Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1376-1376
Author(s):  
Soumyadeep Dey ◽  
David J. Curtis ◽  
Stephen Jane ◽  
Stephen J. Brandt
Keyword(s):  
Transcription Factor ◽  
Real Time ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Critical Role ◽  
Pcr Analysis ◽  
Bhlh Transcription Factor ◽  
Lacz Gene ◽  
Macrophage Differentiation ◽  
In Cells

Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.

siRNA Targeting DNA Methyltransferase I Increases ε- and γ-Globin Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 973-973
Author(s):  
Virryan Banzon ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Kenneth Peterson ◽  
Joseph DeSimone ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Methyltransferase ◽  
Globin Gene ◽  
Pcr Analysis ◽  
Globin Promoter ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
In Cells

Abstract Abstract 973 Pharmacological inhibitors of DNA methyltransferase (DNMT) increase fetal hemoglobin (HbF) levels in experimental primates and patients with sickle cell disease. Therefore we hypothesize that DNMT is directly involved in maintaining repression of the γ-globin gene in adult stage erythroid cells. To test this hypothesis, levels of DNMT1 in mouse chemical-of-dimerization (CID) bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and primary cultured erythroid progenitor cells (EPC) derived from baboon CD34+ BM cells were reduced by treatment with siRNA targeting DNMT1 (siDNMT1) and the effect on globin gene expression determined. Nucleofection conditions that achieved 80-90% transfection efficiency were used to introduce siRNA into CID-dependent mouse βYAC BM cells. Real time PCR analysis showed that expression of DNMT1 was decreased 70-80% in cells treated with siDNMT1 compared to cells transfected with nonsilencing control siRNA while DNMT3A and 3B were not decreased. Results of real time PCR analysis of six independent experiments showed that ε-globin expression was increased 65.3+/−37.8 fold, γ-globin 230.3+/−147.8 fold, and β-globin 6.0+/−3.3 fold in cells treated with siDNMT1 compared to untreated controls, while cells treated with nonsilencing siRNA showed minimal (<2 fold) changes. The difference in ε- and γ-globin expression between cells treated with siDNMT1 and nonsilencing RNA was significant (p<.01). Bisulfite sequence analysis showed that DNA methylation of the ε-globin promoter and γ-globin promoters were reduced to 25.7 and 53.3% dmC, respectively, in cells treated with siDNMT1 compared to 68.8 and 98.8% dmC, respectively, in cells treated with nonsilencing siRNA. Nucleofection of cultured baboon EPC with siDNMT1 or nonsilencing siRNA was performed on d7 and d8 of culture. Transfection efficiencies of 45-50 % were achieved. Expression of DNMT1 was decreased >80% in cells treated with siDNMT1 compared to those treated with nonsilencing siRNA. Real time PCR analysis of duplicate samples showed that γ-globin expression was increased 2.06 and 2.25 fold relative to untreated controls following treatment of cells with siDNMT1 while nonsilencing siRNA had no effect. Expression of ε-globin was increased 35.26 and 25.4 fold relative to untreated controls in cells treated with siDNMT1 while a lesser effect was observed in cells treated with nonsilencing siRNA (7.41 and 8.16 fold). HPLC analysis of biosynthetically radiolabelled globin chains showed that γ-globin chain synthesis (γ/γ+β ratio) was increased in cells treated with siDNMT1 (0.59 and 0.63) compared to cells treated with nonsilencing siRNA (0.41 and 0.37), untreated controls (0.39) and mock-transfected controls (0.43). DNA methylation of 3 CpG residues within the 5' ε-globin promoter region and 5 CpG residues within the 5' γ-globin promoter region was reduced to 52.8 and 45.0% dmC, respectively, in cells treated with siDNMT1, compared to 100 and 85.4% dmC, respectively, in cells treated with nonsilencing siRNA. Our results demonstrate that siDNMT1 reduces DNMT1, reduces levels of DNA methylation of the ε- and γ-globin gene promoters, and increases ε- and γ-globin gene expression and γ-globin chain synthesis in CID-dependent mouse BM cells and in primary baboon EPC cultures derived from CD34+ BM cells. We conclude that DNMT1 is critically involved in the mechanism responsible for repression of γ-globin expression in adult-stage erythroid cells and therefore inhibition of DNMT1 activity by pharmacological inhibitors of DNA methyltransferase likely plays a fundamental role in the ability of these drugs to increase HbF in vivo. Disclosures: No relevant conflicts of interest to declare.

Inversely Association of the Aiolos Transcription Factor with Clinical Progression in Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2069-2069
Author(s):  
Holger Nückel ◽  
Ulrich H Frey ◽  
Ludger Sellmann ◽  
Crista H Collins ◽  
Ulrich Duehrsen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
Mononuclear Cells ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Cd38 Expression ◽  
Important Regulator

Abstract Introduction: Aiolos encode a hemopoietic-specific zinc-finger transcription factor that is an important regulator of lymphocyte differentiation and plays a critical role in regulating B-cell development. RT-PCR analysis of the Aiolos gene expression revealed 16 Aiolos splicing variants, which have been named according to the exons missing from the full-length isoform. Recent data suggest that over 80% of expressed Aiolos in normal as well as in malignant B-cells is of the hAio1 type. Therefore, we investigated Aiolos mRNA expression (hAio1 type) in a large cohort with 155 patients along with the most commonly used biological markers in order to assess its role in risk prediction in B-CLL. Methods and Results: The total amount of Aiolos transcripts in B-cells of 155 CLL patients using normal peripheral blood mononuclear cells represented a continuum ranging from 2.5- to 37-fold upregulation compared to that of normal B-cells, with a median of 20- fold upregulation. Moreover, Aiolos expression in B-CLL was significantly upregulated compared to cells of AML (113-fold; p&lt;0.0001), ALL (14-fold; p=0.0024), CML (154- fold; p&lt;0.0001), multiple myeloma (38-fold; p=0.0018) or NHL (16-fold; p&lt;0.0001) suggesting that this Aiolos transcript is highly expressed specifically in B-CLL cells Patients with high Aiolos expression (according to ROC-analysis) had a significantly longer treatment-free survival (TFS) and overall survival (OS) than patients with low Aiolos expression (median TFS: 119 versus 45 months, p=0.005; median OS: 321 versus 244 months, p=0.0065). Evaluation of several disease characteristics in association with the Aiolos expression status of the patients’ B-CLL cells showed no significant differences for ZAP-70 expression (p=0.28), CD38 expression (p=0.067), IgVH status (p=0.49) and Binet stage (p=0.13) suggesting no correlation of Aiolos expression with these already established adverse prognostic factors. In multivariate analysis low Aiolos expression was an independent prognostic factor with significance for trend (hazard ratio 1.413; p=0.069). Sequential analyses in a subset of 10 CLL patients revealed that Aiolos expression was relatively stable over time in the majority of patients. Conclusions: Here we demonstrate for the first time that the level of Aiolos expression is correlated with prognosis in B-CLL. The exact causes and consequences of this upregulation on the survival of CLL B-cells and the demonstrated better prognosis have yet to be determined. However, Aiolos may function as a tumor suppressor by controlling the cell cycle and DNA replication.

scholarly journals Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Toshio Hattori ◽  
Naomi Ogura ◽  
Miwa Akutsu ◽  
Mutsumi Kawashima ◽  
Suguru Watanabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Temporomandibular Joint ◽  
Real Time ◽  
Real Time Pcr ◽  
Protein Production ◽  
Critical Role ◽  
Synovial Fibroblasts ◽  
Temporomandibular Joint Disorders ◽  
Pcr Analysis ◽  
Dependent Manner

Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD.

DEF8 and Autophagy-Associated Genes are Altered in Mild Cognitive Impairment, Probable Alzheimer’s Disease Patients and a Transgenic Model of the Disease

2021 ◽  
pp. 1-16
Author(s):  
Esteban Leyton ◽  
Diego Matus ◽  
Sandra Espinoza ◽  
José Matías Benitez ◽  
Bastián I. Cortes ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cognitive Impairment ◽  
Mild Cognitive Impairment ◽  
Real Time ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Differentially Expressed ◽  
Protein Levels ◽  
5Xfad Mice

Background: Disturbances in the autophagy/endolysosomal systems are proposed as early signatures of Alzheimer’s disease (AD). However, few studies are available concerning autophagy gene expression in AD patients. Objective: To explore the differential expression of classical genes involved in the autophagy pathway, among them a less characterized one, DEF8 (Differentially expressed in FDCP 8), initially considered a Rubicon family member, in peripheral blood mononuclear cells (PBMCs) from individuals with mild cognitive impairment (MCI) and probable AD (pAD) and correlate the results with the expression of DEF8 in the brain of 5xFAD mice. Method: By real-time PCR and flow cytometry, we evaluated autophagy genes levels in PBMCs from MCI and pAD patients. We evaluated DEF8 levels and its localization in brain samples of the 5xFAD mice by real-time PCR, western blot, and immunofluorescence. Results: Transcriptional levels of DEF8 were significantly reduced in PBMCs of MCI and pAD patients compared with healthy donors, correlating with the MoCA and MoCA-MIS cognitive tests scores. DEF8 protein levels were increased in lymphocytes from MCI but not pAD, compared to controls. In the case of brain samples from 5xFAD mice, we observed a reduced mRNA expression and augmented protein levels in 5xFAD compared to age-matched wild-type mice. DEF8 presented a neuronal localization. Conclusion: DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially expressed in PBMCs of MCI and pAD and neurons of 5xFAD mice. These results suggest a potential role for DEF8 in the pathophysiology of AD.

ID: 316 Real-time PCR analysis of BCL2L12, a novel member of BCL2 family of the apoptosis-related genes, in human leukemia.

2006 ◽  
Vol 4 (s1) ◽  
pp. 82-82
Author(s):  
K. Floros ◽  
H. Thomadaki ◽  
S. Pavlovic ◽  
M. Talieri ◽  
M. Colovic ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Bcl2 Family

An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis

2011 ◽  
Vol 70 (6) ◽  
pp. 1115-1121 ◽  
Author(s):  
Veronica Codullo ◽  
Helen M Baldwin ◽  
Mark D Singh ◽  
Alasdair R Fraser ◽  
Catherine Wilson ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Mononuclear Cells ◽  
Platelet Rich Plasma ◽  
Critical Role ◽  
Serum Levels ◽  
Pcr Analysis ◽  
Peripheral Blood Mononuclear ◽  
Matrix Deposition ◽  
Disease Subtype ◽  
Inflammatory Chemokines

ObjectivesSystemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to sites of tissue damage; their removal from inflamed sites is essential for resolution of the inflammatory response. The atypical chemokine receptor D6 has a critical role in this physiological balance. To explore potential deregulation of this system in SSc, inflammatory chemokine and D6 expression were compared with that in healthy controls (HC).MethodsSerum levels of inflammatory mediators were assessed by luminex analysis. Peripheral blood mononuclear cells (PBMCs) were used in molecular and immunocytochemical analysis. Platelet-rich plasma was collected and assessed by western blotting for D6 expression levels. Sex-matched HC were used for comparison.Results72 patients with SSc and 30 HC were enrolled in the study. The chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and IL-8/CXCL8 were significantly increased in patients with SSc, regardless of disease subtype and phase. Quantitative PCR analysis revealed a significant 10-fold upregulation of D6 transcripts in patients with SSc compared with controls, and this was paralleled by increased D6 protein expression in the PBMCs of patients with SSc. Platelet lysates also showed strong D6 expression in patients with SSc but not in controls. Importantly, high levels of D6 expression correlated with reduced levels of its ligands in serum.ConclusionsInflammatory chemokines and the regulatory receptor D6 are significantly upregulated in SSc and high D6 levels are associated with lower systemic chemokine levels, indicating that some patients control systemic chemokine levels using D6. These results suggest that chemokines may represent a therapeutic target in SSc.

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