PLAG1 and USF2 Regulate Primitive Hematopoietic Expression of Musashi-2

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3583-3583
Author(s):  
Muluken S Belew ◽  
Stefan Rentas ◽  
Laura de Rooij ◽  
Kristin J Hope
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Hematopoietic Cell ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Self Renewal ◽  
Model Cell

Abstract The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

1996 ◽  
Vol 16 (7) ◽  
pp. 3245-3254 ◽  
Author(s):  
V Ngô ◽  
D Gourdji ◽  
J N Laverrière
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Cell Lines ◽  
Anterior Pituitary ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Site Specific ◽  
Cpg Dinucleotides ◽  
Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

scholarly journals Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1331-1331
Author(s):  
Mianmian Yin ◽  
Yang Jo Chung ◽  
R. Coleman Lindsley ◽  
Yeulin Zhu ◽  
Robert L. Walker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Regulatory Elements ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Indel Mutation

Abstract Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Disclosures Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

scholarly journals Epigenomic profiling of primate lymphoblastoid cell lines reveals the evolutionary patterns of epigenetic activities in gene regulatory architectures

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raquel García-Pérez ◽  
Paula Esteller-Cucala ◽  
Glòria Mas ◽  
Irene Lobón ◽  
Valerio Di Carlo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Regulatory Element ◽  
Regulation Of Gene Expression ◽  
Regulatory Elements ◽  
Lymphoblastoid Cell Lines ◽  
Whole Genome ◽  
Specific Expression ◽  
Gene Regulatory ◽  
Human Specific

AbstractChanges in the epigenetic regulation of gene expression have a central role in evolution. Here, we extensively profiled a panel of human, chimpanzee, gorilla, orangutan, and macaque lymphoblastoid cell lines (LCLs), using ChIP-seq for five histone marks, ATAC-seq and RNA-seq, further complemented with whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS). We annotated regulatory elements (RE) and integrated chromatin contact maps to define gene regulatory architectures, creating the largest catalog of RE in primates to date. We report that epigenetic conservation and its correlation with sequence conservation in primates depends on the activity state of the regulatory element. Our gene regulatory architectures reveal the coordination of different types of components and highlight the role of promoters and intragenic enhancers (gE) in the regulation of gene expression. We observe that most regulatory changes occur in weakly active gE. Remarkably, novel human-specific gE with weak activities are enriched in human-specific nucleotide changes. These elements appear in genes with signals of positive selection and human acceleration, tissue-specific expression, and particular functional enrichments, suggesting that the regulatory evolution of these genes may have contributed to human adaptation.

MLL-Fusion Proteins Induce a Stem Cell Program in Committed Progenitors To Generate Leukemia Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 465-465
Author(s):  
Andrei V. Krivstov ◽  
David Twomey ◽  
Zhaohui Feng ◽  
Matthew C. Stubbs ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Expression Profiles ◽  
Cellular Reprogramming ◽  
Gene Set Enrichment Analysis ◽  
Myelogenous Leukemia ◽  
Self Renewal ◽  
Leukemia Stem Cell

Abstract Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIhi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R− Lin− Sca-1− c-Kit+ CD34int. FcγRII/IIIint. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin− sca-1+ c-kit+ HSC-enriched population, IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs), IL-7R− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL-rearranged AMLs including HOXA5, HOXA7, HOXA9, HOXA10, MEIS1 and genes not previously known to have a role in MLL-mediated leukemogenesis such as myocyte enhancer factor 2C (MEF2C). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene

2008 ◽  
Vol 294 (5) ◽  
pp. C1261-C1276 ◽  
Author(s):  
Lior Adler ◽  
Edna Efrati ◽  
Israel Zelikovic
Keyword(s):  
Epithelial Cell ◽  
Inner Ear ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Acidic Ph ◽  
Alkaline Ph ◽  
Specific Expression

Pendrin, a Cl−/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5′-flanking DNA sequence and consecutive 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0–7.1) decreased and alkaline pH (7.6–7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10−8 M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3− secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter

1998 ◽  
Vol 274 (3) ◽  
pp. F602-F610 ◽  
Author(s):  
Shinichi Uchida ◽  
Tatemitsu Rai ◽  
Hiroshi Yatsushige ◽  
Yoshihiro Matsumura ◽  
Masanobu Kawasaki ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chloride Channel ◽  
Gene Promoter ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Start Codon ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Isolation And Characterization ◽  
Flanking Region

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle’s loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5′-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5′-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the ∼2.5-kb pair 5′-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the ∼2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5′-flanking region but was lost in the −29 construct, clearly demonstrating that the 22 bp from −51 to −30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.

Intersecting High-Throughput Screens Identifies SERCA As a Target for Modulating NOTCH1 In Hematopoietic Malignancies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 555-555
Author(s):  
Giovanni Roti ◽  
Annie Carlton ◽  
Kenneth N. Ross ◽  
Michele Markstein ◽  
Kostandin Pajcini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Stem Cell ◽  
Cell Lines ◽  
High Throughput ◽  
Lymphoblastic Leukemia ◽  
Cyclopiazonic Acid ◽  
Luciferase Reporter ◽  
Gamma Secretase ◽  
High Throughput Screens

Abstract Abstract 555 While long-term survival rates for children with acute lymphoblastic leukemia (ALL) are approximately 80%, we have reached a plateau over the past decade with combination chemotherapy. ALL remains a leading cause of cancer-related death in children, and even for those children cured, morbidity is significant. Moreover, prognosis for adults with ALL is poor. Alternative approaches to treating this disease are needed. NOTCH1 is a well-validated target in hematopoietic malignancy, with NOTCH1 activating mutations identified in over 50% of T-cell acute lymphoblastic leukemia (T-ALL) and in 12% of chronic lymphocytic leukemias. NOTCH1 is a heterodimeric protein, normally activated by a series of proteolytic cleavages upon ligand stimulation, which forms a transcription factor complex altering gene expression. We intersected the results of two high-throughput screens focused on modulating NOTCH1 to identify new NOTCH1 inhibitors. First, we applied an unbiased, cell-based gene expression signature screening approach to identify new NOTCH1 inhibitors (Gene Expression-based High-throughput Screening (GE-HTS)) in which a gene signature serves as a surrogate for inactive NOTCH1. Among top-scoring compounds inducing the NOTCH1 inactivation signature, we identified inhibitors of SERCA (Sarco/endoplasmic Reticulum Ca++ATPase) channels, such as the natural products thapsigargin and cyclopiazonic acid. We integrated these results with a cDNA library screen for NOTCH1 activation in which U2OS cells expressing a leukemic NOTCH1 allele, L1601PδP, were scored for NOTCH1-sensitive RBPJ luciferase reporter activity. This screen also identified the SERCA genes ATP2A2 and ATP2A3 as top candidates, whose expression activates NOTCH1, complementing our original observation of SERCA inhibition as an approach to inhibit NOTCH1. SERCA proteins reside on the endoplasmic reticulum and are responsible for the transfer of calcium from the cytosol of the cell to the lumen of the endoplasmic reticulum. We focused our attention on the more potent molecule, thapsigargin, and confirmed that it induces the NOTCH1 off signatures across multiple T-ALL cell lines and targets NOTCH1 signaling by demonstrating its inhibitory effects on the RBPJ luciferase reporter in heterologous U2OS cells expressing the L1601PδP mutation. The anti-leukemic phenotype induced by thapsigargin was consistent with that previously reported with inhibition of gamma secretase, the enzyme involved in the final activation step of NOTCH1. Decreased viability and cell size and a G1/G0 arrest were observed upon low nanomolar thapsigargin treatment in multiple T-ALL cell lines. These effects were on-target for NOTCH1 inhibition. Overexpression of the activated form of NOTCH1, (ICN1) restored the expression of the NOTCH1 target genes c-MYC and DTX1 upon thapsigargin treatment and rescued the effects of thapsigargin on T-ALL viability. As in gamma secretase inhibitor (GSI) treatment, we observed that thapsigargin altered the level of active ICN1 protein in T-ALL cell lines. However, in contrast to treatment with GSI, thapsigargin also decreased levels of the furin-processed transmembrane forms of NOTCH1, while the full length NOTCH1 precursor accumulated as demonstrated by western blot and immunofluorescence studies. To further confirm the role of SERCA activity in NOTCH1 maturation, we inhibited Ca++ATPase activity with SERCA-directed shRNA in T-ALL cell lines and demonstrated that genetically-driven inhibition of ATP2A2 results in loss of ICN1 and trans-membrane NOTCH1 and the expected phenotypic consequences: reduced viability and G1/G0 arrest. To confirm that both chemical and genetic inhibition of SERCA leads to NOTCH inactivation in vivo, we adopted a Drosophila intestinal cancer stem cell model in which the inhibition of NOTCH1 prevents differentiation of stem cell daughter cells resulting in an expansion of the stem cell population. Treating Drosophila with thapsigargin or cyclopiazonic acid resulted in an expansion of the stem cell pool consistent with NOTCH inhibition. Indeed, a transgenic RNAi hairpin directed against Ca-P60A (the Drosophila SERCA orthologue) recapitulated the NOTCH phenotype observed with chemical treatment. In summary, these pre-clinical studies suggest a role for SERCA modulation of NOTCH1 maturation and identify SERCA as a potentially druggable target to inhibit NOTCH1 in T-ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Methylation of the Cyclin A1 Promoter Correlates with Gene Silencing in Somatic Cell Lines, while Tissue-Specific Expression of Cyclin A1 Is Methylation Independent

2000 ◽  
Vol 20 (9) ◽  
pp. 3316-3329 ◽  
Author(s):  
Carsten Müller ◽  
Carol Readhead ◽  
Sven Diederichs ◽  
Gregory Idos ◽  
Rong Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cpg Island ◽  
Trichostatin A ◽  
Specific Gene ◽  
Cyclin A1 ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression

ABSTRACT Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

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