The effects of a globin blocker on the resolution of 3'mRNA sequencing data in porcine blood.

BACKGROUND: Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of QuantSeq 3'mRNA sequencing (QuantSeq) combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of QuantSeq using a novel specific globin blocker (GB) that is included in the library preparation step of QuantSeq. RESULTS: In data set 1, four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads compared to non-GB (NGB) samples (P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads compared to the NGB (from 56.4 to 10.1%). The second highest concentration C2, which showed very similar globin depletion rates (12%) as C1 but a better correlation of the expression of non-globin genes between NGB and GB (r = 0.98), allowed the expression of an additional 1295 non-globin genes to be detected, although 40 genes that were detected in the NGB sample (at a low level) were not present in the GB library. Concentration C2 was applied in the rest of the study. In data set 2, the distribution of the percentage of globin reads for NGB (n = 184) and GB (n = 189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB, similar to results from data set 1. Data set 3 (n = 84) revealed that the proportion of globin reads that remained in GB samples was significantly and positively correlated with the reticulocyte count in the original blood sample (P < 0.001). CONCLUSIONS: The effect of the GB on reducing the proportion of globin reads in porcine blood QuantSeq was demonstrated in three data sets. In addition to increasing the efficiency of sequencing non-globin mRNA, the GB for QuantSeq has an advantage that it does not require an additional step prior to or during library creation. Therefore, the GB is a useful tool in the quantification of whole gene expression profiles in porcine blood.

Identification of a novel distal regulatory element of the human Neuroglobin gene by the chromosome conformation capture approach.

Neuroglobin (NGB) is predominantly expressed in the brain and retina. Studies suggest that NGB exerts protective effects to neuronal cells and is implicated in reducing the severity of stroke and Alzheimer's disease. However, little is known about the mechanisms which regulate the cell type-specific expression of the gene. In this study, we hypothesized that distal regulatory elements (DREs) are involved in optimal expression of the NGB gene. By chromosome conformation capture we identified two novel DREs located -70 kb upstream and +100 kb downstream from the NGB gene. ENCODE database showed the presence of DNaseI hypersensitive and transcription factors binding sites in these regions. Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the -70 kb region upstream of the NGB gene contained a neuronal-specific enhancer and GATA transcription factor binding sites. Knockdown of GATA-2 caused NGB expression to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of NGB expression. The crucial role of the DRE in NGB expression activation was further confirmed by the drop in NGB level after CRISPR-mediated deletion of the DRE. Taken together, we show that the NGB gene is regulated by a cell type-specific loop formed between its promoter and the novel DRE.

Neuroglobin upregulation induced by 17ß-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells.

The sex steroid hormone 17ß-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H(2)O(2)-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H(2)O(2)-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H(2)O(2) treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB-cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERß) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERß activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H(2)O(2) toxicity.

Neuroglobin involvement in respiratory chain function and retinal ganglion cell integrity.

Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.

Cytoglobin, a novel member of the globin family, protects kidney fibroblasts against oxidative stress under ischemic conditions.

Cytoglobin (Cygb) is a novel member of the vertebrate globin superfamily. Although it is expressed in splanchnic fibroblasts of various organs, details of its function remain unknown. In the present study, kidney ischemia-reperfusion (I/R) increased the number of Cygb-positive cells per area and up-regulated Cygb mRNA and protein expression in kidney cortex tissues. Similarly, hypoxia up-regulated Cygb expression in cultured rat kidney fibroblasts. The biological function of Cygb in vivo was evaluated in Cygb-overexpressing transgenic rats. Renal dysfunction and histologic damage after renal I/R were ameliorated (mean [SE] serum urea nitrogen concentration after I/R injury, 260.6 [44.9] mg/dL in wild-type rats versus 101.0 [36.0] mg/dL in transgenic rats; P < 0.05) in association with improvement of oxidative stress. Primary cultured fibroblasts from Cygb transgenic rat kidney were resistant to exogenous oxidant stimuli, and treatment of immortalized kidney fibroblasts with Cygb-small interfering RNA (siRNA) enhanced cellular oxidant stress and subsequently decreased cell viability (cell count ratio after exposure to hydrogen peroxide, 35.9% [1.6%] in control-siRNA-treated cells versus 25.5% [2.0%] in Cygb-siRNA-treated cells; P < 0.05). Further, chemical or mutant disruption of heme in Cygb impaired its antioxidant properties, which suggests that the heme of Cygb per se possesses a radical scavenging function. These findings show for the first time, to our knowledge, that Cygb serves as a defensive mechanism against oxidative stress both in vitro and in vivo.

Inhibition of alpha-globin gene expression by RNAi.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.

Restoration of the balanced alpha/beta-globin gene expression in beta654-thalassemia mice using combined RNAi and antisense RNA approach.

The beta-thalassemia is associated with abnormality in beta-globin gene, leading to imbalanced synthesis of alpha-/beta-globin chains. Consequently, the excessive free alpha-globin chains precipitate to the erythrocyte membrane, resulting in hemolytic anemia. We have explored post-transcriptional strategies aiming at alpha-globin reduction and beta-globin enrichment on beta(654) (Hbb(th-4)/Hbb(+)) mouse, carrying a human splicing-deficient beta-globin allele (Hbb(th-4)). Lentiviral vectors of short hairpin RNA (shRNA) targeting alpha-globin and/or antisense RNA facilitating beta-globin correct splicing were microinjected into beta(654) single-cell embryos. Three transgenic strains were generated, as alpha(i)-Hbb(th-4)/Hbb(+)(shRNA), beta(a)-Hbb(th-4)/Hbb(+)(antisense) and alpha(i)beta(a)-Hbb(th-4)/Hbb(+)(both shRNA and antisense). Without notable abnormalities, all the founders and their offsprings showed sustained amelioration of hematologic parameters, ineffective erythropoiesis and extramedullary hematopoiesis. Augmented effects appeared in alpha(i)beta(a)-Hbb(th-4)/Hbb(+), which correlated with a better-balanced alpha-/beta-globin mRNA level. Among the transgenic mice integrated with shRNA and antisense RNA, one homozygous mouse (Hbb(th-4)/Hbb(th-4)) had been viable, and the 3-week survival rate for heterozygotes (Hbb(th-4)/Hbb(+)) was 97%, compared with 45.4% for untreated. Our data have demonstrated the feasibility of techniques for beta-thalassemia therapy by balancing the synthesis of alpha-/beta-globin chains.

Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins.

Previous work has established that stably transfected human MCF7 cells over-expressing high mobility group A1 proteins (HMGA1) are deficient in global genomic repair (GGR) following exposure to either UV light or cisplatin. To investigate whether HMGA1 over-expression also interferes with gene-specific repair, we employed a rapid and convenient quantitative polymerase chain reaction assay for measuring repair in unique DNA sequences. Efficiency of UV-induced lesion removal was assessed for two genes in MCF7 cells either induced, or not, to over-express transgenic HMGA1 proteins: the constitutively active HPRT gene and the transcriptionally silent beta-globin gene. As controls, similar experiments were also performed in non-transgenic MCF7 cells that do not express detectable levels of HMGA1 and in normal human embryonic fibroblasts that naturally over-express HMGA1 proteins. Our results indicate that exposure of cells to a UV dose of 20 J/m2 produced an average of 0.21+/-0.03 and 0.19+/-0.02 lesions/kb in the HPRT and beta-globin genes, respectively, with no significant difference between HMGA1 over-expressing cells and non-expressing cells. On the other hand, analysis of repair following UV exposure revealed that, compared to controls, HMGA1 over-expressing cells take considerably longer to repair photo-lesions in both the active HPRT and the silent beta-globin loci, with non-expressing cells repairing 50% of lesions in HPRT 3-4 h faster than HMGA1 over-expressing cells. Interestingly, the delay in repair is even more prolonged in the silent beta-globin locus in HMGA1 over-expressing cells compared to control cells. To our knowledge, this is the first report of HMGA1 proteins inhibiting nucleotide excision repair (NER) within specific genes located in either transcriptionally active "open", or inactive "closed", chromatin domains. Furthermore, taken together with previous findings, these results suggest that HMGA1 over-expression interferes with repair processes common to both the GGR and transcription-coupled repair pathways.

Neuroglobin protects the brain from experimental stroke in vivo.

Neuroglobin (Ngb) is an O(2)-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O(2), detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.

Disruption of Smad5 gene leads to enhanced proliferation of high-proliferative potential precursors during embryonic hematopoiesis.

SMAD proteins are downstream signal transducers of the transforming growth factor beta (TGF-beta) superfamily, which serve as pleiotropic regulators in embryonic and adult hematopoiesis. SMAD5, initially considered to mediate bone morphogenetic proteins (BMPs) signals, can also transduce the inhibitory signal of TGF-beta1 on proliferation of hematopoietic progenitors derived from human bone marrow. To define its specific role in regulation of primitive multipotential progenitors during early embryonic hematopoiesis, we examined Smad5(-/-) yolk sacs at E9.0 to 9.5 and detected an elevated number of high-proliferative potential colony-forming cells (HPP-CFCs) with enhanced replating potential. To exclude the possible influence of microenvironmental deficit on embryonic hematopoiesis in vivo, we performed in vitro embryonic stem (ES) cell differentiation assay and investigated the HPP-CFCs in particular. Smad5(-/-) embryoid bodies (EBs) contained an elevated number of blast colony-forming cells (BL-CFCs), the in vitro equivalent of hemangioblast, in contrast to reduced proliferation of primitive erythroid precursors (Ery/Ps) within the mutant EBs. More importantly, profoundly increased frequency of HPP-CFCs, featured with a gene-dosage effect, was detected within day 6 Smad5(-/-) EBs compared with the wild type. In addition, Smad5(-/-) HPP-CFCs displayed enhanced self-renewal capacity and decreased sensitivity to TGF-beta1 inhibition, suggesting a critical role of Smad5 in TGF-beta1 regulation of embryonic HPP-CFCs. Consistently, reverse transcription-polymerase chain reaction analysis detected alterations of the transcription factors including GATA-2 and AML1 as well as cytokine receptors in Smad5(-/-) HPP-CFC colonies. Together, these data define an important function of SMAD5 in negative regulation of high-proliferative potential precursors during embryonic hematopoiesis.

Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides.

Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization.

Restrictocin is a toxin produced by the fungus Aspergillus restritus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture. Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences. Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.

Comparative activity of alpha- and beta-anomeric oligonucleotides on rabbit beta globin synthesis: inhibitory effect of cap targeted alpha-oligonucleotides.

Alpha-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of alpha- and beta-oligonucleotides targeted against various mRNA regions on the rabbit beta globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected beta-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and alpha-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by alpha-oligonucleotides. This occurs provided they are targeted against the cap region in 5' of the mRNA.

The effect of INH-inhibited heme synthesis on globin synthesis.

In the rabbit reticulocyte cell-free system, optimal globin synthesis is dependent upon an adequate level of heme. The effect of heme is mediated by an inhibitor of globin synthesis initiation termed HCR. In addition to marked inhibition of total globin synthesis, HCR results in a decreased alpha/beta globulin synthesis ratio. We describe here the use of INH as a relatively nontoxic inhibitor of heme synthesis in intact rabbit reticulocytes with a resultant inhibition of globin synthesis. In parallel with the inhibition of globin synthesis in reticulocytes, an inhibitor of globin synthesis in the hemin-supplemented cell-free system is generated. No INH-induced alterations in alpha/beta synthesis ratio could be found in "stress" reticulocytes from phenylhydrazine-treated rabbits, but "normal" reticulocytes from untreated rabbits showed a decreased alpha/beta ratio. Inhibition of heme synthesis and the resulting decrease in globin synthesis and intracellular hemoglobin concentration may have application as a potential treatment of homozygous sickle cell disease.

Control of globin synthesis in cell-free preparations of reticulocytes by formation of a translational repressor that is inactivated by hemin.

The control of globin synthesis by hemin in cell-free preparations of rabbit reticulocytes is mediated by an inhibitor (translational repressor) of globin chain initiation that is inactivated by hemin. When the ribosome-free supernatant fraction is warmed at 34 degrees without hemin, it quickly acquires the ability to inhibit a fresh cell-free preparation. If the warmed supernatant fraction is further incubated with hemin, its inhibitory activity is lost. This reversible inhibitor, which is observable only during the early period of incubation without hemin, is distinct from an irreversible inhibitor that becomes prevalent during longer incubations. The reversible inhibitor may be an intermediate in the formation of the irreversible inhibitor. The sensitivity of the reversible inhibitor to inactivation by hemin, which permits resumption of globin synthesis in both intact cells and lysates, indicates that the inhibitor is a physiological regulator.