Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription

BACKGROUND: HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. RESULTS: In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. CONCLUSION: Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription

Reduced Representation Bisulfite Sequencing Determination of Distinctive DNA Hypermethylated Genes in the Progression to Colon Cancer in African Americans

Background and Aims. Many studies have focused on the determination of methylated targets in colorectal cancer. However, few analyzed the progressive methylation in the sequence from normal to adenoma and ultimately to malignant tumors. This is of utmost importance especially in populations such as African Americans who generally display aggressive tumors at diagnosis and for whom markers of early neoplasia are needed. We aimed to determine methylated targets in the path to colon cancer in African American patients using Reduced Representation Bisulfite Sequencing (RRBS). Methods. Genomic DNA was isolated from fresh frozen tissues of patients with different colon lesions: normal, a tubular adenoma, a tubulovillous adenoma, and five cancers. RRBS was performed on these DNA samples to identify hypermethylation. Alignment, mapping, and confirmed CpG methylation analyses were performed. Preferential hypermethylated pathways were determined using Ingenuity Pathway Analysis (IPA). Results. We identified hypermethylated CpG sites in the following genes: L3MBTL1, NKX6-2, PREX1, TRAF7, PRDM14, and NEFM with the number of CpG sites being 14, 17, 10, 16, 6, and 6, respectively, after pairwise analysis of normal versus adenoma, adenoma versus cancer, and normal versus cancer. IPA mapped the above-mentioned hypermethylated genes to the Wnt/β-catenin, PI3k/AKT, VEGF, and JAK/STAT3 signaling pathways. Conclusion. This work provides insight into novel differential CpGs hypermethylation sites in colorectal carcinogenesis. Functional analysis of the novel gene targets is needed to confirm their roles in their associated carcinogenic pathways

Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-I in Tat-activated HIV-I transcription

Background: HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. Results: In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. Conclusion: Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription. © 2005 Ammosova et al; licensee BioMed Central Ltd

Morphometric analysis in ethnic neonates from multiple substance exposure

In the United States, approximately 10% of newborn infants are exposed prenatally to alcohol and/or illicit substances. However, no studies have evaluated the compounding effects of multiple illicit substances exposure in utero as potential teratogen (s). The potential teratogenic effects of nicotine and illicit substances (e.g. cocaine, marijuana and heroin) have previously been studied but there has been no documentation of facial landmark dislocation (s). Our goal is to investigate whether morphometric analysis could differentiate facial landmark dislocations in neonates of African descent, when exposed to alcohol, nicotine and illicit substances, either singly or in combination. Craniofacial features from a cohort of 493 African-American neonates less than 48 hours of age were analyzed by Multivariate Hotelling\u27s T 2 analysis of 99 relevant facial landmark triangles. Morphometric analysis discriminated unique asymmetries in groups of certain illicit exposure(s). Neonates with multiple prenatal exposures had fewer facial landmark dislocation(s) compared to single exposures. Deviation from normal facial features has the potential to be used as a screening tool for prenatal exposure to some illicit substances

Status of LEPR gene in PCB-exposed population: A quick look

Earlier, we have reported that Polychlorinated Biphenyls (PCBs) exposure in Slovak population has made differential gene expression that has linked to the possibilities of some diseases and disorder development in the studied population. Here we report that down-regulation of LEPR (Leptin receptor) gene in the 45-month children may have been following consequences in developing obesity later in life. A pilot high-throughput qRT-PCR [Taqman Low Density Array (TLDA)] study in a small population also corroborated the gene-expression results, and their pathways underlying the consequences of the diseases, amid further detailed large-scale population validation. The study shows the opportunity of predicting long-term effects of chemical exposures using selected genomic classifiers may reflect exposure effect and risk from environmental toxicants

Protein phosphatase-1 dephosphorylates the C-terminal domain of RNa polymerase-II

Transcription by RNA polymerase-II (RNAPII) is controlled by multisite phosphorylation of the heptapeptide repeats in the C-terminal domain (CTD) of the largest subunit. Phosphorylation of CTD is mediated by the cyclin-dependent protein kinases Cdk7 and Cdk9, whereas protein serine/threonine phosphatase FCP1 dephosphorylates CTD. We have recently reported that human immunodeficiency virus-1 (HIV-1) transcription is positively regulated by protein phosphatase-1 (PP1) and that PP1 dephosphorylates recombinant CTD. Here, we provide further evidence that PP1 can dephosphorylate RNAPII CTD. In vitro, PP1 dephosphorylated recombinant CTD as well as purified RNAPII CTD. HeLa nuclear extracts were found to contain a species of PP1 that dephosphorylates both serine 2 and serine 5 of the heptapeptide repeats. In nuclear extracts, PP1 and FCP1 contributed roughly equally to the dephosphorylation of serine 2. PP1 co-purified with RNAPII by gel filtration and associated with RNAPII on immunoaffinity columns prepared with anti-CTD antibodies. In cultured cells treated with CTD kinase inhibitors, the dephosphorylation of RNAPII on serine 2 was inhibited by 45% by preincubation with okadaic acid, which inhibits phosphatases of PPP family, including PP1 but not FCP1. Our data demonstrate that RNAPII CTD is dephosphorylated by PP1 in vitro and by PPP-type phosphatase, distinct from FCP1, in vivo

Protein Phosphatase-1 Regulates Expression of Neuregulin-1

Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner

Protein phosphatase-1 regulates expression of neuregulin-1

Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner

Long-term vector integration site analysis following retroviral mediated gene transfer to hematopoietic stem cells for the treatment of HIV infection.

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV. Here we report an assessment of retroviral integration sites (RISs) recovered out to 3 years post-transplantation. We identified 213 unique RISs from the patient's peripheral blood samples by linear amplification-mediated PCR (LAM-PCR). While vector integration patterns were similar to that previously reported, only 3.76% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). Additionally, common integration sites were enriched among late samples (14.9% vs. 36.8%, respectively). Three RISs were found near or within known oncogenes, but 2 were limited to early timepoints. Interestingly, an integration site near the MDS1 gene was detected in long-term follow-up samples; however, the overall contribution of MDS1 integrated clone remained stably low during follow-up