Elevation of miR-146a Inhibits BTG2/BAX Expression to Ameliorate Postoperative Cognitive Dysfunction Following Probiotics (VSL#3) Treatment.

It has been reported that the gut microbiome modulates postoperative cognitive dysfunction (POCD), and that administration of probiotics (VSL#3) may effectively relieve POCD. In this study, we aimed to identify the underlying mechanism of VSL#3 in POCD. A mouse model of POCD was constructed in adult male C57BL/6 mice, which were then treated with VSL#3. VSL#3 exerted a protective role against POCD and resultant neuronal apoptosis. The expression of miR-146a was found to be downregulated in hippocampal tissues of POCD mice, while VSL#3 could restore its expression. Loss- and gain-function approaches were conducted to determine the roles of microRNA (miR)-146a, B-cell translocation gene 2 (BTG2), and Bcl-2-associated X protein (Bax) in post-operative effects on cognitive function and neuronal apoptosis. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured to determine oxidative stress in brain tissue. The dual-luciferase reporter gene assay identified that miR-146a could target BTG2 and negatively regulate its expression. BTG2 knockdown suppressed neuronal apoptosis and contributed to shortened time of latency, prolonged time of mice spent in the target quadrant, and reduced oxidative stress through downregulating Bax expression. Finally, VSL#3 treatment upregulated the expression of miR-146a to block BTG2/Bax axis and consequently inhibited neuronal apoptosis and reduced oxidative stress in POCD mice. Taken together, the study suggested that miR-146a-mediated suppression of BTG2/Bax contributed to the protective role of probiotics treatment against POCD.

Inhibitory effects of piceatannol on human cytomegalovirus (hCMV) in vitro.

Human cytomegalovirus (hCMV) is a ubiquitous herpesvirus, which results in the establishment of a latent infection that persists throughout the life of the host and can be reactivated when the immunity is low. Currently, there is no vaccine for hCMV infection, and the licensed antiviral drugs mainly target the viral enzymes and have obvious adverse reactions. Thus, it is important to search for compounds with anti-hCMV properties. The present study aimed to investigate the suppressive effects of piceatannol on hCMV Towne strain infection and the putative underlying mechanisms using human diploid fibroblast WI-38 cells. Piceatannol supplementation prevented the lytic changes induced by hCMV infection in WI-38 cells. Furthermore, piceatannol suppressed the expression of hCMV immediate-early (IE) and early (E) proteins as well as the replication of hCMV DNA in a dose-dependent manner. Moreover, hCMV-induced cellular senescence was suppressed by piceatannol, as shown by a decline in the senescence-associated ß-galactosidase (SA-ß-Gal) activity and decreased production of intracellular reactive oxygen species (ROS). p16INK4a, a major senescence-associated molecule, was dramatically elevated by current hCMV infection that was attenuated by pre-incubation with piceatannol in a dose-dependent manner. These results demonstrated that piceatannol suppressed the hCMV infection via inhibition of the activation of p16INK4a and cellular senescence induced by hCMV. Together, these findings indicate piceatannol as a novel and potent anti-hCMV agent with the potential to be developed as an effective treatment for chronic hCMV infection.

miR-487a performs oncogenic functions in osteosarcoma by targeting BTG2 mRNA.

Aberrant microRNA (miRNA) expression plays a critical role in osteosarcoma (OS) pathogenesis. In this study, we elucidated the involvement of miR-487a in OS and the underlying molecular mechanisms. We found that miR-487a was upregulated in OS clinical samples and cell lines. Knockdown of miR-487a suppressed OS cell growth and invasion and induced apoptosis; however, overexpression of miR-487a promoted OS cell growth and invasion. Accordingly, downregulation of miR-487a significantly suppressed tumor growth of OS xenografts in vivo. Furthermore, B-cell translocation gene 2 (BTG2) mRNA was found to be a novel target of miR-487a. Knockdown of BTG2 using small interfering RNA (siRNA) recapitulated the oncogenic effects of miR-487a, whereas BTG2 overexpression partially reversed these effects. Finally, miR-487a levels were found to be negatively correlated with BTG2 expression in OS clinical samples. Collectively, our data suggest that miR-487a is an oncogenic miRNA in OS and it lowers BTG2 expression.

Serum- and glucocorticoid-inducible kinase 1 activity reduces dendritic spines in dorsal hippocampus.

The hippocampus has a well-known role in mediating learning and memory, and its function can be directly regulated by both stress and glucocorticoid receptor activation. Hippocampal contributions to learning are thought to be dependent on changes in the plasticity of synapses within specific subregions, and these functional changes are accompanied by morphological changes in the number and shape of dendritic spines, the physical correlates of these glutamatergic synapses. Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates dendritic spine morphology in the prefrontal cortex, and modulation of SGK1 expression in mouse hippocampus regulates learning. However, the role of SGK1 in dendritic spine morphology within the CA1 and dentate gyrus regions of the hippocampus are unknown. Thus, herpes simplex viral vectors expressing GFP and various SGK1 constructs, including wild type SGK1, a catalytically inactive version of SGK1 (K127Q), and a phospho-defective version of SGK1 (S78A), were infused into the hippocampus of adult mice and confocal fluorescent microscopy was used to visualize dendritic spines. We show that increasing expression of SGK1 in the dentate gyrus increased the total number of spines, driven primarily by an increase in mushroom spines, while decreasing SGK1 activity (K127Q) in the CA1 region increased the total number of dendritic spines, driven by a significant increase in mushroom and stubby spines. The differential effects of SGK1 in these regions may be mediated by the interactions of SGK1 with multiple pathways required for spine formation and stability. As the formation of mature synapses is a crucial component of learning and memory, this indicates that SGK1 is a potential target in the pathway underlying stress-associated changes in cognition and memory.

Effects of the Ethanol Extract of Dipterocarpus alatus Leaf on the Unpredictable Chronic Mild Stress-Induced Depression in ICR Mice and Its Possible Mechanism of Action.

Treatment of the unpredictable chronic mild stress (UCMS) mice with the ethanol extract of Dipterocarpus alatus leaf attenuated anhedonia (increased sucrose preference) and behavioral despair (decreased immobility time in tail suspension test (TST) and forced swimming test (FST)). The extract not only decreased the elevation of serum corticosterone level and the index of over-activation of the hypothalamic-pituitary-adrenal (HPA) axis, caused by UCMS, but also ameliorated UCMS-induced up-regulation of serum- and glucocorticoid-inducible kinase 1 (SGK1) mRNA expression and down-regulation of cyclic AMP-responsive element binding (CREB) and brain-derived neurotrophic factor (BDNF) mRNAs in frontal cortex and hippocampus. In vitro monoamine oxidase (MAO) inhibition assays showed that the extract exhibited the partial selective inhibition on MAO-A. HPLC analysis of the extract showed the presence of flavonoids (luteolin-7-O-glucoside, kaempferol-3-glucoside, rutin) and phenolic acids (gallic acid, ferulic acid, and caffeic acid) as major constituents.

Characterization and Transcriptional Activation of the Immediate Early Gene ARC During a Neural Correlate of Classical Conditioning.

Plasticity and learning genes require regulatory mechanisms that have the flexibility to respond to a variety of sensory stimuli to generate adaptive behavioral responses. The immediate early gene (IEG) activity-regulated cytoskeleton-associated protein (ARC) is rapidly induced not only by neuronal stimulation but also during a variety of learning tasks. How ARC is regulated in response to complex stimuli during associative learning remains to be fully detailed. Here, we characterized the structure of the ARC gene in the pond turtle and mechanisms of its transcriptional activation during a neural correlate of eyeblink classical conditioning. The tARC gene is regulated in part by the presence of paused polymerase (RNAPII) that is poised at the promoter for rapid gene induction. Conditioning induces permissive chromatin modifications in the tARC promoter that allows binding by the transcription factor cAMP response element-binding protein (CREB) within 5 min of training. During learning acquisition, the pausing factor negative elongation factor (NELF) dissociates from the promoter thereby releasing RNAPII for active transcription. Data additionally suggest that the DNA insulator protein CCCTC-binding factor (CTCF) is required for transcription by mediating a learning-induced interaction of the ARC promoter with an enhancer element. Our study suggests that the learning-inducible IEG tARC utilizes both paused RNAPII and rapid chromatin modifications that allow for dynamic gene responsiveness required when an organism is presented with a variety of environmental stimuli.

Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27.

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.

MiR-6875-3p promotes the proliferation, invasion and metastasis of hepatocellular carcinoma via BTG2/FAK/Akt pathway.

BACKGROUND: Increasing evidence supports the association of microRNA with tumor occurrence and development. However, the expression of miR-6875-3p and its role in cell proliferation, invasion and metastasis in hepatocellular carcinoma (HCC) remains elusive. METHODS: The expression of miR-6875-3p and BTG2 in HCC tissues and cell lines was detected by using in situ hybridization, immunohistochemistry and qRT-PCR, respectively. A western blot assay, qRT-PCR and Luciferase reporter assay were employed to study the interaction between miR-6875-3p and BTG2. Cell proliferation invasion and metastasis were measured by MTT, transwell and matrigel analyses in vitro. In vivo, tumorigenicity and metastasis assays were performed in nude mice. RESULTS: We found that miR-6875-3p were elevated expressed in HCC tissues and cell lines, and negatively correlated with BTG2 expression, while positively correlated with tumor staging, size, degree of differentiation, and vascular invasion of HCC. Moreover, in vitro and in vivo assays showed that miR-6875-3p regulates EMT and improve the proliferation, metastasis and stem cell-like properties of HCC cells. BTG2 was identified as a direct and functional target of miR-6875-3p via the 3'-UTR of BTG2. We also confirmed that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway. CONCLUSION: Our study provides evidence that high expression of miR-6875-3p can promote tumorigenesis of HCC in vitro and in vivo, so as to function as a novel oncogene in HCC. In mechanism, we found that miR-6875-3p plays its biological functions via the BTG2/FAK/Akt pathway.

PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma.

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, has roles in cell growth regulation and cancer development. However, the role of PRMT5 in hepatocellular carcinoma (HCC) progression remains unclear. Here, we showed that PRMT5 expression was frequently upregulated in HCC tissues, and its expression was inversely correlated with overall survival in HCC patients. PRMT5 knockdown markedly inhibited in vitro HCC proliferation and in vivo tumorigenesis. We revealed that the mechanism of PRMT5-induced proliferation was partially mediated by BTG downregulation, leading to cell cycle arrest during the G1 phase in HCC cells. Ectopic BTG2 overexpression decreased HCC growth, caused cell cycle arrest at the G1 phase, and downregulated Cyclin D1 and Cyclin E1 protein expression. Furthermore, we found that PRMT5-induced ERK phosphorylation regulated BTG2 expression in HCC cells, whereas pretreatment with a selective ERK1/2 inhibitor (PD184352) significantly reversed the effect of PRMT5 on BTG2 expression. Our results indicated that PRMT5 promotes HCC proliferation by downregulating BTG2 expression via the ERK pathway.

The 5' Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication.

The human cytomegalovirus (HCMV) immediate early 1 (IE1) and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the posttranscriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5' untranslated region (5' UTR) plays an important role in regulating mRNA translation. Therefore, to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared 5' UTR of the IE1 and IE2 mRNAs (MIE 5' UTR) in regulating translation. In a cell-free system, the MIE 5' UTR repressed translation, as predicted based on its length and sequence composition. However, in transfected cells we found that the MIE 5' UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5' UTR has a positive role in translation control. We also found that the MIE 5' UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5' UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild-type MIE 5'-UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5' UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5' UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date, most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore, this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation and thus inhibit lytic replication or prevent HCMV reactivation.

Antiviral activity of arbidol hydrochloride against herpes simplex virus I in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) causes significant human diseases ranging from skin lesions to encephalitis, especially in neonates and immunocompromised hosts. The discovery of novel anti-HSV-1 drugs with low toxicity is required for public health. Arbidol hydrochloride (ARB) is an indole derivative molecule with broad-spectrum antiviral activity. In this study, the antiviral effects of ARB against HSV-1 infection were evaluated in vitro and in vivo. The results showed that ARB presents significant inhibitory effect on HSV-1 plaque formation and generation of progeny virus, with EC50 values (50% effective concentration) of 5.39 µg/mL (10.49 µM) and 2.26 µg/mL (4.40 µM), respectively. Moreover, time-of-addition and time-of-removal assays further suggested that ARB has viral inhibitory effects when added up to 12 h post-infection (p.i.), which could be further corroborated by determining the expression of viral immediate-early (ICP4, ICP22 and ICP27), early (ICP8 and UL42) and late (gB, gD, gH, VP1/2 and VP16) genes by real-time quantitative PCR as well as the expression of viral protein ICP4 and ICP8 at 6 h and 12 h p.i. Results of the in vivo study showed that ARB could reduce guinea pig skin lesions caused by HSV-1 infection. Conclusively, this report offers new perspectives in the search for therapeutic measures in the treatment of HSV-1 infection.

Inducible miR-145 expression by HIF-1a protects cardiomyocytes against apoptosis via regulating SGK1 in simulated myocardial infarction hypoxic microenvironment.

BACKGROUND: Myocardial infarction (MI) is partly due to myocardial cell damage caused by hypoxia. MicroRNAs (miRNAs) have been proved to be closely related to the development and progression of many cardiovascular diseases. This study investigated the role of miR-145 in cardiomyocytes under hypoxic condition. METHODS: The quantitative real-time polymerase chain reaction (qRT-PCR) was performed to test miR-145 expression in H9c2 cells with hypoxia-inducible factor (HIF)-a abnormal expression under hypoxic condition. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT), Tran-swell assay and flow cytometry were used to investigate the effects of miR-145 on cell viability, migration and apoptosis under normoxic or hypoxic condition, respectively. Meanwhile, reactive oxygen species (ROS) content in hypoxic H9c2 cells was analyzed. Western blotting was used to explore the potential mechanism of miR-145 protective effects on cardiomyocytes. Expression levels of miR-145 and SGK1 in rat MI model were also assessed. RESULTS: Results showed that miR-145 was upregulated in H9c2 and HL-1 cells under hypoxic condi-tion, which was promoted by HIF-1a. MiR-145 overexpression enhanced cell viability and migration under normoxic condition. Under hypoxic condition, miR-145 overexpression promoted cell viability, inhibited apoptosis and ROS activity. Western blotting results proved that miR-145 overexpression inhibited the activation of apoptotic related factors, and promoted activation of PI3K/AKT signaling pathway via SGK1 upregulation. Expression levels of miR-145 and SGK1 were both upregulated in rat MI models. CONCLUSIONS: HIF-1a could induce miR-145 upregulation in hypoxic H9c2 and HL-1 cells. MiR-145 protected H9c2 cells against hypoxic damage. SGK1 upregulation and activated PI3K/AKT may have participated in the protective effects of miR-145 on cardiomyocytes.

Knockdown of the long non-coding RNA HOTTIP inhibits colorectal cancer cell proliferation and migration and induces apoptosis by targeting SGK1.

More and more long non-coding RNA (lncRNA) might be serve as molecular biomarkers for tumor cell progression. HOTTIP has been recently revealed as oncogenic regulator in several cancers. However, it remains unclear about whether and how HOTTIP regulates Colorectal cancer (CRC). In the present study, we assayed the expression of HOTTIP in CRC tissues and cell lines, and detected CRC cells (HCT-116 and SW620) proliferation, migration, and apoptosis when HOTTIP was knocked down. Furthermore, we discovered the underlying mechanism. The level of HOTTIP was higher in CRC tissues and in CRC cells compared with adjacent normal tissues and normal colon tissue cell. Knockdown of HOTTIP inhibited the cell proliferation migration and induced apoptosis in HCT-116 and SW620 cell lines. In addition, luciferase reporter assay suggested that knockdown of HOTTIP could target decreasing the expression of Serum- and glucocorticoid-inducible kinase 1 (SGK1) gene, and we subsequently verified that up-regulation of the SGK1 gene promoted cell proliferation and migration and inhibited cell apoptosis in HCT-116 and SW620 cell lines. Furthermore, Knockdown of HOTTIP significantly suppressed the expression of GSK3ß, ß-catenin, c-myc, Vimentin and MMP-7, and increased the expression of E-cadherin, FoxO3a, p27 and Bim proteins in HCT-116 and SW620 cell lines, and up-regulation of the SGK1 emerged the opposite effect with knockdown of HOTTIP. The data described in this study suggest that HOTTIP may be an oncogene and a potential target in CRC.

Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1.

VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 was the same in cells infected with the parent and VP22-deficient viruses. Hence, VP22 appears to promote synthesis of ICP4 post-transcriptionally.

Characterization of an anti-varicella-zoster virus compound that targets the portal protein encoded by ORF54.

An anti-varicella-zoster virus compound, a 5-chlorobenzo[b]thiophen derivative (45B5), was characterized. Its 50% effective concentration against the cell-free vaccine Oka strain and 50% cytotoxic concentration in human fibroblasts were 16.9 µM and more than 100 µM, respectively. Treatment with 45B5 decreased viral DNA synthesis and IE62 expression weakly but significantly. All 45B5-resistant viral clones isolated were found to have at least one mutation in ORF54 that encodes the portal protein. There were no effects on interaction between the portal and scaffold proteins. Thus, 45B5 may inhibit nuclear delivery of viral DNA.

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

The Inhibitory Effect of Eplerenone on Cell Proliferation in the Contralateral Kidneys of Rats with Unilateral Ureteral Obstruction.

BACKGROUND: The unilateral ureteral obstruction (UUO) model not only induces renal interstitial fibrosis in the obstructed kidney but also induces injury in the contralateral kidney. We hypothesized that activation of the mineralocorticoid receptor (MR) may induce fibrosis in the early stage of UUO. METHODS: Thirty male Sprague-Dawley rats weighting 200 ± 10 g were used in this study and randomly divided into 3 groups: a UUO group, a UUO and eplerenone group, and a sham group. The contralateral kidney and plasma were harvested for further study 10 days after surgery. RESULTS: The level of plasma aldosterone (869.95 ± 55.851 pg/mL) was significantly higher in the UUO group than that in the sham group (478.581 ± 36.186 pg/mL vs. UUO, p < 0.05). The infiltrated inflammatory cells (F4/80) and deposited collagens were increased significantly in the contralateral kidneys in the UUO group compared to those in the sham group, which were decreased by eplerenone. However, proliferating cell nuclear antigen was increased 2.47 times in the UUO group compared to the sham group in the contralateral kidney (p < 0.01), and those changes are attenuated by eplerenone. The expression of SGK-1 protein and mRNA was upregulated in the contralateral kidney in the UUO group, which is suppressed by eplerenone treatment. NF-κB pathway effecters were also changed markedly in the contralateral kidney in the UUO group and partly reversed by eplerenone. CONCLUSION: Aldosterone induces inflammatory cell proliferation via the MR/SGK-1 and NF-κB pathways and eventually leads to fibrosis in the contralateral kidney.

BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

The transcriptional modulator Ifrd1 controls PGC-1α expression under short-term adrenergic stimulation in brown adipocytes.

Sympathetic tone activates the function of classical brown adipocytes, which constitutively exist in the brown adipose tissue (BAT), and inducible brown adipocytes (so-called beige adipocytes), which sporadically reside within the white adipose tissue (WAT). Here we identified the transcriptional modulator interferon-related developmental regulator 1 (Ifrd1) as a negative regulator of thermogenic and mitochondrial gene expression in brown adipocytes. Ifrd1 expression was markedly induced by cold exposure and administration of CL-316243 (a ß3 adrenergic agonist) in interscapular brown adipose and inguinal subcutaneous WATs, but not in epididymal visceral WAT, in vivo. Adrenergic stimulation also induced Ifrd1 expression in brown adipocytes in a cAMP responsive element binding protein-dependent manner in vitro. CL-316243 injection markedly elevated thermogenic and mitochondrial gene expression, including peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1a) in the subcutaneous WAT of Ifrd1 knockout mice compared with gene expression in wild-type mice. Pgc1a promoter activity enhanced by the transcription factor specificity protein 1 (Sp1) was markedly repressed by co-introduction of Ifrd1 in brown adipocytes, whereas the repression was markedly prevented by the addition of trichostatin A, a histone deacetylase inhibitor. Moreover, adrenergic stimulation induced complex formation between Ifrd1, Sp1 and mSIN3B, which is a component of the SIN complex containing histone deacetylase, in brown adipocytes. These findings, therefore, suggest that Ifrd1 could be a pivotal negative regulator of sympathetic regulation of thermogenic and mitochondrial gene expression in brown adipocytes by interacting with Sp1 and the mSIN3 complex.

Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins.

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production.