CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

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.

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

Blood ◽

10.1182/blood.v92.9.3416.421k16_3416_3421 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3416-3421 ◽

Cited By ~ 2

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.

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Direct interaction of NF-E2 with hypersensitive site 2 of the β-globin locus control region in living cells

Blood ◽

10.1182/blood.v96.1.334.013k17_334_339 ◽

2000 ◽

Vol 96 (1) ◽

pp. 334-339 ◽

Cited By ~ 4

Author(s):

E. Camilla Forsberg ◽

Karen M. Downs ◽

Emery H. Bresnick

Keyword(s):

Gene Expression ◽

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Erythroid Cell ◽

Hypersensitive Site ◽

Intact Cells ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia ◽

Globin Gene Expression

The human β-globin locus control region (LCR) confers high-level, tissue-specific expression to the β-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for β-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in - and β-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of β-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate β-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of β-globin gene expression through activation of the LCR.

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S1 nuclease analysis of alpha-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.84.8.2387 ◽

1987 ◽

Vol 84 (8) ◽

pp. 2387-2390 ◽

Cited By ~ 5

Author(s):

J. Helder ◽

A. Deisseroth

Keyword(s):

Gene Expression ◽

Globin Gene ◽

S1 Nuclease ◽

Erythroleukemia Cells ◽

Alpha Globin ◽

Globin Gene Expression ◽

Hemoglobin H Disease ◽

Mouse Erythroleukemia

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Developmental programs of human erythroleukemia cells: globin gene expression and methylation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4917 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4917-4926 ◽

Cited By ~ 19

Author(s):

T Enver ◽

J W Zhang ◽

N P Anagnou ◽

G Stamatoyannopoulos ◽

T Papayannopoulou

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Expression Profiles ◽

Globin Gene ◽

Beta Globin ◽

Gamma Globin ◽

Erythroleukemia Cells ◽

Erythroleukemic Cell ◽

High Level ◽

Globin Gene Expression

We investigated the programs of globin gene expression in three known (K562, HEL, and KMOE) and three novel (OCI-M1, OCI-M2, and HEL-R) human erythroleukemic cell lines of adult origin. RNAs from induced and uninduced cells were analyzed for epsilon-, gamma-, delta-, and beta-, zeta-globin-specific transcripts. While high-level gamma-globin expression was common, the lines differed in their expression of embryonic (epsilon, zeta) and adult (delta, beta) globin mRNAs. The patterns of globin gene methylation were generally consistent with their observed expression profiles, with many of the same correlations being seen in normal cells. Although the programs of globin gene expression and methylation displayed by the lines appeared to be diverse, they were not random; rather, they made developmental sense, mimicking defined globin gene programs observed during normal human development. The characteristics exhibited by several of these lines suggest that they may have been derived from the transformation of multi- or oligopotent hematopoietic progenitor cells. We speculate that the expression of fetal or embryonic globins in these adult erythroleukemic cell lines is not an aberration of neoplastic transformation but is indicative of a fetal or embryonic potential in normal adult hematopoietic progenitors.

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Direct interaction of NF-E2 with hypersensitive site 2 of the β-globin locus control region in living cells

Blood ◽

10.1182/blood.v96.1.334 ◽

2000 ◽

Vol 96 (1) ◽

pp. 334-339 ◽

Cited By ~ 72

Author(s):

E. Camilla Forsberg ◽

Karen M. Downs ◽

Emery H. Bresnick

Keyword(s):

Gene Expression ◽

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Erythroid Cell ◽

Hypersensitive Site ◽

Intact Cells ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia ◽

Globin Gene Expression

Abstract The human β-globin locus control region (LCR) confers high-level, tissue-specific expression to the β-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for β-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in - and β-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of β-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate β-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of β-globin gene expression through activation of the LCR.

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

Blood ◽

10.1182/blood.v92.9.3416 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3416-3421 ◽

Cited By ~ 15

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

Abstract 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.

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