Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

Epiblast-specific loss of HCF-1 leads to failure in anterior-posterior axis specification.

Mammalian Host-Cell Factor 1 (HCF-1), a transcriptional co-regulator, plays important roles during the cell-division cycle in cell culture, embryogenesis as well as adult tissue. In mice, HCF-1 is encoded by the X-chromosome-linked Hcfc1 gene. Induced Hcfc1(cKO/+) heterozygosity with a conditional knockout (cKO) allele in the epiblast of female embryos leads to a mixture of HCF-1-positive and -deficient cells owing to random X-chromosome inactivation. These embryos survive owing to the replacement of all HCF-1-deficient cells by HCF-1-positive cells during E5.5 to E8.5 of development. In contrast, complete epiblast-specific loss of HCF-1 in male embryos, Hcfc1(epiKO/Y), leads to embryonic lethality. Here, we characterize this lethality. We show that male epiblast-specific loss of Hcfc1 leads to a developmental arrest at E6.5 with a rapid progressive cell-cycle exit and an associated failure of anterior visceral endoderm migration and primitive streak formation. Subsequently, gastrulation does not take place. We note that the pattern of Hcfc1(epiKO/Y) lethality displays many similarities to loss of ß-catenin function. These results reveal essential new roles for HCF-1 in early embryonic cell proliferation and development.

Regulation of cyclin T1 and HIV-1 Replication by microRNAs in resting CD4+ T lymphocytes.

The replication of integrated human immunodeficiency virus type 1 (HIV-1) is dependent on the cellular cofactor cyclin T1, which binds the viral Tat protein and activates the RNA polymerase II transcription of the integrated provirus. The activation of resting CD4(+) T cells upregulates cyclin T1 protein levels independently of an increase in cyclin T1 mRNA levels, suggesting a translational repression of cyclin T1 in resting CD4(+) T cells. Hypothesizing that microRNAs (miRNAs) repress cyclin T1 translation in resting CD4(+) T cells and that this inhibition is lifted upon cell activation, we used microarray expression analysis to identify miRNAs miR-27b, miR-29b, miR-150, and miR-223 as being significantly downregulated upon CD4(+) T cell activation. The overexpression of these miRNAs decreased endogenous cyclin T1 protein levels, while treatment with the corresponding antagomiRs increased cyclin T1 protein levels. An miR-27b binding site within the cyclin T1 3' untranslated region (3'UTR) was identified and confirmed to be functional after the mutation of key resides abrogated the ability of miR-27b to decrease the expression of a luciferase reporter upstream of the cyclin T1 3'UTR. Ago2 immunoprecipitation revealed an association with cyclin T1 mRNA that was decreased following treatment with miR-27b and miR-29b antagomiRs. Cells overexpressing miR-27b showed decreased viral gene expression levels of the HIV-1 reporter virus and a decreased replication of strain NL4.3; a partial rescue of viral transcription could be seen following the transfection of cyclin T1. These results implicate miR-27b as a novel regulator of cyclin T1 protein levels and HIV-1 replication, while miR-29b, miR-223, and miR-150 may regulate cyclin T1 indirectly.

miR-198 inhibits HIV-1 gene expression and replication in monocytes and its mechanism of action appears to involve repression of cyclin T1.

Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3' untranslated region (3'UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression.

55K isoform of CDK9 associates with Ku70 and is involved in DNA repair.

Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

PPARG Pro12Ala Polymorphism with CKD in Asians: A Meta-Analysis Combined with a Case-Control Study-A Key for Reaching Null Association.

BACKGROUND: So far, numerous meta-analyses have been published regarding the correlation between peroxisome proliferator-activated receptor gamma (PPARG) proline 12 alanine (Pro12Ala) gene polymorphism and chronic kidney disease (CKD); however, the results appear to be contradictory. Hence, this study is formulated with the objective of using existing meta-analysis data together with our research population to study the correlation between PPARG Pro12Ala gene polymorphism and CKD and evaluate whether an accurate result can be obtained. METHODS: First, literature related to CKD and PPARG Pro12Ala available on the PubMed and EMBASE databases up to December 2016 was gathered from 20 publications. Then, the gathered results were combined with our case-control study of 1693 enrolled subjects and a trial sequential analysis (TSA) was performed to verify existing evidence and determine whether a firm conclusion can be drawn. RESULTS: The TSA results showed that the cumulative sample size for the Asian sample was 6078 and was sufficient to support a definite result. The results of this study confirmed that there is no obvious correlation between PPARG Pro12Ala and CKD for Asians (OR = 0.82 (95% CI = 0.66-1.02), I2 = 63.1%), but this was not confirmed for Caucasians. Furthermore, the case-control sample in our study was shown to be the key for reaching this conclusion. CONCLUSIONS: The meta-analysis results of this study suggest no significant correlation between PPARG Pro12Ala gene polymorphism and CKD for Asians after adding our samples, but not for Caucasian.

Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride.

Aging is a complex process involving declines in various cellular and physical functionalities, including regenerative ability. Telomere maintenance is thought to be necessary for regeneration, and telomere attrition is one mechanism that contributes to aging. However, it is unclear if aging affects regeneration owing to deterioration of telomeric maintenance. We introduce Aeolosoma viride-a freshwater annelid with strong regenerative abilities-as a new model for studying the effects of aging on telomere functions and regeneration. We show that the anterior regenerative ability of A. viride declines with age. We characterized the A. viride telomere sequence as being composed of TTAGGG repeats and identifyied the telomerase gene Avi-tert. In adult A. viride, telomerase was constantly active and telomere lengths were similar among different body sections and stably maintained with age. Notably, we found that regeneration did not result in telomere shortening at regenerating sites. Moreover, transient up-regulation of Avi-tert expression and telomerase activity was observed at regenerating sites, which might promote telomere lengthening to counteract telomere erosion resulting from cell proliferation. Our study suggests that although aging affects A. viride regeneration independent of steady-state telomere length, timely regulation of telomerase functions is critical for the regeneration process in A. viride.

Vitrectomy with or without internal limiting membrane peeling for idiopathic epiretinal membrane: A meta-analysis.

BACKGROUND: Studies on vitrectomy with and without internal limiting membrane (ILM) peeling for idiopathic epiretinal membrane (ERM) have yielded uncertain results regarding clinical outcomes and recurrence rates. OBJECTIVE: To compare the clinical outcomes of vitrectomy with and without ILM peeling for idiopathic ERM. METHODS: Databases, including PubMed, Embase, Cochrane, Web of Science, Google Scholar, CNKI databases, FDA.gov, and ClinicalTrials.gov, published until July 2016, were searched to identify studies comparing the clinical outcomes following vitrectomy with ERM and ILM peeling and with only ERM peeling, for treating idiopathic ERM. Studies with sufficient data were selected. Pooled results were expressed as mean differences (MDs) and risk ratios (RRs) with corresponding 95% confidence intervals (CI) for vitrectomy with and without ILM peeling with regard to postoperative best corrected visual acuity (BCVA), central retinal thickness (CRT), and ERM recurrence rate. RESULTS: Eleven retrospective studies and one randomized controlled trial involving 756 eyes were identified. This demonstrated that the postoperative BCVA within 12 months was significantly better in the non-ILM peeling group (MD = 0.04, 95% CI: 0.00 to 0.08; P = 0.0460), but that the patients in the ILM peeling group had significantly better postoperative BCVA after 18 months (MD = -0.13, 95% CI: -0.23 to -0.04; P = 0.0049) than did those in the non-ILM peeling group. The non-ILM peeling group exhibited a higher reduction in postoperative CRT (MD = 51.55, 95% CI:-84.23 to -18.88; P = 0.0020) and a higher recurrence rate of ERM (RR = 0.34, 95% CI:0.16 to 0.72; P = 0.0048) than did the ILM peeling group. However, the improvement rates of BCVA (RR = 1.03, 95% CI:0.72 to 1.47; P = 0.8802) and postoperative CRTs (MD = 18.15, 95% CI:-2.29 to 38.60; P = 0.0818) were similar between the two groups. CONCLUSIONS: Vitrectomy with ILM peeling results in better visual improvement in long-term follow-ups and lower ERM recurrence rates, and vitrectomy with only ERM peeling is more efficacious in reduction of CRT than is vitrectomy with ILM peeling.

Extrachromosomal telomere repeat DNA is linked to ALT development via cGAS-STING DNA sensing pathway.

Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNß production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX-Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells.

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4+ T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively.

Cyclin T1-dependent genes in activated CD4 T and macrophage cell lines appear enriched in HIV-1 co-factors.

HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4(+) T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4(+) T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that appear to be Cyclin T1-dependent for their induction in activated CD4(+) T Jurkat T cells and during differentiation and activation of MM6 cells, a human monocytic cell line. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021). The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication. It is likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

Phosphatase PPM1A regulates phosphorylation of Thr-186 in the Cdk9 T-loop.

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to approximately 50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.

Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay.

While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive- and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive- and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.

Sequence diversity of the capsid gene and the nonstructural gene NS2B of dengue-3 virus in vivo.

Previously, we studied the envelope (E) gene of dengue virus and reported that dengue-3 virus is present as a quasispecies. To investigate the extent of intrahost sequence variation of other dengue viral genes, we examined in this study the capsid (C) gene and the nonstructural gene, NS2B, derived directly from plasma dengue viruses from 18 confirmed dengue-3 patients. Using reverse transcription-PCR, multiple clones of a 360-nucleotide region covering the C gene and of a 404-nucleotide region covering the NS2B gene from each patient were completely sequenced and analyzed. Our findings of the intrahost sequence variation of the C and the NS2B genes (mean pairwise p-distance: 0.12 to 1.02%, and 0.16 to 1.20%, respectively) demonstrate the quasispecies structure of dengue virus in vivo. A linear relationship was found between the extent of sequence variation of the C and NS2B proteins, suggesting that intrahost sequence variation of dengue-3 virus is likely to reflect genetic drift. The extent of intrahost sequence variation observed is in the same range as that of acute human immunodeficiency virus or hepatitis C virus infection, indicating that the random mutation frequency of dengue virus is similar to that of other RNA viruses in vivo. Consistent with a previous report of the E gene, the observations of genome-defective clones in both the C and the NS2B genes (3.9 and 5.0% of the clones, respectively) suggest a higher frequency of defective viruses in vivo. These findings would add to our understanding of the evolution of dengue-3 virus.