Kareem Washington

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

BackgroundHIV-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.ResultsIn 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.ConclusionOur 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.

Development of a Human Immunodeficiency Virus Type 1-Based Lentiviral Vector That Allows Efficient Transduction of both Human and Rhesus Blood Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5α and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (χHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34+ hematopoietic stem cells (HSCs), the χHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34+ HSCs, the χHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the χHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.

5% Dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO

BACKGROUND: Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system.

Biomarkers Linking PCB Exposure and Obesity

Recently the prevalence of obesity has increased dramatically across much of the world. Obesity, as a complex, multifactorial disease, and its health consequences probably result from the interplay of environmental, genetic, and behavioral factors. Several lines of evidence support the theory that obesity is programmed during early development and that environmental exposures can play a key role. We therefore hypothesize that the current epidemic might associated with the influence of chemical exposures upon genetically controlled developmental pathways, leading to metabolic disorders. Some environmental chemicals, such as PCBs and pesticide residues, are widespread in food, drinking water, soil, and they exert multiple effects including estrogenic on cellular processes; some have been shown to affect the development of obesity, insulin resistance, type 2 diabetes, and metabolic syndrome. To bring these lines of evidence together and address an important health problem, this narrative review has been primarily designed to address PCBs exposures that have linked with human disease, obesity in particular, and to assess the effects of PCBs on gene expression in a highlyexposed population. The results strongly suggest that further research into the specific mechanisms of PCBs-associated diseases is warranted.

Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.

Transient In Vivo β-Globin Production After Lentiviral Gene Transfer to Hematopoietic Stem Cells in the Nonhuman Primate

Inherited disorders of globin synthesis remain desirable targets for hematopoietic stem cell (HSC)-based therapies. Gene transfer using retroviral vectors offers an alternative to allogeneic HSC transplantation by the permanent integration of potentially therapeutic genes into primary autologous HSCs. Although proof of principle has been demonstrated in humans, this approach has been met by formidable obstacles, and large-animal models have become increasingly important for the preclinical development of gene addition strategies. Here we report lentiviral gene transfer of the human beta-globin gene under the control of the globin promoter and large fragments of the globin locus control region (LCR) in the nonhuman primate. Using an HIV-1, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped vector, modified to overcome a species-specific restriction to HIV-1, gene transfer to colony-forming units (CFU) derived from mobilized peripheral blood (PB) rhesus CD34+ cells was 84.4 +/- 2.33%. Erythroid cells derived from transduced rhesus CD34+ cells expressed human beta-globin at high levels as assessed by flow cytometry with a human beta-globin-specific antibody. Two rhesus macaques (RQ3586 and RQ3583) were transplanted with mobilized PB CD34+ cells transduced with our modified HIV vector at a multiplicity of infection of 80. High gene transfer rates to CFUs were achieved in vitro (RQ3586, 87.5%; RQ3583, 83.3%), with efficient human beta-globin expression among erythroid progeny generated in vitro. Early posttransplantation, gene transfer rates of 5% or higher were detectable and confirmed by genomic Southern blotting, with equivalent-level human beta-globin expression detected by flow cytometry. Long-term gene marking levels among mononuclear cells and granulocytes assessed by quantitative polymerase chain reaction gradually decreased to about 0.001% at 2 years, likely due to additional HIV-1 restrictive elements in the rhesus macaque. No evidence of clonal hematopoiesis has occurred in our animals in up to 2 years. Current efforts are aimed at developing a lentiviral vector capable of efficiently transducing both human and rhesus HSCs to allow preclinical modeling of globin gene transfer.

Efficient transduction of human hematopoietic repopulating cells with a chimeric HIV1-based vector including SIV capsid

Innate immune factors, such as TRIM5α and cyclophilin A (CypA), act as a major restriction factor of retroviral infection among species. When HIV1 infects human cells, HIV1 capsid binds to human CypA to escape from human TRIM5α restriction. However, in rhesus cells, the mismatch between HIV1 capsid and rhesus CypA is recognized by rhesus TRIM5α to reduce HIV1 infectivity through proteasomal degradation. To circumvent this block, we previously developed a chimeric HIV1 vector (χHIV) that substituted HIV1 capsid with SIV capsid, and it significantly increased transduction efficiency for nonhuman primate cells. In this study, we evaluated whether the χHIV vector efficiently transduces human cells, and the transduction efficiency might increase by a CypA inhibitor (cyclosporine) and a proteasome inhibitor (MG132). The χHIV vector could transduce human CD34⁺ cells, as efficiently as the HIV1 vector, in vitro and in xenograft mice, even in the mismatch between SIV capsid and human CypA. Cyclosporine decreased transduction efficiency with the HIV1 vector, whereas it slightly increased transduction efficiency with the χHIV vector in human CD34⁺ cells. MG132 increased transduction efficiency with both χHIV and HIV1 vectors in the same manner. However, MG132 was toxic to human CD34⁺ cells at high concentrations, and both drugs had a small range of effective dosage. These findings demonstrate that both χHIV and HIV1 vectors have similar transduction efficiency for human hematopoietic repopulating cells, suggesting that the χHIV vector escapes from TRIM5α restriction, which is independent of human CypA.

RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells

Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5 splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5 splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

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.

440. Optimization of RNA Trans-Splicing for the β-Globin Gene; Detecting Trans-Splicing Events Targeting Endogenous β-Globin Pre-mRNA in Human Erythroid Cells

Sickle cell disease is caused by a point mutation in exon 1 of the β-globin gene (3 exons and 2 introns). RNA splicing between two distinct pieces of pre-mRNA, known as trans-splicing, represents a potential therapeutic strategy allowing replacement of the mutated exon 1 with a normal exon. Induction of exogenous mRNA splicing using spliceosome-mediated trans-splicing requires an RNA trans-splicing molecule (RTM) which imitates endogenous cis-splicing elements. However, clinical application of trans-splicing for globin disorders will require high efficiency, thus we sought to optimize the efficiency of trans-splicing targeting the β-globin gene to replace the exon 1 among human erythroid cells.To optimize the binding domain targeting the β-globin gene, the randomized binding domains of 20-600b were generated by sonicating the β-globin gene, and these were inserted into RTM-expressing plasmids downstream of the 5’ half of GFP and a 5’ splice site. In addition, we designed a target plasmid which encodes the β-globin gene and an artificial 3’ splice site connected to 3’ site of the other half of GFP. In transfection of both RTM and target plasmids, we selected 6 candidates from several thousand RTMs, which produced the brightest GFP-positive cells (maximal trans-splicing), and interestingly, all 6 binding domains targeted the β-globin intron 2.To evaluate efficiency of trans-splicing for human endogenous β-globin RNA, we constructed lentiviral vectors encoding RTMs which contain the γ-globin cDNA connected to a 5’ splice site and 3 candidates (1E1; 0.6kb, 2D10; 0.7kb, and 13-1; 0.8kb) of β-globin binding domains (selected from the 6 candidates). Human CD34+ cells were transduced with these RTM-encoding lentiviral vectors, and these cells were differentiated to erythroid cells in vitro. Trans-splicing was detected by RT-PCR and sequencing in RTM 2D10 and 13-1 but not in RTM 1E1, suggesting that longer binding domains improve trans-splicing efficiency. To elongate the binding domain in RTM 13-1, we designed RTM BGin2 (containing 5’ splice site of β-globin intron 2; 0.9kb) and BGex1-in2 (containing whole β-globin sequence except exon 3; 1.3kb). In both RTM BGin2 and BGex1-in2, ~4-fold higher trans-splicing was detected by RT-qPCR than RTM 13-1, which resulted in ~0.07% trans-splicing efficiency as compared to endogenous human β-globin RNA, implying that the 5’ splice site of intron 2 should be included as a binding domain for efficient trans-splicing.In summary, we developed a screening system to select more efficient binding domains for trans-splicing. For the first time, we obtained detectable levels of spliceosome-mediated trans-splicing (~0.07%) for endogenous β-globin RNA in human erythroid cells which were transduced with RTM-expressing lentiviral vectors. Further optimization is required to develop trans-splicing based gene therapy.