Hemodynamic correlates of low umbilical cord vitamin D and ionized calcium.

Vitamin D deficiency and hypocalcemia are associated with gestational hypertension. Therefore, we hypothesized that umbilical cord [Ca(2+)] and [vitamin D] are correlated with perinatal blood pressures. Mothers and newborns comprised vitamin D sufficient (vitamin D ≥ 50 nM, range 52-111 nM, n = 14), and vitamin D deficient groups (vitamin D < 50 nM, range 13-49 nM, n = 29). Cord [Ca²âº] was negatively correlated with maternal systolic pressure (SBP) (r = -0.56, p < 0.01) and positively correlated with neonatal SBP (r = +0.55, p < 0.01) in the vitamin D deficient group. We conclude that low umbilical cord [vitamin D] and [Ca²âº] may predispose mothers to higher and newborns to lower blood pressures.

Chicken HS4 insulators have minimal barrier function among progeny of human hematopoietic cells transduced with an HIV1-based lentiviral vector.

Position effects limit the curative potential of gene transfer strategies for the hemoglobinopathies by inducing clonal variability of transgene expression. We evaluated the mitigating effects of the chicken hypersensitivity site 4 (HS4) insulator among lentiviral vector-transduced human hematopoietic cells. We constructed various lentiviral vectors using a green fluorescent protein (GFP) reporter under the control of a reverse-oriented murine stem cell virus (MSCV)-long-term repeat (LTR) promoter or a reverse-oriented ß-globin expression cassette. A full-length HS4, a tandem HS4 core, and a single core insulator were inserted into the 3' LTR in both forward and reverse orientation. All but the reverse single core insulator significantly decreased titers. All reduced %GFP without increasing mean fluorescence intensity (MFI) among erythroid progeny of transduced human CD34(+) cells. A lower coefficient of variation (CV) was observed only among progeny of the full-length vector-transduced cells, yet a fivefold reduction in transduction efficiency was observed. In xenografted mice, the single core insulator decreased both the %GFP and the MFI at 4 and 8 weeks after transplantation with no difference in CVs. These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction, and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo.

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.

Development of a human immunodeficiency virus type 1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells.

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha 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 (chiHIV) 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 chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs, the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the chiHIV 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. STUDY DESIGN AND METHODS: We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice. RESULTS: CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. CONCLUSION: Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.

Determination of distinctive hypomethylated genes in African American colorectal neoplastic lesions.

BACKGROUND: Few studies have analyzed progressive demethylation in the path to cancer. This is of utmost importance, especially in populations such as African Americans, who display aggressive tumors at diagnosis, and for whom markers of early neoplastic transformation are needed. Here, we determined hypomethylated targets in the path to colorectal cancer (CRC) using Reduced Representation Bisulfite Sequencing (RRBS). METHODS: DNA was extracted from fresh frozen tissues of patients with different colon lesions (normal, tubular adenoma, tubulovillous adenoma, and five cancers). RRBS was performed on these DNA extracts to identify hypomethylated gene targets. Alignment, mapping, and methylation analyses were performed. Pathways affected by the hypomethylated gene targets were determined using Ingenuity Pathway Analysis (IPA). RESULTS: Pairwise analyses of samples led to the identification of the following novel hypomethylated genes: ELMO3 (Engulfment and cell motility 3), SLC6A2 (Solute carrier family 6 member 2), SYNM (Synemin), and HMX2 (Homeobox 2). The ELMO3 promoter was significantly hypomethylated at five CpG sites, SYNM at two CpG sites, SLC6A2 at one CpG site, and the HMX2 gene at one CpG site. IPA placed these genes within important carcinogenic pathways. CONCLUSIONS: This work provides insight into the role of hypomethylation in colon carcinogenesis in African Americans. The identified targets affected many important pathways, as demonstrated through IPA. These targets might serve as biomarkers for early diagnosis and potential targets for therapy.

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's T2 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.

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

Sickle cell disease results from a point mutation in exon 1 of the ß-globin gene (total 3 exons). Replacing sickle ß-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 ß-globin pre-messenger RNA among human erythroid cells. Binding domains from random ß-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 ß-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 ß-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.

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/ß-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-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.

Apoptotic Effects of Novel Dithiocarbamate Analogs of Emetine in Prostate Cancer Cell Lines.

BACKGROUND/AIM: Prostate cancer is one of the leading causes of death in American males. Emetine, a naturally-derived alkaloid from the Ipecacuanha plant, has been shown to have potential for anti-tumorigenic effects for cancer treatments. The objective of this study was to characterize novel emetine dithiocarbamate (EMTDTC) analogs for potent anti-tumorigenic activity with minimal toxicity to normal prostate cells and identify targeted apoptotic regulatory genes. The leading key compounds, EMTDTC-55 and EMTDTC-56 were studied. MATERIALS AND METHODS: Established methods of cell flow cytometry were used to analyze apoptotic potential in prostate cancer cell lines (DU145, PC3 and LNCaP) and real time-polymerase chain reaction (PCR) for identifying key genes mediating apoptosis. RESULTS: The effect of EMTDTC-55 on DU145, LNCaP and PC3 revealed significant anti-tumorigenic activities. Both compounds showed highly significant apoptotic potential on days 3 and 5 in the prostate cancer cells. Key apoptotic genes were differentially regulated suggestive of cell-cycle arrest and apoptotic induction in androgen-independent cell lines, DU145 and PC3, by both compounds. However, in the androgen-dependent cell line LNCaP, cells were marginally affected by EMTDTC-55, but significant apoptosis was observed by EMTDTC-56 leading to cell-cycle arrest. CONCLUSION: Both dithiocarbamate compounds EMTDTC-55 and EMTDTC-56 have significant chemotherapeutic potential in moderately metastatic DU145 and highly metastatic PC3 cells.

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.

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.

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.

The assessment of human erythroid output in NOD/SCID mice reconstituted with human hematopoietic stem cells.

The third-generation NOD/LtSz-scid/IL2Rγ(null) (NOD/SCID IL2Rγ(null)) mouse represents a significantly improved xenograft model allowing high levels of human leukocyte engraftment over extended follow up. One remaining limitation of this mouse model, however, is the low level of circulating human erythrocytes. We established a practical ex vivo erythroid culture system of xenograft marrow progenitors to enrich for human erythroid progeny. At various time points after transplant, erythroid cells were easily assayed after 17 days of ex vivo culture of xenograft marrow, with nearly all nucleated cells of human origin and approximately 60% human GPA or CD71 positive. We then transplanted cord blood CD34(+) cells marked with a lentiviral vector encoding green fluorescent protein (GFP). Three months later, ex vivo culture of xenograft marrow progenitors showed 41.3% of the cultured erythroid cells were positive for GFP and human CD71, and 56.2% were positive for GFP and human GPA, similar to that of circulating leukocytes at the same time point. Next, G-CSF mobilized peripheral blood CD34(+) cells from a sickle cell trait subject were infused in this mouse model to determine if the hemoglobin pattern could be modeled. CD34(+) cells from the sickle cell trait subject engrafted equally compared to CD34(+) cells from normal subjects, establishing the sickle cell trait phenotype. Lastly, a comparison of adult-derived peripheral blood CD34(+) cells and cord blood-derived CD34(+) cells xenografted mice was made, and long term follow-up demonstrated a recapitulation of the fetal to adult hemoglobin switch. This approach should prove a useful tool for testing strategies for genetic manipulation of erythroid progeny and the study of hemoglobin switching.

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.

Transient in vivo beta-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.

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.