scholarly journals Transcriptional regulation and chromatin structure of the human CD34 gene promoter region

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1822-1830 ◽  
Author(s):  
XY He ◽  
PN Cockerill ◽  
D Cen ◽  
BR Davis
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Promoter Region ◽  
Hematopoietic Cells ◽  
Dnase I ◽  
Molecular Control ◽  
Dnase I Hypersensitivity ◽  
Human Cd34 ◽  
Cd34 Cells

The human CD34 surface antigen is selectively expressed on stem/progenitor cells within the hematopoietic system. Because CD34 expression is tightly linked to the immature status of hematopoietic cells, with expression being rapidly lost as hematopoietic cells mature and differentiate, an examination of its regulation may provide important insights into the molecular control of blood cell development. A comparison of the CD34 nuclear transcription rate in CD34+ and CD34- cells indicated that the CD34 gene is transcriptionally regulated in hematopoietic cell lines. In a previous report, we had identified two major clusters of CD34 transcription initiation sites by 5′ RACE (rapid amplification of cDNA ends) analysis. In transient transfection experiments, we now demonstrate the ability of sequences encompassing each of these clusters to function as promoters of transcription in CD34+ cells. These promoters functioned at equivalent levels in CD34+ and CD34- cells, and the addition of 5′ flanking sequences, extending as far as 3.7 kb upstream, to the core promoters did not differentially modify the level of expression in CD34+ versus the CD34- cell lines. An examination of DNase I hypersensitivity sites within an 18-kb segment of DNA, extending 9 kb either side of the proximal promoter, showed six sites that were primarily associated with CD34- expressing cells. Taken together, these data indicate that the CD34 promoter sequences alone do not confer tissue-or stage-specific expression. Appropriate transcriptional regulation of the CD34 gene must be controlled by chromatin structure, as identified by DNase I hypersensitivity, and/or by other tissue- and stage-specific elements present outside of the promoter region.

scholarly journals Transcriptional regulation and chromatin structure of the human CD34 gene promoter region

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1822-1830 ◽  
Author(s):  
XY He ◽  
PN Cockerill ◽  
D Cen ◽  
BR Davis
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Promoter Region ◽  
Hematopoietic Cells ◽  
Dnase I ◽  
Molecular Control ◽  
Dnase I Hypersensitivity ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The human CD34 surface antigen is selectively expressed on stem/progenitor cells within the hematopoietic system. Because CD34 expression is tightly linked to the immature status of hematopoietic cells, with expression being rapidly lost as hematopoietic cells mature and differentiate, an examination of its regulation may provide important insights into the molecular control of blood cell development. A comparison of the CD34 nuclear transcription rate in CD34+ and CD34- cells indicated that the CD34 gene is transcriptionally regulated in hematopoietic cell lines. In a previous report, we had identified two major clusters of CD34 transcription initiation sites by 5′ RACE (rapid amplification of cDNA ends) analysis. In transient transfection experiments, we now demonstrate the ability of sequences encompassing each of these clusters to function as promoters of transcription in CD34+ cells. These promoters functioned at equivalent levels in CD34+ and CD34- cells, and the addition of 5′ flanking sequences, extending as far as 3.7 kb upstream, to the core promoters did not differentially modify the level of expression in CD34+ versus the CD34- cell lines. An examination of DNase I hypersensitivity sites within an 18-kb segment of DNA, extending 9 kb either side of the proximal promoter, showed six sites that were primarily associated with CD34- expressing cells. Taken together, these data indicate that the CD34 promoter sequences alone do not confer tissue-or stage-specific expression. Appropriate transcriptional regulation of the CD34 gene must be controlled by chromatin structure, as identified by DNase I hypersensitivity, and/or by other tissue- and stage-specific elements present outside of the promoter region.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3590-3590
Author(s):  
Samuel A Stoner ◽  
Russell Dekelver ◽  
Miao-Chia Lo ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Human Cd34 ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors

2004 ◽  
Vol 286 (6) ◽  
pp. G922-G931 ◽  
Author(s):  
Lingling Jiang ◽  
Jiafang Wang ◽  
R. Sergio Solorzano-Vargas ◽  
Hugh V. Tsai ◽  
Edgar M Gutierrez ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Promoter Region ◽  
Core Promoter ◽  
Dnase I ◽  
Fc Receptor ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Core Promoter Region

The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor ( FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at −157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.

Effects of Elevated BCR/ABL Expression Level on Cellular Transformation and Sensitivity to Imatinib in Primitive Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1998-1998
Author(s):  
Hardik Modi ◽  
Su Chu ◽  
Tinisha McDonald ◽  
Stephen Forman ◽  
Ravi Bhatia
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Transformation ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Cellular Transformation ◽  
Pcr Analysis ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Elevated Expression

Abstract Increased levels of BCR/ABL (BA) expression in CML hematopoietic cells have been associated with disease progression and resistance to the tyrosine kinase inhibitor imatinib mesylate (IM). Although cell lines with varying levels of BA expression have been studied, the role of elevated BA expression in cell transformation and drug resistance has not been directly evaluated in the primitive human hematopoietic cells in which the disease arises. Here we have used a human transduction model of CML to determine the effects of varying BA expression levels on cellular transformation (proliferation, apoptosis and differentiation) as well as responsiveness to IM. Cord blood (CB) CD34+ cells were transduced with MSCV vectors expressing BA and GFP, or control vectors expressing GFP alone followed by CD34+GFP+ cells selection by flow cytometry sorting. For BA expressing cells, two separate populations were selected based on low or high GFP expression (BAlo and BAhi). Quantitative RT-PCR analysis confirmed increased expression of BA in high GFP expressing cells (5.9±1.5 fold increase in BA:B2M levels in BAhi compared with the BAlo cells, n=3). The proliferation rate of BAhi cells, measured by fold expansion after 3 days of growth factor (GF) culture, was 2.0±0.2 fold higher than BAlo cells, and 6.7±0.1 fold higher than control (n=3). Upon GF deprivation, BAhi cells demonstrated increased resistance to apoptosis (24.4±11.4%, n=3) compared with BAlo (42±12%, n=3) or control cells (45±12%, n=3). BA transduced CD34+ cells generated higher numbers of glycophorin A+ cells than control (35.6%) following GF culture for 7 days. This effect was enhanced in BAhi (91.5%) compared with BAlo cells (77.1%). This was accompanied by an increase in CD33+ myeloid cells and a decrease in CD11b+ cells (36.3, 60.1 and 65.2% CD33+ cells and 6.0, 1.8 and 0.5% CD11b+ cells for control, BAlo and BAhi cells respectively). In addition the frequency of CD41a+ megakaryocytic cells was higher in BAhi (7.0%) relative to BAlo (3.1%) and control cells (1.0%). Next, we asked whether elevated expression of BA resulted in altered sensitivity to IM (0.025μM to 1μM) in an MTS assay. We observed that BAhi cells were more sensitive (83% inhibition at 1μM) to the IM compared with BAlo cells (14% inhibition at 1μM). We also investigated whether elevated levels of expression of two BA kinase mutants, M351T and E255K, were associated with altered IM sensitivity in CD34+ cells. The M351T mutation leads to intermediate level of IM resistance in cell lines. As was observed for wild type BA, CD34+ cells expressing higher levels of M351T demonstrated increased sensitivity to IM. On the other hand, cells expressing high and low levels of the E255K mutant, which is associated with high levels of IM resistance, demonstrated similar levels of IM sensitivity. In conclusion, increased levels of BA expression in human CD34+ cells results in enhanced proliferation; increased resistance to apoptosis following GF withdrawal, and altered differentiation with increased expression of erythroid, megakaryocytic and early myeloid markers and reduced expression of mature myeloid markers. Interestingly, expression of high levels of BA was associated with enhanced rather than reduced sensitivity to IM. Taken together these observations suggest that the effects of varying levels of BA expression on imatinib sensitivity in primitive human hematopoietic cells are determined primarily by increased proliferation rather than reduced apoptosis resulting from enhanced BA expression.

scholarly journals Chromatin structure and transcriptional regulation of human RAG-1 gene

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3785-3791 ◽  
Author(s):  
T Kitagawa ◽  
K Mori ◽  
H Kishi ◽  
H Tagoh ◽  
T Nagata ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chromatin Structure ◽  
Promoter Region ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Dnase I ◽  
Upstream Region ◽  
Specific Expression ◽  
Rag 1

The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5′ upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Anna Jetmore ◽  
P. Artur Plett ◽  
Xia Tong ◽  
Frances M. Wolber ◽  
Robert Breese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
G1 Cells ◽  
Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P < .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

scholarly journals Topoisomerase activity is linked to altered nucleosome positioning and transcriptional regulation in the fission yeast fbp1 gene

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242348
Author(s):  
Ryuta Asada ◽  
Satoshi Senmatsu ◽  
Ben Montpetit ◽  
Kouji Hirota
Keyword(s):  
Transcriptional Regulation ◽  
Fission Yeast ◽  
Chromatin Structure ◽  
Promoter Region ◽  
Nucleosome Positioning ◽  
Dna Topology ◽  
Upstream Region ◽  
Inactive Form ◽  
Topological State ◽  
Transcriptional Output

Chromatin structure, including nucleosome positioning, has a fundamental role in transcriptional regulation through influencing protein-DNA interactions. DNA topology is known to influence chromatin structure, and in doing so, can also alter transcription. However, detailed mechanism(s) linking transcriptional regulation events to chromatin structure that is regulated by changes in DNA topology remain to be well defined. Here we demonstrate that nucleosome positioning and transcriptional output from the fission yeast fbp1 and prp3 genes are altered by excess topoisomerase activity. Given that lncRNAs (long noncoding RNAs) are transcribed from the fbp1 upstream region and are important for fbp1 gene expression, we hypothesized that local changes in DNA topological state caused by topoisomerase activity could alter lncRNA and fbp1 transcription. In support of this, we found that topoisomerase overexpression caused destabilization of positioned nucleosomes within the fbp1 promoter region, which was accompanied by aberrant fbp1 transcription. Similarly, the direct recruitment of topoisomerase, but not a catalytically inactive form, to the promoter region of fbp1 caused local changes in nucleosome positioning that was also accompanied by altered fbp1 transcription. These data indicate that changes in DNA topological state induced by topoisomerase activity could lead to altered fbp1 transcription through modulating nucleosome positioning.

scholarly journals Platelet Factor 4 and Other CXC Chemokines Support the Survival of Normal Hematopoietic Cells and Reduce the Chemosensitivity of Cells to Cytotoxic Agents

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2328-2335 ◽  
Author(s):  
Zhong Chao Han ◽  
Min Lu ◽  
Junmin Li ◽  
Mai Defard ◽  
Bernadette Boval ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Cell Lines ◽  
Cancer Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Platelet Factor 4 ◽  
Cytotoxic Agents ◽  
Platelet Factor ◽  
Cd34 Cells

Abstract The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 μg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 μg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor β1 (TGFβ1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFβ1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFβ1 for 12 days in hematopoietic growth factor–rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 μg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU–granulocyte/macrophage (CFU-GM), and burst-forming units–erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 μg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

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