scholarly journals Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line

2020 ◽  
Vol 21 (22) ◽  
pp. 8658
Author(s):  
Erik Maronde
Keyword(s):  
Cell Line ◽  
Transcriptional Activation ◽  
Organic Anion ◽  
Neuronal Cell ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Phosphodiesterase Inhibition ◽  
Activity Maximum ◽  
Hippocampal Cell

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp-8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

scholarly journals Mutations in the tat Gene Are Responsible for Human Immunodeficiency Virus Type 1 Postintegration Latency in the U1 Cell Line

1998 ◽  
Vol 72 (2) ◽  
pp. 1666-1670 ◽  
Author(s):  
Stephane Emiliani ◽  
Wolfgang Fischle ◽  
Melanie Ott ◽  
Carine Van Lint ◽  
Carol Ann Amella ◽  
...  
Keyword(s):  
Cell Line ◽  
Transcriptional Activation ◽  
Jurkat Cells ◽  
Luciferase Reporter ◽  
Tat Protein ◽  
Protein Sequence Analysis ◽  
Luciferase Reporter Gene ◽  
Necrosis Factor Alpha ◽  
Severe Reduction ◽  
Factor Alpha

ABSTRACT Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13→L). Bothtat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13→L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.

scholarly journals Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

2021 ◽  
Vol 22 (10) ◽  
pp. 5221
Author(s):  
Danqi Zeng ◽  
Jaime A. Teixeira da Silva ◽  
Mingze Zhang ◽  
Zhenming Yu ◽  
Can Si ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Flower Development ◽  
Regulatory Elements ◽  
Negative Control ◽  
Dendrobium Officinale ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Genome Wide ◽  
Ap2 Transcription Factor

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

scholarly journals Estrogen Receptor-β Mediates Dihydrotestosterone-Induced Stimulation of the Arginine Vasopressin Promoter in Neuronal Cells

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3371-3382 ◽  
Author(s):  
Toni R. Pak ◽  
Wilson C. J. Chung ◽  
Laura R. Hinds ◽  
Robert J. Handa
Keyword(s):  
Androgen Receptor ◽  
Cell Line ◽  
Arginine Vasopressin ◽  
Promoter Activity ◽  
Neuronal Cell ◽  
Luciferase Reporter ◽  
Adult Males ◽  
Primary Metabolite ◽  
Neuronal Cell Line ◽  
The Brain

Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17β-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5α-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5α-androstane-3β,17β-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERβ, and ERβ splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5α-androstane-3β,17β-diol stimulate AVP promoter activity through ERβ in a neuronal cell line.

scholarly journals ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Lei Zheng ◽  
Hongmei Xu ◽  
Ya Di ◽  
Lanlan Chen ◽  
Jiao Liu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Transcriptional Activation ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
M2 Polarization ◽  
Lysine Specific Demethylase ◽  
Dependent Inhibition ◽  
Kinase Suppressor Of Ras ◽  
The Stability

Abstract Background We tried to elaborate the molecular mechanism of ETS-like transcription factor 4 (ELK4) affecting gastric cancer (GC) progression through M2 polarization of macrophages mediated by lysine-specific demethylase 5A (KDM5A)-Praja2 (PJA2)-kinase suppressor of ras 1 (KSR1) axis. Methods GC expression dataset was obtained from GEO database, and the downstream regulatory mechanism of ELK4 was predicted. Tumor-associated macrophages (TAMs) were isolated from GC tissues. The interaction among ELK4, KDM5A, PJA2 and KSR1 was analyzed by dual luciferase reporter gene, ChIP and Co-IP assays. The stability of KSR1 protein was detected by cycloheximide (CHX) treatment. After TAMs were co-cultured with HGC-27 cells, HGC-27 cell biological processes were assessed through gain- and loss-of function assays. Tumorigenicity was detected by tumorigenicity test in nude mice. Results In GC and TAMs, ELK4, KDM5A and KSR1 were highly expressed, while PJA2 was lowly expressed. M2 polarization of macrophages promoted the development of GC. ELK4 activated KDM5A by transcription and promoted macrophage M2 polarization. KDM5A inhibited the expression of PJA2 by removing H3K4me3 of PJA2 promoter, which promoted M2 polarization of macrophages. PJA2 reduced KSR1 by ubiquitination. ELK4 promoted the proliferative, migrative and invasive potentials of GC cells as well as the growth of GC xenografts by regulating KSR1. Conclusion ELK4 may reduce the PJA2-dependent inhibition of KSR1 by transcriptional activation of KDM5A to promote M2 polarization of macrophages, thus promoting the development of GC.

Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5206-5206
Author(s):  
Irene Ganan-Gomez ◽  
Yue Wei ◽  
Hui Yang ◽  
Maria Carmen Boyano-Adánez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Conflicts Of Interest ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Mirna Cluster ◽  
Luciferase Reporter Gene ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Gene Assay

Abstract Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation. For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide. Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures: No relevant conflicts of interest to declare.

Expression of Core-Binding Factor-β Is Regulated By Mir-143 and Mir-223 during Granulopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2721-2721
Author(s):  
Maria Torp Larsen ◽  
Anette Borgkilde ◽  
Stine Novrup Clemmensen ◽  
Niels Borregaard ◽  
Jack B. Cowland
Keyword(s):  
Cell Proliferation ◽  
Transcription Factors ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Conflicts Of Interest ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Strong Suppression ◽  
Genes Encoding ◽  
Granule Proteins

Abstract Granulopoiesis is a strictly regulated process governed to a large extent by an ordered temporal expression of transcription factors. One of the earliest expressed transcription factors, obligatory for correct granulopoiesis, is the Runx1:CBF-beta heterodimer that induces cell division and transcription of genes encoding azurophile granule proteins. The expression level of Runx1:CBF-beta peaks in myeloblasts (MBs) and promyelocytes (PMs) and declines when cells enter the myelocyte (MC) stage, where cell proliferation ceases and terminal differentiation commences. Runx1 binds DNA directly and provides the transcriptional activation domain whereas CBF-beta binds to Runx1 and increases its DNA-binding affinity. CBF-beta is in most cases required for Runx1-mediated transcriptional activity. microRNAs (miRNAs) post-transcriptionally regulate protein expression and are important for proper granulopoiesis. Using a combined density gradient and immunomagnetic purification protocol, we have isolated neutrophil precursors from human bone marrow and identified 135 differentially regulated miRNAs. Two of these, miR-143 and miR-223, were predicted to target CBFB mRNA that encodes the CBF-beta subunit. Both miRNAs are expressed at low levels in MBs and PMs and increases 5-fold when the cells mature to MCs and remain high in the following stages of neutrophil differentiation. This expression pattern supports a role for miR-143 and miR-223 as translational inhibitors of CBF-beta that diminish the transcriptional capacity of Runx1 and thus aid in terminating cell proliferation in MCs. Transient transfection of HEK293 cells, that express CBF-beta endogenously, with pre-miR-143 and pre-miR-223 demonstrated a strong suppression of CBF-beta synthesis when both miRNAs were present. A luciferase reporter assay where the 3’-UTR of CBFB mRNA is inserted in the 3’-UTR of the firefly luciferase transcript confirmed that both miRNAs affect translation. Mutation of either the miR-143 or miR-223 miRNA recognition element (MRE) partly relieved repression of the luciferase mRNA and mutation of both MREs completely abolished translational repression. shRNA against CBFB mRNA was used to detect transcriptional targets affected by downregulation of CBF-beta in a murine promyelocyte cell line (MPRO) and in the myeloblastic cell line 32Dcl3 and expression of a number of genes encoding azurophilic granule proteins were found to be significantly diminished. Together these data demonstrate that miR-143 and miR-223 affect CBF-beta expression and indicate that correct timing of expression of these two miRNAs is a prerequisite for proper granulopoiesis. Grant acknowledgments: The Danish Medical Research Council, Novo Nordisk Foundation, Brøchner Mortensen Foundation, and the Danish Cancer Society. MTL and AB contributed equally to the work Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

2009 ◽  
Vol 23 (11) ◽  
pp. 1746-1757 ◽  
Author(s):  
Hyereen Kang ◽  
Yoon Suk Kim ◽  
Jesang Ko
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Transmembrane Domain ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Basic Leucine Zipper

Abstract The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as “sLZIP” contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

scholarly journals Mutant MyoD Lacking Cdc2 Phosphorylation Sites Delays M-Phase Entry

2004 ◽  
Vol 24 (4) ◽  
pp. 1809-1821 ◽  
Author(s):  
Lionel A. J. Tintignac ◽  
Valentina Sirri ◽  
Marie Pierre Leibovitch ◽  
Yann Lécluse ◽  
Maria Castedo ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Kinase Activity ◽  
Expression Patterns ◽  
Cyclin B ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cdc2 Kinase ◽  
Specific Expression ◽  
M Phase ◽  
Phase Entry

ABSTRACT The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G2/M transition by controlling the expression of p21Waf1/Cip1 (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G2. In growing myoblasts, MyoD reaccumulates during G2 concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G2 and delays M-phase entry. This G2 arrest is not observed in p21−/− cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G2/M transition.

scholarly journals Transcriptional activation of vesicular monoamine transporter 2 in the pre-B cell line Ea3.123

1999 ◽  
Vol 337 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Fiona WATSON ◽  
Damian G. DEAVALL ◽  
Janet A. MACRO ◽  
Rachel KIERNAN ◽  
Rod DIMALINE
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Vesicular Monoamine Transporter ◽  
Start Site ◽  
Monoamine Transporter ◽  
Vesicular Monoamine Transporter 2

Uptake and storage of monoamines in secretory granules is accomplished by vesicular monoamine transporters, and it is likely that vesicular monoamine transporter 2 (VMAT2) is important for histamine transport in vivo. In the present study we have used the pre-B-cell line Ea3.123 to investigate the mechanisms involved in the transcriptional activation of the VMAT2 gene. In Ea3.123 cells, VMAT2 mRNA abundance was increased following mobilization of intracellular calcium, and this increased mRNA expression was paralleled by changes in l-histidine decarboxylase mRNA, suggesting that VMAT2 may be responsible for sequestration of histamine into secretory vesicles in this cell line. We cloned the 5´-flanking region of the VMAT2 gene and determined its transcriptional start site by primer extension of rat VMAT2 mRNA. There was no TATA or TATA-like sequence upstream of this region; instead there were GC-rich elements, Ca2+/cAMP-response-element- and SP1-binding motifs. Approx. 900 bp upstream of the transcriptional start site was a purine–pyrimidine repeat sequence that may form a Z-DNA structure. A series of 5´-deletional VMAT2-promoter segments cloned upstream of a luciferase reporter were capable of driving transcription and indicated the presence of multiple regulatory elements, while stimulation with ionomycin or PMA resulted in an increased level of the transcriptional activity of the 5´-promoter segments studied.

Transcriptional regulation of the cystathionine-β-synthase gene in Down syndrome and non–Down syndrome megakaryocytic leukemia cell lines

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1551-1557 ◽  
Author(s):  
Yubin Ge ◽  
Tanya L. Jensen ◽  
Larry H. Matherly ◽  
Jeffrey W. Taub
Keyword(s):  
Transcriptional Regulation ◽  
Down Syndrome ◽  
Cell Line ◽  
Cell Lines ◽  
Promoter Activity ◽  
Leukemia Cell ◽  
Chromosome 21 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21–localized gene, cystathionine-β-synthase(CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5′-cDNA ends (5′-RACE) analysis demonstrated exclusive use of the CBS−1b promoter in the cell lines, and transient transfections with the full-length CBS −1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS −1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f– or GT-d–binding site resulted in an approximately 90% decrease of theCBS −1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to theCBS −1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns ofCBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.

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