scholarly journals Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1832-1837 ◽  
Author(s):  
P Vyas ◽  
JA Sharpe ◽  
P Watt ◽  
DR Higgs ◽  
WG Wood
Keyword(s):  
Regulatory Elements ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Promoter Regions ◽  
Regulatory Regions ◽  
Chromosomal Organization ◽  
Erythroleukemia Cells ◽  
Cast Doubt ◽  
Mouse Erythroleukemia

Abstract Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1832-1837 ◽  
Author(s):  
P Vyas ◽  
JA Sharpe ◽  
P Watt ◽  
DR Higgs ◽  
WG Wood
Keyword(s):  
Regulatory Elements ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Promoter Regions ◽  
Regulatory Regions ◽  
Chromosomal Organization ◽  
Erythroleukemia Cells ◽  
Cast Doubt ◽  
Mouse Erythroleukemia

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

scholarly journals Regulation of human fetal and adult globin genes in mouse erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1355-1363 ◽  
Author(s):  
BJ Morley ◽  
CA Abbott ◽  
WG Wood
Keyword(s):  
Globin Genes ◽  
Beta Globin ◽  
Chromosome 11 ◽  
Globin Mrna ◽  
Chromosomal Organization ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
Regulatory Information ◽  
Beta Gene ◽  
Mouse Erythroleukemia

Abstract We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta- globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5′ end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

scholarly journals Regulation of human fetal and adult globin genes in mouse erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1355-1363 ◽  
Author(s):  
BJ Morley ◽  
CA Abbott ◽  
WG Wood
Keyword(s):  
Globin Genes ◽  
Beta Globin ◽  
Chromosome 11 ◽  
Globin Mrna ◽  
Chromosomal Organization ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
Regulatory Information ◽  
Beta Gene ◽  
Mouse Erythroleukemia

We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta- globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5′ end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

scholarly journals Trans-activation of human globin genes by HTLV-I tax1

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2749-2754 ◽  
Author(s):  
HB Fox ◽  
PD Gutman ◽  
HP Dave ◽  
SX Cao ◽  
M Mittelman ◽  
...  
Keyword(s):  
Chromosomal Location ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Beta Globin ◽  
Flanking Sequence ◽  
Promoter Regions ◽  
Base Pairs ◽  
Activation Response ◽  
Insight Into

Abstract We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5′- deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5′-flanking sequence or 177 bp of epsilon-globin 5′ flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.

scholarly journals Trans-activation of human globin genes by HTLV-I tax1

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2749-2754
Author(s):  
HB Fox ◽  
PD Gutman ◽  
HP Dave ◽  
SX Cao ◽  
M Mittelman ◽  
...  
Keyword(s):  
Chromosomal Location ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Beta Globin ◽  
Flanking Sequence ◽  
Promoter Regions ◽  
Base Pairs ◽  
Activation Response ◽  
Insight Into

We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5′- deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5′-flanking sequence or 177 bp of epsilon-globin 5′ flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.

scholarly journals Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 705-712 ◽  
Author(s):  
NP Anagnou ◽  
TY Yuan ◽  
E Lim ◽  
J Helder ◽  
S Wieder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Chromosome ◽  
Globin Gene ◽  
Globin Genes ◽  
Chromosome 16 ◽  
Regulatory Factors ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3416-3421 ◽  
Author(s):  
E. Skarpidi ◽  
G. Vassilopoulos ◽  
G. Stamatoyannopoulos ◽  
Q. Li
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Mrna Levels ◽  
Globin Genes ◽  
Position Effects ◽  
Erythroleukemia Cells ◽  
Cell Clones ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

scholarly journals Modeling of Epigenetic Modification-Induced Changes in CRX-dependent Genes cis-Regulatory Elements

2017 ◽  
Author(s):  
Reafa A. Hossain ◽  
Nicholas R. Dunham ◽  
Megan E. Harris ◽  
Taylor L. Hutchinson ◽  
Justin M. Kidd ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Binding Sites ◽  
Epigenetic Modification ◽  
Inverse Correlation ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Ocular Tissues ◽  
Regulatory Regions

AbstractPurposeDNA methylation is a well characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of synthesizing a strand of RNA from DNA, or transcription, is controlled by proteins that regulate RNA polymerase activity by binding to specific gene regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell specific gene expression. While much is known about the functions and DNA binding specificity of CRX, less is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements.MethodsWe used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO, PDE6B, PAX6, and LINE. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences.ResultsIn this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to our DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate that changes to the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX dependent transcription.ConclusionCollectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

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