scholarly journals Patient-derived gene and protein expression signatures of NGLY1 deficiency

2021 ◽  
Author(s):  
Benedikt Rauscher ◽  
William F Mueller ◽  
Sandra Clauder-Münster ◽  
Petra Jakob ◽  
M Saiful Islam ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Web Application ◽  
Ribosome Biogenesis ◽  
Genetic Disorder ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
Complex Genetic Disorder

Abstract N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

scholarly journals Patient-derived gene and protein expression signatures of NGLY1 deficiency

2021 ◽  
Author(s):  
Benedikt Rauscher ◽  
William F. Mueller ◽  
Sandra Clauder-Muenster ◽  
Petra Jakob ◽  
M. Saiful Islam ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Web Application ◽  
Genetic Disorder ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
Proteasomal Genes ◽  
Complex Genetic Disorder

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient- derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5957-5957
Author(s):  
Marie-Magdelaine Coudé ◽  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Mélanie Dupont ◽  
Raphael Itzykson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Mrna Expression ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Antileukemic Activity ◽  
Protein Levels ◽  
Gene And Protein Expression

Abstract Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

scholarly journals Dihydropyrimidine Dehydrogenase Levels in Colorectal Cancer Cells Treated with a Combination of Heat Shock Protein 90 Inhibitor and Oxaliplatin or Capecitabine

2019 ◽  
Vol 9 (3) ◽  
pp. 439-444 ◽  
Author(s):  
Mahshid Mohammadian ◽  
Shima Zeynali-Moghaddam ◽  
Mohammad Hassan Khadem Ansari ◽  
Yousef Rasmi ◽  
Anahita Fathi Azarbayjani ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Dihydropyrimidine Dehydrogenase ◽  
Heat Shock Protein 90 ◽  
Water Soluble ◽  
Chemotherapy Response ◽  
Expression Levels ◽  
Protein Levels ◽  
Gene And Protein Expression

Purpose: Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme in the catabolism of fluoropyrimidine drugs including capecitabine. A recent report has suggested that oxaliplatin chemotherapy is associated with elevated DPD levels and chemoresistance pattern. As a newly developed chemotherapeutic agent, 17-allyloamino-17-demethoxy-geldanamycin (17-AAG) can be effective in combination therapy with oxaliplatin and capecitabine in colorectal cancer (CRC). DPD expression level can be a predictive factor in oxaliplatin and capecitabine-based chemotherapy. We evaluated DPD in mRNA and protein levels with new treatments: 17-AAG in combination with oxaliplatin and capecitabine in HT-29 and HCT-116 cell lines. Methods: Drug sensitivity was determined by the water-soluble tetrazolium-1 assay in a previous survey. Then, we evaluated the expression levels of DPD and its relationship with the chemotherapy response in capecitabine, oxaliplatin, and 17-AAG treated cases in single and combination cases in two panels of CRC cell lines. DPD gene and protein expression levels were determined by real-time polymerase chain reaction and western blotting assay, respectively. Results: DPD gene expression levels insignificantly increased in single-treated cases versus untreated controls in both cell lines versus controls. Then, the capecitabine and oxaliplatin were added in double combinations, where DPD gene and protein expression increased in combination cases compared to pre-chemotherapy and single drug treatments. Conclusion: The elevated levels of cytotoxicity in more effective combinations could be related to a different mechanism apart from DPD mediating effects or high DPD level in the remaining resistance cells (drug-insensitive cells), which should be investigated in subsequent studies.

scholarly journals CellO: Comprehensive and hierarchical cell type classification of human cells with the Cell Ontology

2019 ◽  
Author(s):  
Matthew N. Bernstein ◽  
Zhongjie Ma ◽  
Michael Gleicher ◽  
Colin N. Dewey
Keyword(s):  
Single Cell ◽  
Web Application ◽  
Cell Types ◽  
Rna Seq ◽  
Cell Type ◽  
Training Set ◽  
Sequence Read Archive ◽  
Cell Ontology ◽  
Cell Type Specific ◽  
Type Classification

SummaryCell type annotation is a fundamental task in the analysis of single-cell RNA-sequencing data. In this work, we present CellO, a machine learning-based tool for annotating human RNA-seq data with the Cell Ontology. CellO enables accurate and standardized cell type classification by considering the rich hierarchical structure of known cell types, a source of prior knowledge that is not utilized by existing methods. Furthemore, CellO comes pre-trained on a novel, comprehensive dataset of human, healthy, untreated primary samples in the Sequence Read Archive, which to the best of our knowledge, is the most diverse curated collection of primary cell data to date. CellO’s comprehensive training set enables it to run out-of-the-box on diverse cell types and achieves superior or competitive performance when compared to existing state-of-the-art methods. Lastly, CellO’s linear models are easily interpreted, thereby enabling exploration of cell type-specific expression signatures across the ontology. To this end, we also present the CellO Viewer: a web application for exploring CellO’s models across the ontology.HighlightWe present CellO, a tool for hierarchically classifying cell type from single-cell RNA-seq data against the graph-structured Cell OntologyCellO is pre-trained on a comprehensive dataset comprising nearly all bulk RNA-seq primary cell samples in the Sequence Read ArchiveCellO achieves superior or comparable performance with existing methods while featuring a more comprehensive pre-packaged training setCellO is built with easily interpretable models which we expose through a novel web application, the CellO Viewer, for exploring cell type-specific signatures across the Cell OntologyGraphical Abstract

scholarly journals The expression of cytokines in the milk somatic cells, blood leukocytes and serum of goats infected with small ruminant lentivirus

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Justyna Jarczak ◽  
Danuta Słoniewska ◽  
Jarosław Kaba ◽  
Emilia Bagnicka
Keyword(s):  
Protein Expression ◽  
Small Ruminant ◽  
Somatic Cells ◽  
Immunological Response ◽  
Dairy Goats ◽  
Blood Leukocytes ◽  
Protein Levels ◽  
Tnf Α ◽  
Gene And Protein Expression ◽  
Milk Somatic Cells

Abstract Background The present study aimed to determine the expression of cytokines, which is associated with the immunological response of dairy goats against small ruminant lentivirus (SRLV). The study was conducted on 26 dairy goats in their second to sixth lactation, which were divided by breed and parity into two groups: SRLV naturally infected (N = 13) and non-infected (N = 13) animals. All goats in the study were asymptomatic. The milk and blood samples, which served as studied material were taken on days 7, 30, 120 and 240 of the lactation. The gene and protein expression of several cytokines was studied using Real-Time PCR and ELISA methods. Results INF-β and INF-γ expression was down-regulated in the milk somatic cells (MSC) of SRLV-infected goats. However, an increased concentration of INF-β was observed in the MSC in SRLV-infected goats, while INF-γ expression was not observed in both SRLV-infected and non-infected animals The SRLV-infected goats also displayed decreased expression of IL-1α, IL-1β, IL-6 and INF-γ genes in the blood leukocytes,with IL-1α, IL-1β and IL-6 protein levels also being decreased in the sera. TNF-α was the only gene that demonstrated increased expression in both the MSC and the blood of infected animals; however, no such overexpression was observed at the protein level. Conclusions SRLV probably influences the immune system of infected animals by deregulating of the expression of cytokines. Further, epigenetic studies may clarify the mechanisms by which SRLV regulates the gene and protein expression of the host.

Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2796-2796
Author(s):  
Christof Schneider ◽  
Dirk Winkler ◽  
Meike Loddenkemper ◽  
Alexander Krober ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Candidate Genes ◽  
Cell Lines ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Expression Levels ◽  
Protein Levels ◽  
Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

The Histone Deacetylase (HDAC) Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1562-1562 ◽  
Author(s):  
Noor M Khaskhely ◽  
Daniela Buglio ◽  
Jessica Shafer ◽  
Catherine M. Bollard ◽  
Anas Younes
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Protein Expression ◽  
Cell Lines ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Apoptosis Pathway ◽  
Gene And Protein Expression ◽  
Combination Studies

Abstract Abstract 1562 Poster Board I-585 Purpose SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay. Results SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced. Conclusions Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing. Disclosures Younes: MethylGene: Honoraria, Research Funding.

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3296-3296
Author(s):  
Raul Teruel Montoya ◽  
Xianguo Kong ◽  
Shaji Abraham ◽  
Lin Ma ◽  
Leonard C. Edelstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Binding Sites ◽  
Transgene Expression ◽  
Cell Types ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematological Disorders

Abstract Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The carboxy-terminal transactivation domain of Oct-4 acquires cell specificity through the POU domain.

1997 ◽  
Vol 17 (1) ◽  
pp. 154-162 ◽  
Author(s):  
A Brehm ◽  
K Ohbo ◽  
H Schöler
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Regulatory Mechanism ◽  
Cell Types ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Cell Type ◽  
Pou Domain ◽  
Cell Type Specific ◽  
Carboxy Terminal

The POU transcription factor Oct-4 is expressed in totipotent and pluripotent cells of the early mouse embryo and the germ cell lineage. Transactivation capacities of regions flanking the DNA binding domain of Oct-4 were analyzed in undifferentiated and differentiated cell lines. The amino- and carboxy-terminal regions (N domain and C domain) fused to the Gal4 DNA binding domain both functioned as transactivation domains in all cell lines tested. However, the C domain failed to activate transcription in some cell lines in the context of the native protein. The underlying regulatory mechanism appears to involve the POU domain of Oct-4 and can discriminate between different POU domains, since constructs in which the C domain was instead fused to the POU domain of Pit-1 were again equally active in all cell lines. These results indicate that the C domain is subject to cell-type-specific regulation mediated by the Oct-4 POU domain. Phosphopeptide analysis revealed that the cell-type-specific difference of C-domain activity correlates with a difference in Oct-4 phosphorylation status. Since Oct-4 is expressed in a variety of distinct cell types during murine embryogenesis, these results suggest an additional regulatory mechanism for determining Oct-4 function in rapidly changing cell types during development.

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