Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring.

A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum.

Protein-protein interaction domains of Bacillus subtilis DivIVA.

DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.

Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network.

BACKGROUND: A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter. RESULTS: We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity. CONCLUSION: These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.

Combined RNA-seq and RAT-seq mapping of long noncoding RNAs in pluripotent reprogramming.

Pluripotent stem cells hold great investigative potential for developmental biology and regenerative medicine. Recent studies suggest that long noncoding RNAs (lncRNAs) may function as key regulators of the maintenance and the lineage differentiation of stem cells. However, the underlying mechanisms by which lncRNAs affect the reprogramming process of somatic cells into pluripotent cells remain largely unknown. Using fibroblasts and induced pluripotent stem cells (iPSCs) at different stages of reprogramming, we performed RNA transcriptome sequencing (RNA-Seq) to identify lncRNAs that are differentially-expressed in association with pluripotency. An RNA reverse transcription-associated trap sequencing (RAT-seq) approach was then utilized to generate a database to map the regulatory element network for lncRNA candidates. Integration of these datasets can facilitate the identification of functional lncRNAs that are associated with reprogramming. Identification of lncRNAs that regulate pluripotency may lead to new strategies for enhancing iPSC induction in regenerative medicine.

Approximal carious lesion depth assessment with insight and ultraspeed films.

This study evaluated the efficiency of a new E/F-speed film, Insight, at the determination of approximal carious lesion depths compared with Ultraspeed. Radiographs of 80 extracted human molars and premolars were taken with both films under standardized conditions. The presence or absence of caries and depth of lesions was determined by three observers using a predetermined scale. The actual status of each surface was determined histologically. Observer responses were assessed with the Gamma measure of association test. Differences between the observers' agreement levels were not significant. The efficiency of Insight and Ultraspeed at true depth diagnosis was found to be 54.9%; 55.8% and Gamma values were found to be 0.883 and 0.922, respectively, at p<0.001. The difference between the two films was not statistically significant (p=0.852). This study suggested that there was no statistically significant difference between the two films at detecting the depths of approximal carious lesions.

Common errors on panoramic radiographs taken in a dental school.

The aim of this study is to evaluate the frequency of common errors seen on panoramic radiographs taken in the Radiology Department of a dental school by trained assistants. Four hundred and sixty radiographs were evaluated for 20 categories of common errors. Out of the evaluated radiographs, 37.61% were found to be error-free. The most common errors were found to be the palatoglossal airspace shadow of air above the tongue due to the patient not raising the tongue against the palate (46.30%) and the superimposition of hyoid bone with the mandible (26.30%) respectively. The least common error was found to be dirty or bent films (0.21%). The quality of panoramic radiographs could be enhanced by improving radiographic technique.