Clinical characteristics of preterm and term infants with Ureaplasma in gastric fluid.

BACKGROUND: Ureaplasma spp. is an endemic microorganism that causes placental chorioamnionitis or preterm delivery in pregnant women, and the occurrence of bronchopulmonary dysplasia or intraventricular hemorrhaging in preterm infants after birth, although the pathogenicity of Ureaplasma remains controversial. The association between Ureaplasma exposure and the symptoms or outcomes of infected mothers or their infants born at term remains poorly understood. We investigated the clinical characteristics of preterm and term infants with or without Ureaplasma in their gastric fluid. METHODS: Gastric fluid samples were collected from 47 newborns in the neonatal intensive-care unit immediately after birth and tested using multiplex polymerase chain reaction (PCR) assays targeting Ureaplasma spp., Ureaplasma parvum, and Ureaplasma urealyticum. The clinical findings and outcomes of the neonates and their mothers were retrospectively evaluated. RESULTS: Ureaplasma spp. were detected in 9/47 samples (19%) by multiplex PCR assays. In all cases, the subspecies was U. parvum. The Ureaplasma-positive group had a significantly higher incidence of chorioamnionitis in utero than the Ureaplasma-negative group. Regarding preterm infants, the IgM levels in the Ureaplasma-positive group were significantly higher than in the Ureaplasma-negative group. In contrast, in term infants, the rates of a non-reassuring fetal status, a maternal fever, and maternal leukocyte counts and maternal C-reactive protein levels within five days before delivery in the Ureaplasma-positive group were significantly higher than those in the Ureaplasma-negative group. All three extremely-low-birth-weight infants with Ureaplasma developed bronchopulmonary dysplasia. The length of hospitalization in the Ureaplasma-positive group was almost same as that in the Ureaplasma-negative group for term infants. CONCLUSION: Mothers or their fetuses with exposure to Ureaplasma expressed characteristic clinical features during pregnancy and after birth.

Cross-contamination risk and decontamination during changeover after cell-product processing.

Introduction: During changeover in cell-product processing, it is essential to minimize cross-contamination risks. These risks differ depending on the patient from whom the cells were derived. Human error during manual cell-product processing increases the contamination risk in biosafety cabinets. Here, we evaluate the risk of cross-contamination during manual cell-processing to develop an evidence-based changeover method for biosafety cabinets. Methods: Contaminant coverage was analyzed during simulated medium preparation, cell seeding, and waste liquid decanting by seven operators, classified by skill. Environmental bacteria were surveyed at four participating facilities. Finally, we assessed the effect of conventional UV irradiation in biosafety cabinets on bacteria and fungi that pose a cross-contamination risk. Results: Under simulated conditions, scattered contamination occurred via droplets falling onto the surface from heights of 30 cm, and from bubbles rupturing at this height. Visible traces of contaminants were distributed up to 50 cm from the point of droplet impact, or from the location of the pipette tip when the bubble ruptured. In several facilities, we detected Bacillus subtilis, of which the associated endospores are highly resistant to disinfection. Irradiation at 50 mJ/cm2 effectively eliminated Bacillus subtilis vegetative cells and Aspergillus brasiliensis, which is highly resistant to UV. Bacillus subtilis endospores were eliminated at 100 mJ/cm2. Conclusions: Under these simulated optimal conditions, UV irradiation successfully prevents cross-contamination. Therefore, following cell-product processing, monitoring the UV dose in the biosafety cabinet during cell changeover represents a promising method for reducing cross-contamination.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes.

PURPOSE: Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-µl samples. DESIGN: Multicenter prospective evaluation of a diagnostic PCR test. PARTICIPANTS: A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. METHODS: After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. MAIN OUTCOME MEASURES: The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. RESULTS: Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ = 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. CONCLUSIONS: The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Evaluation of a Multiplex Strip PCR Test for Infectious Uveitis: A Prospective Multicenter Study.

PURPOSE: A novel multiplex polymerase chain reaction (PCR) test (Strip PCR) for 24 common ocular infectious disease pathogens was established. Solid-phase techniques provide stable, prompt, and accurate results while using less sample amount with lower cost than conventional quantitative real-time PCR (qPCR). Strip PCR for infectious uveitis was optimized and evaluated using intraocular samples. DESIGN: Evaluation of diagnostic testing. METHODS: We examined 722 samples at 14 institutions. Genomic DNA from aqueous humor and vitreous fluid was analyzed by qPCR and Strip PCR. Clinical diagnosis was determined based on symptoms, clinical findings, and laboratory tests. MainOutcomeMeasures: The diagnostic parameters of the Strip PCR were based on qPCR results. RESULTS: Strip PCR showed low intra- and inter-institutional variability even when performed by technicians with various PCR skill levels. The targets of Strip PCR for infectious uveitis were optimized for 9 major pathogens (herpes simplex virus [HSV] 1, HSV2, varicella-zoster virus, human T-cell lymphotropic virus 1, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, Toxoplasma gondii, and Treponema pallidum) with 772 intraocular samples. The Strip PCR successfully detected pathogen DNA at concentrations ranging from 100 to 109 copies/mL in 252 of the 255 qPCR-positive samples. It yielded negative results for all the 191 qPCR-negative samples. Strip PCR had higher sensitivity (98.8%), specificity (98.5%), positive predictive value (98.8%), and negative predictive value (98.5%) than qPCR, with distinct primers. The Strip PCR results had strong correlation with that of the qPCR (r = 0.838) and they were consistent with the clinical diagnosis. CONCLUSIONS: Easy-to-use Strip PCR is recommended for rapid diagnosis of infectious uveitis, as its results are equivalent to that of conventional qPCR.

Enterovirus D68 respiratory infection in a children's hospital in Japan in 2015.

BACKGROUND: Outbreaks of enterovirus D68 (EV-D68) respiratory infections in children were reported globally in 2014. In Japan, there was an EV-D68 outbreak in the autumn of 2015 (September-October). The aim of this study was to compare EV-D68-specific polymerase chain reaction (PCR)-positive and EV-D68-specific PCR-negative patients. METHODS: Pediatric patients admitted for any respiratory symptoms between September and October 2015 were enrolled. Nasopharyngeal swabs were tested for multiplex respiratory virus PCR and EV-D68-specific reverse transcription-PCR. EV-D68-specific PCR-positive and -negative patients were compared regarding demographic data and clinical information. RESULTS: A nasopharyngeal swab was obtained from 76 of 165 patients admitted with respiratory symptoms during the study period. EV-D68 was detected in 40 samples (52.6%). Median age in the EV-D68-specific PCR-positive and -negative groups was 3.0 years (IQR, 5.5 years) and 3.0 years (IQR, 4.0 years), respectively. The rates of coinfection in the two groups were 32.5% and 47.2%, respectively. There was no significant difference in the history of asthma or recurrent wheezing, length of hospitalization, or pediatric intensive care unit admission rate between the groups. The median days between symptom onset and admission was significantly lower for the EV-D68-positive group (3.0 days vs 5.0 days, P = 0.001). EV-D68 was identified as clade B on phylogenetic analysis. No cases of acute flaccid myelitis were encountered. CONCLUSIONS: More than half of the samples from the children admitted with respiratory symptoms were positive for EV-D68-specific PCR during the outbreak. Asthma history was not associated with the risk of developing severe respiratory infection.

Induction of Hepatic Metabolic Functions by a Novel Variant of Hepatocyte Nuclear Factor 4γ.

Hepatocyte nuclear factor 4α (HNF4α) is a critical factor for hepatocyte differentiation. HNF4α expression is decreased in hepatocellular carcinoma (HCC), which suggests a role in repression of hepatocyte dedifferentiation. In the present study, hepatic expression of HNF4γ was increased in liver-specific Hnf4a-null mice. The HNF4γ whose expression was increased contained two variants, a known short variant, designated HNF4γ1, and a novel long variant, designated HNF4γ2. HNF4G2 mRNA was highly expressed in small intestine, and the transactivation potential of HNF4γ2 was the strongest among these variants, but the potential of HNF4γ1 was the lowest. Cotransfection experiments revealed that HNF4γ1 repressed HNF4α- and HNF4γ2-dependent transactivation, while HNF4γ2 promoted HNF4α-dependent transactivation. HNF4γ1 and HNF4γ2 were able to bind to the HNF4α binding sites with an affinity similar to that of HNF4α. Furthermore, HNF4γ2, but not HNF4γ1, robustly induced the expression of typical HNF4α target genes to a greater degree than HNF4α. Additionally, HNF4γ2 suppressed proliferation of hepatoma cells as well as HNF4α and HNF4γ1 did, and HNF4γ2 induced critical hepatic functions, such as glucose and urea production, and cytochrome P450 1A2 activity more strongly than HNF4α and HNF4γ1 did. These results indicate that HNF4γ2 has potential for redifferentiation of HCC and thus may be explored as a target for HCC therapy.

Diagnosing superinfection keratitis with multiplex polymerase chain reaction.

PURPOSE: To report the potential usefulness of multiplex polymerase chain reaction (mPCR) for diagnosing superinfection keratitis caused by herpes simplex virus-1 (HSV-1), bacteria and fungus. METHODS: Case series. Corneal scrapings were analyzed with mPCR for human herpes virus 1-8, bacterial 16S ribosomal DNA (rDNA) and fungal 28S rDNA. RESULTS: Case 1 was a 69-year-old man who presented with refractory infectious keratitis. PCR examination was positive for bacterial 16S rDNA and negative for fungal 28S rDNA. HSV-1 was not examined at this time. A geographic ulcer arose after 2 months of intensive antibacterial treatment. Herpes simplex keratitis (HSK) was suspected; PCR analysis was positive for HSV-1. Corneal scrapings obtained at the initial visit were re-analyzed and found to be HSV-1 positive. Thus, it turned out that this was a case of superinfection keratitis caused by bacteria and HSV-1. Case 2 was a 60-year-old man with corneal ulcer who had received unsuccessful treatment with antibiotics. mPCR analysis was positive for HSV-1, bacterial 16S rDNA and fungal 28S rDNA. The patient was diagnosed with superinfection keratitis caused by HSV-1, bacteria and fungus. Case 3 was an 82-year-old woman who had been treated for HSK and then developed bacterial keratitis during treatment. mPCR analysis was positive for HSV-1 and bacterial 16S rDNA. The patient was diagnosed with superinfection keratitis caused by HSV-1 and bacteria. CONCLUSION: Superinfection keratitis is hard to diagnose because of its atypical manifestation. mPCR has the potential to allow prompt diagnosis and appropriate treatment in these cases.

[Ovum collection following ovarian stimulation in patients with chronic active Epstein-Barr virus infection].

The only curative treatment for chronic active Epstein-Barr virus infection (CAEBV) is hematopoietic stem cell transplantation. For young female patients, ovulation induction and oocyte cryopreservation may be performed prior to transplantation to provide for future pregnancies. However, the effects of this ovum treatment on CAEBV and EBV infections have not been reported. Attempts were made to collect ova from three female CAEBV patients before transplantation conditioning, but this was only successful in two cases. Ovarian stimulation did not induce disease progression, and there was no change in the peripheral blood EBV DNA load. In one patient, 460 copies/ml of EBV DNA were detected in the follicular fluid by real-time PCR. Red blood cells were also present in the follicular fluid but not mononuclear cells. EBV protein mRNA was not detected in the RNA extracted from the same fluid, suggesting that the EBV DNA resulted from peripheral blood contamination. Moreover, there were no EBV-infected cells in the follicular fluid. Therefore cryopreservation of oocytes from CAEBV patients is possible and may be used to provide for future pregnancies.

Multiplex polymerase chain reaction for pathogen detection in donor/recipient corneal transplant tissue and donor storage solution.

Corneal transplantation is a safe, reliable method of restoring visual acuity in patients with corneal disorders. Although it has a very high success rate, rejection can still occur, especially if the site is infected. Therefore, seeking to find better ways to manage infection risk, this study investigated a new technique, based on multiplex polymerase chain reaction (mPCR), to identify pathogens, including viruses, bacteria, and fungi, in corneal transplantation recipient sites, donor corneas and the donor cornea storage solution. The subjects comprised 50 patients who underwent corneal transplantation at Tohoku University Hospital between July 2014 and April 2015. We obtained extracted (recipient) cornea samples in 37 cases, donor cornea samples in 50 cases, and corneal storage solution samples in 50 cases (18 of these 50 samples contained DNA). Herpes simplex virus type 1 DNA was detected in four recipient corneas, Parvovirus B19 DNA was detected in two recipient corneas, Human herpes virus type 6 was detected in two donor corneas, and Aspergillus DNA was detected in one corneal storage solution sample. Thus, mPCR successfully identified pathogenic DNA in corneal tissues and storage solution, suggesting that evaluation with mPCR may improve the ability to predict the risk of infection after corneal transplantation.

Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens.

Purpose: To establish and evaluate a new multiplex solid-phase strip polymerase chain reaction (strip PCR) for concurrent detection of common ocular infectious disease pathogens. Methods: A new multiplex strip PCR was established to detect 24 common ocular infectious disease pathogens: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, human T-cell lymphotropic virus (HTLV)-1, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, Propionibacterium acnes (P. acnes), bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, Aspergillus, Fusarium, fungal 28S rRNA, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), and Acanthamoeba. Strip PCR was tested with a negative control (distilled water) and standard positive control DNA. Cutoffs of quantification cycle (Cq) values were determined with noninfectious ocular samples to avoid false-positives caused by contamination with P. acnes, bacterial 16S, and fungal 28S from reagents and ocular surfaces. A pilot study to evaluate the strip PCR was performed using infectious ocular samples (aqueous humor, vitreous, cornea, and tears) by strip PCR and previously developed capillary-type multiplex PCR and quantitative real-time PCR (qPCR). Results: Strip PCR was verified with negative and positive controls. Strip PCR rapidly detected HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, HTLV-1, adenovirus, P. acnes, bacterial 16S, Candida sp., C. glabrata, Aspergillus, fungal 28S, T. gondii, C. trachomatis, and Acanthamoeba in patient samples. The sensitivity was comparable to that of qPCR. Conclusions: Our novel strip PCR assay is a simple, rapid, and high-sensitivity method for detecting ocular infectious disease pathogens.

Telomerase reverse transcriptase regulates microRNAs.

MicroRNAs are small non-coding RNAs that inhibit the translation of target mRNAs. In humans, most microRNAs are transcribed by RNA polymerase II as long primary transcripts and processed by sequential cleavage of the two RNase III enzymes, DROSHA and DICER, into precursor and mature microRNAs, respectively. Although the fundamental functions of microRNAs in RNA silencing have been gradually uncovered, less is known about the regulatory mechanisms of microRNA expression. Here, we report that telomerase reverse transcriptase (TERT) extensively affects the expression levels of mature microRNAs. Deep sequencing-based screens of short RNA populations revealed that the suppression of TERT resulted in the downregulation of microRNAs expressed in THP-1 cells and HeLa cells. Primary and precursor microRNA levels were also reduced under the suppression of TERT. Similar results were obtained with the suppression of either BRG1 (also called SMARCA4) or nucleostemin, which are proteins interacting with TERT and functioning beyond telomeres. These results suggest that TERT regulates microRNAs at the very early phases in their biogenesis, presumably through non-telomerase mechanism(s).

A transient disruption of fibroblastic transcriptional regulatory network facilitates trans-differentiation.

Transcriptional Regulatory Networks (TRNs) coordinate multiple transcription factors (TFs) in concert to maintain tissue homeostasis and cellular function. The re-establishment of target cell TRNs has been previously implicated in direct trans-differentiation studies where the newly introduced TFs switch on a set of key regulatory factors to induce de novo expression and function. However, the extent to which TRNs in starting cell types, such as dermal fibroblasts, protect cells from undergoing cellular reprogramming remains largely unexplored. In order to identify TFs specific to maintaining the fibroblast state, we performed systematic knockdown of 18 fibroblast-enriched TFs and analyzed differential mRNA expression against the same 18 genes, building a Matrix-RNAi. The resulting expression matrix revealed seven highly interconnected TFs. Interestingly, suppressing four out of seven TFs generated lipid droplets and induced PPARG and CEBPA expression in the presence of adipocyte-inducing medium only, while negative control knockdown cells maintained fibroblastic character in the same induction regime. Global gene expression analyses further revealed that the knockdown-induced adipocytes expressed genes associated with lipid metabolism and significantly suppressed fibroblast genes. Overall, this study reveals the critical role of the TRN in protecting cells against aberrant reprogramming, and demonstrates the vulnerability of donor cell's TRNs, offering a novel strategy to induce transgene-free trans-differentiations.

Temporal dynamics and transcriptional control using single-cell gene expression analysis.

BACKGROUND: Changes in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event. RESULTS: Here we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages. We systematically measure temporal regulation of expression and variation by profiling 120 single cells at eight distinct time points, and infer highly controlled regulatory modules through which signaling operates with stochastic effects. This reveals dynamic and specific rewiring as a cellular strategy for differentiation. The integration of both positive and negative co-expression networks further identifies the proto-oncogene MYB as a network hinge to modulate both the pro- and anti-differentiation pathways. CONCLUSIONS: Compared to averaged cell populations, temporal single-cell expression profiling provides a much more powerful technique to probe for mechanistic insights underlying cellular differentiation. We believe that our approach will form the basis of novel strategies to study the regulation of transcription at a single-cell level.

Identification of ZNF395 as a novel modulator of adipogenesis.

Adipogenesis is the process of cell differentiation by which mesenchymal stem cells (MSC) become adipocytes. Investigating the transcriptional regulatory process during adipogenesis may provide strategies to prevent obesity and other metabolic disorders. In recent years, numerous zinc finger proteins (ZFPs) have been implicated in regulating differentiation and cell fate determination. To investigate the regulatory role of ZFPs involved in adipogenesis, we performed genome-wide microarray expression profiling of an adipogenesis time series. Particularly focusing on the transiently responsive ZFPs, we identified and characterized the functional role of ZNF395 in adipogenesis. A systematic ablation of the ZNF395 transcript during adipogenesis revealed 40% reduction of adipocytes when compared to control. Furthermore, the number of adipocytes as well as the expression of key adipocyte markers were greatly induced when MSC were co-transduced with ZNF395 and PPARG2. To further elucidate the functional role of ZNF395 during adipogenesis, we attempted to trans-differentiate human dermal fibroblasts with PPARG2. The test remarkably revealed that ZNF395 in conjunction with PPARG2 greatly induced adipogenesis from dermal fibroblasts when compared to PPARG2 alone. These loss and gain of function experiments firmly establish that ZNF395 coordinate the transcriptional regulatory pathway with PPARG2, which may be necessary for the genesis of adipocytes.

CpG site-specific alteration of hydroxymethylcytosine to methylcytosine beyond DNA replication.

Hydroxymethylcytosines (hmC), one of several reported cytosine modifications, was recently found to be enriched in embryonic stem cells and neuronal cells, and thought to play an important role in regulating gene expression and cell specification. However, unlike methylcytosines (mC), the fate of hmC beyond DNA replication is not well understood. Here, to monitor the status of hmC during DNA replication, we prepared a stable episomal vector-based monitoring system called MoCEV in 293T cells. The MoCEV system containing fully hydroxymethylated-cytosine fragments revealed a significant modification towards mC after several rounds of DNA replication. Strikingly this modification was specifically observed at the CpG sites (71.9% of cytosines), whereas only 1.1% of modified cytosines were detected at the non-CpG sites. Since the unmodified MoCEV did not undergo any DNA methylation during cell division, the results strongly suggest that somatic cells undergo hmC to mC specifically at the CpG sites during cell division.

Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin.

While several studies have focused on the relationship between individual miRNA loci or classes of small RNA with human Argonaute (AGO) proteins, a comprehensive, global analysis of the RNA content associating with different AGO proteins has yet to be performed. We have compared the content of deep sequenced RNA extracted from immunoprecipitation experiments with the AGO1, AGO2, and AGO3 proteins. Consistent with previous observations, sequence tags derived from miRNA loci globally associate in approximately equivalent amounts with AGO1, AGO2, and AGO3. Exceptions include miR-182, miR-222, and miR-223*, which could be coupled to processes targeting the loci for interaction with specific AGO proteins. A closer inspection of the data, however, supports the presence of an unusual sorting mechanism wherein a subset of miRNA loci give rise to distinct isomirs which preferentially associate with distinct AGO proteins in a significantly differential manner. We also identify the complete set of short RNA derived from non-miRNA sources including tRNA, snRNA, snoRNA, vRNA, and mRNA associating with the AGO proteins, many of which are predicted to play roles in post-transcriptional gene silencing. We also observe enrichment of tags mapping to promoter regions of genes, suggesting that a fraction of the recently-identified promoter-associated small RNAs in humans could function through interaction with AGO proteins. Finally, we observe antisense miRNA transcripts are frequently present in low copy numbers across a range of diverse miRNA loci and these transcripts appear to associate with AGO proteins.

Building promoter aware transcriptional regulatory networks using siRNA perturbation and deepCAGE.

Perturbation and time-course data sets, in combination with computational approaches, can be used to infer transcriptional regulatory networks which ultimately govern the developmental pathways and responses of cells. Here, we individually knocked down the four transcription factors PU.1, IRF8, MYB and SP1 in the human monocyte leukemia THP-1 cell line and profiled the genome-wide transcriptional response of individual transcription starting sites using deep sequencing based Cap Analysis of Gene Expression. From the proximal promoter regions of the responding transcription starting sites, we derived de novo binding-site motifs, characterized their biological function and constructed a network. We found a previously described composite motif for PU.1 and IRF8 that explains the overlapping set of transcriptional responses upon knockdown of either factor.

A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

The combination of gene perturbation assay and ChIP-chip reveals functional direct target genes for IRF8 in THP-1 cells.

Gene regulatory networks in living cells are controlled by the interaction of multiple cell type-specific transcription regulators with DNA binding sites in target genes. Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence binding protein (ICSBP), is a transcription factor expressed predominantly in myeloid and lymphoid cell lineages. To find the functional direct target genes of IRF8, the gene expression profiles of siRNA knockdown samples and genome-wide binding locations by ChIP-chip were analyzed in THP-1 myelomonocytic leukemia cells. Consequently, 84 genes were identified as functional direct targets. The ETS family transcription factor PU.1, also known as SPI1, binds to IRF8 and regulates basal transcription in macrophages. Using the same approach, we identified 53 direct target genes of PU.1; these overlapped with 19 IRF8 targets. These 19 genes included key molecules of IFN signaling such as OAS1 and IRF9, but excluded other IFN-related genes amongst the IRF8 functional direct target genes. We suggest that IRF8 and PU.1 can have both combined, and independent actions on different promoters in myeloid cells.