (p < 0.0001), by use of a newly derived minimal promoter based on the core promoter of TgGRA2. In comparison with the SAG1 minimal promoter, use of the GRA2 minimal promoter in stable transfectants has a 23-fold higher Signal to Noise Ratio for EYFP fluorescence in the absence or presence of anhydrotetracycline. We conclude that the performance of TATi for both activation and suppression of transcription can be markedly enhanced by incorporating a GRA2 minimal promoter.

Artificial Protein-Responsive Riboswitches Upregulate Non-AUG Translation Initiation in Yeast.

Artificial control of gene expression is one of the core technologies for engineering biological systems. Riboswitches are cis-acting elements on mRNA that regulate gene expression in a ligand-dependent manner often seen in prokaryotes, but rarely in eukaryotes. Because of the poor variety of such elements available in eukaryotic systems, the number of artificially engineered eukaryotic riboswitches, especially of the upregulation type, is still limited. Here, we developed a design principle for upregulation-type riboswitches that utilize non-AUG initiation induced by ribosomal stalling in a ligand-dependent manner in Saccharomyces cerevisiae. Our design principle simply required the proper positioning of a near-cognate start codon relative to the RNA aptamer. Intriguingly, the CUG codon was the most preferable for non-AUG ON switches in terms of output level and switch performance. This work establishes novel choices for artificial genetic control in eukaryotes with versatile potential for industrial and biomedical applications as well as basic research.

The Transcription Factor Function of Parkin: Breaking the Dogma.

PRKN (PARK2) is a key gene involved in both familial and sporadic Parkinson's disease that encodes parkin (PK). Since its discovery by the end of the 90s, both functional and more recently, structural studies led to a consensual view of PK as an E3 ligase only. It is generally considered that this function conditions the cellular load of a subset of cytosolic proteins prone to proteasomal degradation and that a loss of E3 ligase function triggers an accumulation of potentially toxic substrates and, consequently, a neuronal loss. Furthermore, PK molecular interplay with PTEN-induced kinase 1 (PINK1), a serine threonine kinase also involved in recessive cases of Parkinson's disease, is considered to underlie the mitophagy process. Thus, since mitochondrial homeostasis significantly governs cell health, there is a huge interest of the scientific community centered on PK function. In 2009, we have demonstrated that PK could also act as a transcription factor (TF) and induces neuroprotection via the downregulation of the pro-apoptotic and tumor suppressor factor, p53. Importantly, the DNA-binding properties of PK and its nuclear localization suggested an important role in the control of several genes. The duality of PK subcellular localization and of its associated ubiquitin ligase and TF functions suggests that PK could behave as a key molecular modulator of various physiological cellular signaling pathways that could be disrupted in pathological contexts. Here, we update the current knowledge on PK direct and indirect TF-mediated control of gene expression.

Homeobox genes and tooth development: Understanding the biological pathways and applications in regenerative dental science.

OBJECTIVES: Homeobox genes are a group of conserved class of transcription factors that function as key regulators during the embryonic developmental processes. They act as master regulator for developmental genes, which involves coordinated actions of various auto and cross-regulatory mechanisms. In this review, we summarize the expression pattern of homeobox genes in relation to the tooth development and various signaling pathways or molecules contributing to the specific actions of these genes in the regulation of odontogenesis. MATERIALS AND METHODS: An electronic search was undertaken using combination of keywords e.g. Homeobox genes, tooth development, dental diseases, stem cells, induced pluripotent stem cells, gene control region was used as search terms in PubMed and Web of Science and relevant full text articles and abstract were retrieved that were written in English. A manual hand search in text books were also carried out. Articles related to homeobox genes in dentistry and tissue engineering and regenerative medicine of odontogenesis were selected. RESULTS: The possible perspective of stem cells technology in odontogenesis and subsequent analysis of gene correction pertaining to dental disorders through the possibility of induced pluripotent stem cells technology is also inferred. CONCLUSIONS: We demonstrate the promising role of tissue engineering and regenerative medicine on odontogenesis, which can generate a new ray of hope in the field of dental science.

Engineered UV-A light-responsive gene expression system for measuring sun cream efficacy in mammalian cell culture.

Light-dependent gene regulation systems are advantageous as they allow for precise spatio-temporal control of target gene expression. In this paper, we present a novel UV-A and blue-light-inducible gene control system that is based on the light-dependent heterodimerization of the CRY2 and C1BN domains. Upon their interaction, a transcription factor is released from the cell membrane and initiates target gene expression. Capitalizing on that, sun cream UV-A protection properties were measured intracellularly.

Speed-stability paradox in DNA-scanning by zinc-finger proteins.

Extensive contact with DNA via multiple zinc fingers allows highly specific DNA-binding of zinc-finger-class transcription factors, but can also slow the target search process. Here we introduce recent insights into how zinc-finger proteins can rapidly scan DNA. Potential application of the new knowledge to the zinc-finger-based technology is also discussed.

Hypoxic response elements and Tet-On advanced double-controlled systems regulate hVEGF 165 and angiopoietin-1 gene expression in vitro.

Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic response elements (HRE) and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF165 and angiopoietin-1 (Ang-1) genes in rat cardiomyocytes exposed to hypoxia and pharmacologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE- hVEGF165. Our results indicated that the viral titer was 1×10(12) vg /mL and the viral purity exceeded 98%. hVEGF 165 expression was induced by hypoxia, but not by normoxia (P < 0.001). Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction (P < 0.001). Immunofluorescence staining showed that positively stained hVEGF165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.

MiRNA and breast cancer metastasis / 国际外科学杂志

Most deaths of breast cancer patients may be caused by the development of metastases. The latest research suggest that some miRNAs regulated gene expressions related with breast cancer metastasis at post-transcriptional level, which was closely related to invasion and metastasis of breast cancer. This review mainly dicussed the miRNA involved in regulation of invasion and metastasis of breast cancer and their mo-lecular mechanisms.